Multidrug resistant

Ann N Y Acad Sci, 2001 Dec, 953, 165 - 84
DOTS and DOTS-plus: not the only answer; Farmer P; Multidrug-resistant tuberculosis is already a global pandemic, with focal "hot spots" of ongoing transmission . Although DOTS (directly observed treatment, short course) chemotherapy is the goal of global tuberculosis control, short-course chemotherapy will not cure multidrug-resistant tuberculosis . In settings of high transmission of multidrug-resistant tuberculosis, "DOTS plus" (a complementary DOTS-based strategy with provisions for treating multidrug-resistant tuberculosis) is warranted . DOTS-plus project implementation to date reveals important clinical, epidemiological, and economic lessons . Community-based strategies designed to enhance local capacity are cost effective and make it possible to meet new medical challenges.

Hepatol Res, 2002 Jan, 22(1), 58 - 64
Increased expression of multidrug resistance-associated protein 1 (mrp1) in hepatocyte basolateral membrane and renal tubular epithelia after bile duct ligation in rats; Pei QL et al.; Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver . Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence . The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated . To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL) . Bile duct was ligated in Wistar rats . Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy . Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes . In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL . Furthermore, mrp1 immunofluorescence increased over time after BDL . No specific immunoflurescence of mrp1 was detected in control rat kidney . However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL . This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL . This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

J Bioenerg Biomembr, 2001 Dec, 33(6), 503 - 11
The role of half-transporters in multidrug resistance; Bates SE et al.; ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance . Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance . These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1 . This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan . At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement . Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.

J Bioenerg Biomembr, 2001 Dec, 33(6), 481 - 91
The mechanism of action of multidrug-resistance-linked P-glycoprotein; Sauna ZE et al.; P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis . Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis . The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release . In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle . Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner . Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding . Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.

J Bioenerg Biomembr, 2001 Dec, 33(6), 469 - 74
ABC proteins of Leishmania; Legare D et al.; ABC proteins were first characterized in the protozoan parasite Leishmania while studying mechanisms of drug resistance . PGPA is involved in resistance to arsenite and antimonite and it most likely confers resistance by sequestering metal-thiol conjugates into an intracellular vesicle . PGPA is part of gene family with at least four more members which are in search of a function . Leishmania also contains a P-glycoprotein, homologous to the mammalian MDR1, that is involved in multidrug resistance . The ongoing genome project of Leishmania has pinpointed several novel ABC transporters and experiments are carried out to study the function of the ABC proteins in drug resistance and in host-pathogen interactions.

Bone Marrow Transplant, 2001 Dec, 28(12), 1171 - 3
Pneumonia and sepsis due to fluoroquinolone-resistant Capnocytophaga gingivalis after autologous stem cell transplantation; Geisler WM et al.; Human oral Capnocytophaga species have been only rarely described as a cause of sepsis in patients following stem cell or marrow transplantation, and pneumonia has not been reported in this setting . In addition, fluoroquinolone resistance is rarely seen in these species, and has never been reported in C . gingivalis . We report a case of pneumonia (confirmed by culture of bronchoalveolar lavage fluid) and sepsis due to fluoroquinolone- resistant Capnocytophaga gingivalis in a patient following autologous stem cell transplantation, who responded to treatment with linezolid and metronidazole . Capnocytophaga infections should be considered in patients with fever following stem cell or marrow transplantation, especially those with neutropenia and mucositis . Susceptibility testing is needed given the existence of multidrug-resistant isolates.

Biochem J, 2002 Feb 1, 361(Pt 3), 497 - 503
Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues; Lai L et al.; Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters . Within the MRP subfamily, ABCC4,ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies . All lack the additional transmembrane domain, TMD(0), which is present in the other MRPs . Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH . ABCC4 also facilitates the efflux of cAMP . Depletion of intracellular GSH with DL-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed . ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine . This resistance can be reversed by the presence of DL-buthionine-(S,R)-sulphoximine . We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH . In addition, GSH plays an important role in the function of ABCC4 . Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4 . Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.

Clin Cancer Res, 2002 Jan, 8(1), 221 - 32
Differences in therapeutic indexes of combination metronomic chemotherapy and an anti-VEGFR-2 antibody in multidrug-resistant human breast cancer xenografts; Klement G et al.; One of the greatest barriers to the treatment of cancer with chemotherapeutic drugs is acquisition of drug resistance . This includes multidrug resistance mediated by P-glycoprotein (Pgp) to multiple lipophilic natural compounds such as taxanes, doxorubicin (Adriamycin), and vinblastine . The considerable efforts made thus far to reverse this and other types of drug resistance have had very limited success . We report here that a variety of orthotopic human breast cancer xenografts selected for high levels of Pgp and multidrug resistance respond in a significant and durable manner to different continuous low-dose (e.g., one-tenth the maximum tolerated dose of chemotherapy) chemotherapy regimens, when used in combination with an antivascular endothelial cell growth factor (anti-VEGF) receptor-2 (flk-1)-neutralizing antibody (DC101) . The Pgp substrates paclitaxel (Taxol), Adriamycin, and vinblastine were all effective using this type of combination treatment, although the chemotherapy protocols showed little or no effect as monotherapies . Similar results were also obtained using cisplatinum (a non-Pgp substrate drug) against cisplatinum-resistant tumors . Evidence of significant tumor cell death by the combination treatment was detected within 3 weeks of initiation of therapy by histopathological analysis, in the absence of shrinkage of tumor mass . There were, however, marked differences in the cumulative toxicity of long-term regimens of Adriamycin and cisplatinum, where toxicity was observed, when compared with the tubulin inhibitors, vinblastine and Taxol, where it was not . We conclude that vascular-targeting protocols involving frequent administration of very low doses of certain chemotherapeutic drugs can provide a stable and safe way to circumvent multidrug resistance in established orthotopically growing tumors, as long as these are used in combination with a second antiangiogenic drug, in this case, anti-VEGFR-2 blocking antibodies.

Clin Cancer Res, 2002 Jan, 8(1), 22 - 8
The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells; Kim M et al.; The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines, including the MCF-7 cell line, selected for its resistance to mitoxantrone (MCF-7/MitoR) . Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells . Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine, a substrate for the MDR1 transporter . Other studies have identified a primitive hematopoietic stem cell population, or side population (SP) cells, which are identified by their efflux of the fluorescent dye, Hoechst 33342 . In an attempt to identify the transport genes responsible for this phenotype, we examined the uptake of Hoechst 33342 into MCF-7, MCF-7/MitoR, and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2) . MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells . We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells . Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells . These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells.

J Med Assoc Thai, 2001 Sep, 84(9), 1241 - 5
In vitro susceptibility testing of levofloxacin and ofloxacin by microtiter plate Alamar blue against multidrug and non multidrug resistant Mycobacterium tuberculosis in Thailand; Pracharktam R et al.; Antituberculous drugs for current therapy of multidrugs resistant tuberculosis (MDR-TB) are limited . The in vitro susceptibility of Mycobacterium tuberculosis (MTB) as well as MDR-TB with other antibiotic drugs were determined in order to find alternative drugs for the treatment of MDR-TB . Forty-seven MDR-TB and 62 MTB of clinical isolates were tested against levofloxacin and ofloxacin . The MDR-TB at the MIC90 of levofloxacin and ofloxcain were 1 microg/ml and 2 microg/ml, respectively . Of these MTB, the MIC90 of both drugs were 0.5 microg/ml and 1 microg/ml, respectively . It seemed that levofloxacin MICs of both MDR-TB and MTB were one dilution less than ofloxcin . The promising activity of ofloxacin and levofloxacin against MDR-TB and MTB suggest that both drugs could be used as second-line drugs for MDR-TB or as a good alternative antituberculous drug for patients who have intolerance to the first line drug.

Zhong Yao Cai, 2001 Sep, 24(9), 655 - 7
{Study on active constituents of traditional Chinese medicine reversing multidrug resistance of tumor cells in vitro}; Zhang H et al.; OBJECTIVE: To screen drugs reversing multidrug resistance of tumor cells from active constituents of traditional Chinese medicine and to study the reversal action . METHODS: The kill effects of the drugs on tumor cell lines in vitro were determined with MTT method . The Jin's formula was used to analyse the effect of drug combination . RESULTS: 5 micrograms/ml rhynchophylline, 2 micrograms/ml jatrorrhizine and 1.25 micrograms/ml indirulin could reverse multidrug resistance for vincristine on KBv200 cell line by 16.8, 5.1 and 4 fold respectively . 1.56-12.5 micrograms/ml curcumine combining with vincristine could sensitize antitumor effect both on KB and KBv200 cell lines . CONCLUSION: All rhynchophylline, jatrorrhizine and indirulin could reverse multidrug resistance for vincristine on KBv200 cell line . Curcumine combinating vincristine could sensitize antitumor effect both on kB and kBv200 cell lines.

J Virol, 2002 Feb, 76(4), 1753 - 61
Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection; Brenner BG et al.; This study examines the persistence and fitness of multidrug-resistant (MDR) viruses acquired during primary human immunodeficiency virus infection (PHI) . In four individuals, MDR infections persisted over the entire study period, ranging from 36 weeks to 5 years, in the absence of antiretroviral therapy . In stark contrast, identified source partners in two cases showed expected outgrowth of wild-type (WT) virus within 12 weeks of treatment interruption . In the first PHI case, triple-class MDR resulted in low plasma viremia (1.6 to 3 log copies/ml) over time compared with mean values obtained for an untreated PHI group harboring WT infections (4.1 to 4.3 log copies/ml) . Increasing viremia in PHI patient 1 at week 52 was associated with the de novo emergence of a protease inhibitor-resistant variant through a recombination event involving the original MDR virus . MDR infections in two other untreated PHI patients yielded viremia levels typical of the untreated WT group . A fourth patient's MDR infection yielded low viremia (<50 to 500 copies/ml) for 5 years despite his having phenotypic resistance to all antiretroviral drugs in his treatment regimen . In two of these PHI cases, a rebound to higher levels of plasma viremia only occurred when the M184V mutation in reverse transcriptase could no longer be detected and, in a third case, nondetection of M184V was associated with an inability to isolate virus . To further evaluate the fitness of MDR variants acquired in PHI, MDR and corresponding WT viruses were isolated from index and source partners, respectively . Although MDR viral infectivity (50% tissue culture infective dose) was comparable to that observed for WT viruses, MDR infections in each case demonstrated 2-fold and 13- to 23-fold reductions in p24 antigen and reverse transcriptase enzymatic activity, respectively . In dual-infection competition assays, MDR viruses consistently demonstrated a marked replicative disadvantage compared with WT virus . These results indicate that MDR viruses that are generated following PHI can establish persistent infections as dominant quasispecies despite their impaired replicative competence.

Zhonghua Yi Xue Za Zhi, 2001 Mar 25, 81(6), 328 - 31
{Alteration of subcellular distribution of protein kinase C isoforms in swelling-activated multi-drug-resistant gastric cancer cells and its significance}; Han Y et al.; OBJECTIVE: To study the alteration of expression and subcellular distribution of classical protein kinase (cPKC) isoforms in gastric cancer cells SGC7901 and their multidrug-resistant cell line SGC7901/VCR under condition of swelling activation and to study the significance of such alterations . METHODS: Immuno-fluorescence technique and Western Blotting were used to determine the expression and subcellular distribution of cPKC isoforms in gastric cancer cells SGC7901 and its multidrug-resistant cell line SGC7901/VCR under normal condition and during continuous hypotonic perfusion . The co-expression of p-glycoprotein (Pgp) and protein kinase C alpha (PKC alpha) was visualized by immunofluorescence double labeling and laser confocus microscopy . RESULTS: Under the normal condition, the four isoenzymes of cPKC were expressed in both the cell membrane and the nuclei of the gastric cancer cells SGC7901 and their multidrug-resistant cell line SGC7901/VCR; PKC alpha was strongly positively expressed in the multidrug-resistant cells and positively expressed in the drug-sensitive cells; PKC beta I and PKC beta II were positively expressed in both drug-resistant and drug-sensitive cells, and PKC gamma was strongly positively expressed in both cells . In drug-resistant cell line, cell-swelling and translocation of PKC alpha and PKC gamma were observed ten minutes after continuous hypotonic perfusion . Thirty minutes after the perfusion, almost all the PKCalpha was translocated into the cell membrane of SGC7901/VCR, part of the PKC gamma was translocated into the nucleus, and the cell volume increased to two to three times as much as before . Sixty minutes later, the subcellular distribution of PKCalpha and PKC gamma and the cell volume returned to normal . In drug-sensitive cells, 10 approximately 20 minutes after the continuous hypotonic perfusion nearly all the PKCalpha was translocated into the cell membrane, part of PKC gamma was translocated into the cell membrane, and the cell volume increased to two to three times as much as before . Forty minutes later, PKCalpha and PKCgamma had basically returned to normal . The subcellular distribution of PKCbeta I and PKC beta II remained unchanged in both SGC7901 and SGC7901/VCR . Co-expression of Pgy and PKCalpha was observed in both drug-sensitive and drug-resistant cells, especially in the former . CONCLUSION: Only PKCalpha and PKC gamma isoforms play an important role in the regulation of signal transduction of Pgy and cell volume under continuous perfusion and are related to the expression of PKC alpha and Pgy . PKC beta I and PKC beta II may have nothing to do with such processes.

Zhonghua Yi Xue Za Zhi, 2000 Mar, 80(3), 219 - 21
{Reversal of multidrug resistance in lung adenocarcinoma-resistant cell line A549/R by mdr1 antisense oligodeoxynucleotides in vitro}; Chen L et al.; OBJECTIVE: To investigate the possibility of reversion of multidrug resistance (MDR) in lung adenocarcinoma-resistant cell line A549/R by mdr1 antisense oligodeoxynucleotide . METHODS: A 15-mer phosphorothioate antisense oligodeoxynucleotide (ATCCATCCCGACCTC), complementary to -9 approximately + 6 in mdr1 cDNA sequence, was synthesized . Meanwhile, sense sequence (GAGGTCGGGATGGAT) was taken as control . Both antisense and sense oligodeoxynucleotides were transduced to A549/R cell line by lipofectin . The mdr1 mRNA, expression of Pgp, cellular rhodamine accumulation and sensitivity to daunorubicin were detected in antisense and sense oligodeoxynucleotides treated cells, using RT-PCR, flow cytometry, rhodamine test and MTT method . RESULTS: Treatment with antisense oligodeoxynucleotide in A549/R cells led to a decrease in mdr1 mRNA and Pgp expression, an increase in rhodamine cellular accumulation, and a 20-fold increase of sensitivity to doxorubicin compared with those treated with sense oligodeoxynucleotide or control cells . CONCLUSION: The mdr1 antisense oligodeoxynucleotide possesses an effect of reversing MDR in lung adenocarcinoma-resistant cell A549/R by inhibiting mdr1 transcription and Pgp expression.

Zhonghua Nei Ke Za Zhi, 1999 Nov, 38(11), 760 - 3
{The clinical significance of lung resistance-related protein gene (lrp), multidrug resistance-associated protein gene (mrp) and mdr-1/p170 expression in acute leukemia}; Zhao Y et al.; OBJECTIVE: To evaluate the relationship between lrp, mrp, mdr-1/p170 and multidrug resistance in acute leukemia (AL) . METHODS: 85 AL patients were divided into three groups: untreated (A), complete remission (B) and relapsed/refractory (C) . The expression of lrp, mrp and mdr-1 mRNA was detected with RT-PCR assay and that of p170 measured with immunocytochemistry . RESULTS: The frequency of lrp gene expression in ANLL A, B and C group was 11.11%, 9.09% and 36.36%, in ALL A, B, C group it was 0%, 20.00% and 46.67%; the frequency of mrp gene expression in ANLL A, B, C group was 44.44%, 9.09% and 59.09%, in ALL A, B, C group it was 28.57%, 20.00% and 53 . 33%; the frequency of mdr-1 gene expression in ANLL A, B, C group was 0%, 4.54% and 59.09%, in ALL A, B, C group it was 0%, 0% and 33.33%; p170 expression in ANLL C group was (9.45 +/- 14.66) %, it was the highest value in the three groups; statistics showed that there was no correlationship among the expressions of lrp, mrp and mdr-1 gene, P > 0.05; patients with lrp and mdr-1 expression had a lower complete remission (CR) percentage than those who had not in the untreated group; the combination of lrp, mrp and mdr-1 gene detection turned out to be a more sensitive, specific and accurate way in evaluation of multidrug resistance (MDR) . CONCLUSION: Overexpression of one or more genes of lrp, mrp and mdr-1/p170 is associated with MDR in AL . Expression of lrp, mrp and mdr-1/p170 is good indicators in clinical MDR . Two or three factors of lrp, mrp and mdr-1 are more valuable in the evaluation of MDR than any one of these three factors . The mechanisms of mrp, lrp and mdr-1 causing MDR are different, it means that the mechanisms of MDR are complicated the combination of lrp, mrp and mdr-1/p170 detection may be the best way to evaluate MDR.

Zhonghua Nei Ke Za Zhi, 1999 Jun, 38(6), 373 - 6
{Reversal of multidrug resistance of leukemia cell line in vitro by antisense phosphorothioate oligonucleotide}; Chen Y et al.; OBJECTIVE: To investigate the effectiveness of 14 mer antisense phosphorothiaoate oligonucleotide (AS-SODN) complementary to the published multidrug resistance gene (mdr(1) gene) sequence to reverse multidrug resistance of leukemia cell line . METHODS: 50% inhibitory concentration (IC(50)) of K562 cell line resistant to adriamycin (K562/ADM cell line) was determined by the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the concentration of daunorubicin (DNR) in the cell by flow cytometry, the level of P170 by immunohistochemical staining technique and the level of mdr(1)-mRNA by half-quantitative RT-PCR (the ratio of mdr(1)/beta(2)-MG) . RESULTS: Treatment of K562/ADM cell line with 2 micromol/L AS-SODN reduced the level of both the mdr(1)-mRNA and P170 . The ratio of mdr(1)/beta(2)-MG reduced from (2.07 +/- 0.16) to (1.57 +/- 0.14), and positive rate of P170 expression decreased from (98.26 +/- 1.35)% to (18.30 +/- 6.50)% . Accordingly, the concentration of DNR in cell increased . With these findings, IC(50) to ADM reduced from 25.72 mg/L to 6.97 mg/L and relative effciency of modulation was 73.01% . CONCLUSION: Antisense oligonucleotide targeting mdr(1) gene can reduce md(1)-mRNA, block P170 expression so as to increase the sensitivity of K562/ADM cell line to chemotherapy drugs.

Zhonghua Nei Ke Za Zhi, 1999 Jan, 38(1), 47 - 9
{Expression and clinical study of multidrug resistance gene and multidrug resistance associated protein gene in acute leukemia}; Ma J et al.; OBJECTIVE: To evaluate the relationship between the expression of multidrug resistance gene (mdr1) or multidrug resistance-associated protein gene (MRP) and the prognosis in patients with acute leukemia (AL) . METHODS: The expression of mdr1 and MRP were measured in 55 patients with AL by reverse transcription polymerase chain reaction (RT-PCR) . RESULTS: The mdr1 and MRP gene expression levels in the relapsed AL and the blastic phase of CML group (0.735 +/- 0.249, 1.157 +/- 0.447) were significantly higher than those in the untreated group (0.408 +/- 0.186, 0.465 +/- 0.253) (P < 0.01) . The complete remission (CR) rate in high mdr1 and MRP expression group was significantly lower than that in low mdr1 and MRP expression group (P < 0.01) in a follow up of 40 AL patients . CONCLUSION: Increased expression of mdr1 and MRP gene might be an important factor for predicting drug resistance, relapse and unfavorable prognosis in AL patients.

Stem Cells, 2002, 20(1), 11 - 20
ABC transporters as phenotypic markers and functional regulators of stem cells; Bunting KD; Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment . The multidrug resistance-1 (MDR1) gene product, P-glycoprotein, and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis, with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-), respectively . Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described . However, recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis . Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia, but very low to undetectable in normal myelomonocytic cells . Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice . A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach . Additionally, the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors, makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues . Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation . In light of these findings, it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers.

J Neurochem, 2002 Jan, 80(1), 64 - 72
P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress; Felix RA et al.; During ischaemia/reperfusion, cells of the blood-brain barrier are subjected to oxidative stress . This study uses primary cultured rat brain endothelial cells to examine the effect of such stresses on expression of multidrug transporters . H(2)O(2) up to 500 microm applied to cell monolayers caused a concentration-dependent increase in expression of P-glycoprotein (Pgp) but not of multidrug resistance-associated protein (Mrp1) . Concentrations > 250 microm H(2)O(2) decreased cell viability . Application of 100 microm H(2)O(2) caused a significant increase after 48 h in Pgp functional activity, as assessed from {(3)H}vincristine accumulation experiments . At this concentration, H(2)O(2) produced a transient increase within 10 min followed by a sustained decrease in levels of intracellular reactive oxygen species (iROS), detectable by flow cytometry . Reoxygenation of cell monolayers after 6 h hypoxia gave rise to a similar transient increase in iROS and this also led to increased Pgp expression by 24 h . Increases were also observed within 4 h after both H(2)O(2) and hypoxia/reoxygenation treatments in mdr1a and mdr1b mRNA . Evidence suggests this was due to enhanced transcription rather than mRNA stabilization . Therefore, oxidative stress, by changing Pgp expression, may affect movement of Pgp substrates in and out of the brain.

Antimicrob Agents Chemother, 2002 Feb, 46(2), 443 - 50
Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India; Siddiqi N et al.; The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection . We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M . tuberculosis from India to identify the respective mutations . The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin) . We found known as well as novel mutations at these loci . Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M . tuberculosis isolates and occurred with frequencies different from those reported earlier . Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin . We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions . Mutations were also mapped in the ribosomal binding site of the inhA gene . A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates . A few isolates showed other mutations in this locus . Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121 . While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M . tuberculosis strains prevalent in this region . Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.

Antimicrob Agents Chemother, 2002 Feb, 46(2), 344 - 9
Involvement of multidrug resistance-associated protein 2 in intestinal secretion of grepafloxacin in rats; Naruhashi K et al.; We investigated the contribution of multidrug resistance-associated protein 2 (MRP2) to the secretory transport of grepafloxacin and compared its functional role with that of P-glycoprotein (P-gp) by using Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs), in which MRP2 is hereditarily defective . In intestinal tissue from SDRs mounted in Ussing chambers, the level of transport in the direction from the serosal layer to the mucosal layer was twofold greater than that in the direction from the mucosal layer to the serosal layer . This secretory transport of grepafloxacin was diminished by both probenecid, an MRP2 inhibitor, and cyclosporine, a P-gp inhibitor . In intestinal tissue from EHBRs, the secretory transport of grepafloxacin was lower than that in intestinal tissue from SDRs and was inhibited by cyclosporine but not by probenecid . The absorption of grepafloxacin from intestinal loops in SDRs was in the order of duodenum > jejunum > ileum and was increased by cyclosporine but not by probenecid . The absorption in EHBRs was not higher than that in SDRs . The intestinal secretory clearance in SDRs after intravenous administration of grepafloxacin was shown to be greater for the ileum than for the duodenum, which is in good agreement with the previously reported regional expression profile of MRP2 mRNA . The intestinal secretory clearance was lower in EHBRs than in SDRs . Accordingly, in addition to P-gp, MRP2 might play a role in the secretory transport of grepafloxacin . The function of MRP2 in facilitating grepafloxacin transport in the secretory direction is more pronounced both in vitro and in vivo, while the restriction of entry from the lumen into the cell by MRP2 seems to be negligible, compared with that by P-gp, in the case of grepafloxacin.

Breast Cancer, 2001, 8(4), 333 - 8
Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues; Kubota T et al.; Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane . After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene . Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp . MRP also excretes substrates through the cell membrane using energy from ATP catabolism . The substrate of MRP is conjugated with glutathione before active efflux from cell membrane . Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at and A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively . Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively . Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm . LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp . The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

Int J Oncol, 2002 Feb, 20(2), 255 - 60
Comparative genomic hybridization study of genetic changes associated with vindesine resistance in esophageal carcinoma; Obara K et al.; The acquisition of drug-resistance is a major problem for cancer patients undergoing chemotherapy . To clarify genetic alterations in cancer cells that develop drug-resistance, comparative genomic hybridization (CGH) was applied to esophageal squamous cell carcinoma cell lines (SH-1V1, SH-1V2, SH-1V4 and SH-1V8) and chemoresistance-related genes in altered chromosomal regions were evaluated . These cell lines were derived from the parental SH-1 cell line, after multiple steps of selection by an increasing exposure to vindesine . SH-1V8 cells were strongly resistant to vindesine . DNA copy number at 16p which includes the MRP (multidrug resistance related protein) gene was markedly increased in all cell lines examined . Increased DNA copy numbers were found at the regions of 5q31-32, 10q11.1-23, and 14q32-qter in SH-1V8 cells that acquired resistance to other drugs as well . Both SH-1V4 and SH-1V8 showed increased DNA copy numbers at 7q11.1-22, 16q12.1-qter, 19p13.2-13.3, 19q11-13.2 and 20q13.1-qter . The chromosomal region of 7q11.1-22 including MDR-1 (multidrug resistance-1) gene was highly amplified in SH-1V4 and SH-1V8 . Amplification of the MRP region suggests the prerequisite of developing resistance to vindesine, and further amplification of MDR-1 may play a critical role in acquiring drug-resistance . Several unknown genes related to the induction of chemoresistance might be concealed in other altered chromosomal regions.

Respiration, 2001, 68(6), 590 - 4
Natural killer cell activity in multidrug-resistant pulmonary tuberculosis; Yildiz P et al.; BACKGROUND: Multidrug-resistant pulmonary tuberculosis (MDRTB), a major problem in developing countries, may result from either insufficiency of host cellular immune response or mycobacterial mechanisms which has been more intensively investigated so far . OBJECTIVES: The aim of the study was to investigate natural killer cell activity (NKA) and T lymphocyte subsets in HIV- patients with secondary MDRTB . Methods: 20 male patients with MDRTB (mean age 38 +/- 8 years), 15 nonresistant tuberculosis male patients (NRTB) (mean age 36 +/- 11 years) and 12 healthy male controls (mean age 35 +/- 8 years) were included . The percentages of CD3+, CD4+, CD8+, CD25+, CD11b+ and CD16+56+ cells were measured by flow-cytometric analysis of peripheral blood lymphocytes (PBL) . NKA was evaluated using the anticandidal index method . RESULTS: The mean tuberculin response was higher in MDRTB and NRTB patients compared to controls (15.4 +/- 3.8, 15.1 +/- 3.3 and 10.9 +/- 2.8 mm, respectively; p < 0.001) . There was no significant correlation between PPD response and PBL subsets or NKA . The percentages of both CD3+ and CD3+CD4+ T lymphocytes were significantly lower in MDRTB (62.4 +/- 12.1 and 33.9 +/- 9.0%) compared to NRTB (70.8 +/- 7.5 and 42.9 +/- 8.6%; p < 0.05) . Patients with MDRTB had significantly lower NKA compared to NRTB and controls (30.9 +/- 11.3, 49.7 +/- 15.5 and 40.0 +/- 8.5%, respectively; p < 0.01) . CONCLUSION: This reduction in NKA may suggest a role for impaired NK function in the pathogenesis of MDRTB in HIV- patients .

Med Dosw Mikrobiol, 2001, 53(3), 291 - 5
{Etiologic agents of fungemia in hospitalized patients}; Swoboda-Kopec E et al.; The aim of performed examinations was the analysis of fungi as etiological agents of blood infections in patients hospitalized in surgical wards, internal medicine wards and intensive care units of the Medical Academy Central Clinical Hospital in Warsaw . Blood samples from patients hospitalized in 1997 were examined . Peripheral blood samples were incubated in BacT/Alert system (Organon Teknika, USA) . Positive blood samples were inoculated on Sabouraud medium with chloramphenicol (bioMerieux, France or Oxoid, England) . The time of cultivation was from 48 hours to 7 days at 30 degrees C . Fungal strains were identified by standard mycological procedures with the use of chromogenic medium BBL CHROMagar Candida (Becton Dickinson, USA) and biochemical test ID 32 C (bioMerieux, France) . Susceptibility of strains to antifungal agents was determined by ATB FUNGUS method (bioMerieux, France) . The total number of positive blood cultures in 1997 was 1380 . Forty-two fungal strains were isolated from blood samples (3%) . Strains belonged to the following species: C . albicans (17 isolates), C . parapsilosis (15), C . glabrata (3), melibiosica (2), C . pelliculosa (2), C . guilliermondii (1), C . tropicalis (1) and T . beigelii (1) . Among fungi cultured from patients hospitalized in operative wards dominated C . parapsilosis (11) and C . albicans (10) strains, whereas from patients hospitalized in conservative wards most often C . albicans (6) strains were isolated . Candida strains were mostly susceptible to antifungal agents tested . It was interesting to culture Trichosporon beigelii (T . cutaneum) strain as an etiological agent of fungemia . This strain was multidrug-resistant.

Zhonghua Zhong Liu Za Zhi, 2001 Jul, 23(4), 281 - 4
{Differential display of vincristine-resistant proteins in gastric cancer cell line SGC7901}; Wang X et al.; OBJECTIVE: To find out new multidrug-resistant proteins in gastric cancer cells SGC7901 and to explain the new multidrug-resistant mechanism of gastric cancer cells . METHODS: Two-dimensional gel electrophoresis was used with immobilized pH gradients (IPG) to compare the differential expression of multidrug-resistant proteins in gastric cancer cells SGC7901 and Vincristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate . 2-D gels were used to silver stain the protein . RESULTS: Approximately, 680 protein spots were identified in each of the 2-D gel patterns by silver stain . With most of them showing no difference in composition, shape or density, twenty-five proteins were found to differ in quantity (6 higher in SGC7901/VCR cells; 19 higher in 7901 cells) . Five proteins were seen to be unique in one region or the others (3 in SGC7901/VCR cells, 2 in 7901 cells) . The coordinate position of 30 protein spots in the 2-D gels were listed . CONCLUSION: The results suggest that these differential proteins be related to the vincristine-resistant mechanism in human gastric cancer cell line SGC7901/VCR.

Zhonghua Zhong Liu Za Zhi, 2001 May, 23(3), 184 - 6
{Subcellular distribution of daunorubicin in the P-glycoprotein-mediated multidrug-resistant cell line K562/ADR}; Gong Y et al.; OBJECTIVE: To examine subcellular distribution of daunorubicin (DNR) in P-glycoprotein-mediated multidrug-resistant cell line K562/ADR and its relation to multidrug resistance . METHODS: The subcellular distribution of DNR in K562/ADR was studied by confocal scanning laser microscopy (CSLM), fluorometry, RT-PCR . Rhodamine 123, NBD-ceramide and neutral red as fluorescent probes to stain the mitochondria, Golgi apparatus and lysosomes respectively were used to identify the subcellular compartments wherein DNR was sequestered . Effect of verapamil, chloroquine and brefeldin A on DNR distribution and accumulation was examined . RESULTS: Compared with the drug-sensitive cell K562/S in which DNR fluorescence diffusely appeared in the nucleus and cytoplasm, DNR in K562/ADR cells was distributed to the perinuclear region and peripheral cytoplasm, It was scenty in the nucleus and other cytoplasmic regions, as suggested by the distribution of Rhodamine123 . Only verapamil, but not chloroquine and brefeldin A, could markedly restore diffuse cytoplasmic and nuclear fluorescence distribution in the resistant cell line . CONCLUSION: Altered subcellular distribution of DNR in drug resistant cell line may participate in the generation of multidrug resistance in which P-glycoprotein plays an important role.

Zhonghua Zhong Liu Za Zhi, 2001 Jan, 23(1), 53 - 6
{Immunoelectron microscopic analysis of P-glycoprotein, p53, and Bcl-2 proteins expressions in lung cancer}; Huang X et al.; OBJECTIVE: To determine the ultrastructural localization of P-gp, p53 protein, and Bcl-2 protein in lung cancer cells, their relation to multidrug resistance and possible mechanisms of action . METHODS: Expression of P-gp, p53, and Bcl-2 proteins was examined in 8 NSCLC surgical specimens and 7 SCLC bronchoscopically biopsied specimens using postembedding PAG immunolabelling technique for electron microscopy . RESULTS: P-gp was detected on the cell membrane and the periphery of endoplasmic reticulum (ER) . P53 protein was observed not only in the nuclei associated with heterochromatin but also in the cytosol . Bcl-2 protein immunoreactivity was associated with mitochondria and ER . P-gp, p53, and Bcl-2 were detected in 5(33%), 9(60%), and 4 (26.7%) out of the 15 samples examined, respectively . In 8 normal lung tissues, these three proteins were detected . Of 5 P-gp positive samples 4 were NSCLC, only 1 was SCLC after chemotherapy . CONCLUSION: P-gp, p53, and Bcl-2 proteins are detectable immuno-electron microscopically in lung cancer cells . No correlation in expression existed between of p53 and P-gp, nor did that between p53 and Bcl-2 . The plasma membrane localization of P-gp supports its action as a transmembrane drug efflux pump . P-gp may play a role in MDR in lung cancer.

Zhonghua Zhong Liu Za Zhi, 2001 Mar, 23(2), 103 - 6
{Expression and function of protein kinase C-alpha and beta I isoenzymes in drug-resistant gastric cancer cells}; Han Y et al.; OBJECTIVE: To study the expression and function of PKC-alpha and beta I isoenzymes in drug-resistant cells of gastric cancer . METHODS: Two tumor cell lines were used in the study: gastric cancer SGC7901 and its drug-resistant counterpart SGC7901/VCR stepwise-selected by various concentrations of vincristine . The expression of PKC-alpha and beta I isoenzymes in SGC7901 and SGC7901/VCR was detected by immunohistochemistry, laser confocal scanning microscopy and Western blot . The effects of anti-PKC-alpha or beta I antibody on adriamycin accumulation in SGC7901/VCR cells were determined by flow cytometric analysis . RESULTS: SGC7901 cells exhibited positive staining of PKC-alpha . The staining of PKC-alpha in SGC7901/VCR was stronger than that in SGC7901 cells . The higher the concentrations of vincristine, the stronger staining of PKC-alpha was observed on SGC7901/VCR cells . Both SGC7901 and SGC7901/VCR cells were positive for PKC-beta I, but without difference in staining intensity . Comparing with SGC7901, SGC7901/VCR cells showed decreased adriamycin accumulation; the decrease was more marked with the increase in concentrations of vincristine for selection of cells . Increased adriamycin accumulation in SGC7901/VCR was observed when co-incubated with anti-PKC-alpha but not anti-PKC-beta I antibodies . CONCLUSION: PKC-alpha may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.

J Pharm Sci, 2002 Jan, 91(1), 117 - 28
Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells; Tran CD et al.; The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated . Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 microM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow . Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 microM 1alpha,25-dihydroxyvitamin D(3) to promote cellular CYP3A4 expression . Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 +/- 54.35%) compared to that of parent cells (100 +/- 6.05%) . Furthermore, they retained significantly less {(3)H}vincristine sulphate (p < 0.05), a P-gp substrate, after efflux (272.89 +/- 11.86 fmol/mg protein) than the parent cells (381.39 +/- 61.82 fmol/mg protein) . The expression of CYP3A4 in parental cells after 1alpha,25-dihydroxyvitamin D(3) treatment was quantified to be 76.2 +/- 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 +/- 20.0 pmol/mg protein) . Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting . Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 +/- 20.0 to 1057.0 +/- 46.0 pmol/min/mg protein . Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells . The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics .

J Acquir Immune Defic Syndr, 2002 Jan 1, 29(1), 11 - 20
Dioxolane guanosine, the active form of the prodrug diaminopurine dioxolane, is a potent inhibitor of drug-resistant HIV-1 isolates from patients for whom standard nucleoside therapy fails; Mewshaw JP et al.; Amdoxovir ({-}-beta-D-2,6-diaminopurine dioxolane {DAPD}) is a nucleoside reverse transcriptase inhibitor (NRTI) with activity against HIV-1 . DAPD is deaminated in vivo by adenosine deaminase to (-)-beta-D-dioxolane guanosine (DXG), a highly active anti-HIV compound . The median 50% effective concentrations (EC 50 ) +/- SD (representing antiviral activity against a laboratory-derived HIV-1 isolate) for DAPD and DXG in peripheral blood mononuclear cells were 4.0 +/- 2.2 micromol/L and 0.25 +/- 0.17 micromol/L, respectively . The 50% cytotoxic dose (CC 50 ) of both DAPD and DXG was >500 micromol/L . Recombinant viruses and clinical isolates of HIV-1 from patients for whom NRTI therapy and/or nonnucleoside reverse transcriptase inhibitor (NNRTI) combination therapies failed remained susceptible to inhibition by DXG (less than fourfold change in EC 50) . Similar analysis showed that recombinant viruses harboring mutations known to confer resistance to NRTIs (zidovudine, lamivudine, and abacavir) and NNRTIs (efavirenz and nevirapine) as well as the multidrug resistance-associated mutation Q151M and double codon insertions (SS and SG) were also susceptible to inhibition by DXG . Resistance to DXG was observed only in recombinant isolates containing the 65R and 151M double mutations . Phenotypic analysis of a site-directed mutant containing only the 151M mutation demonstrated moderate resistance to DXG (<10-fold change in EC 50) . We also examined site-directed mutants containing only L74V or K65R, the characteristic resistance mutations for DXG . The L74V mutant remained susceptible to inhibition by DXG, and the K65R mutant demonstrated moderate resistance to DXG.

Blood, 2002 Jan 15, 99(2), 641 - 8
P-glycoprotein-actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin; Luciani F et al.; P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype . However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear . On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump-related proteins . To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function . The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein . These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype . Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.

Blood, 2002 Jan 15, 99(2), 507 - 12
The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors; Scharenberg CW et al.; A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342 . The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side "population" (SP) of bone marrow . This SP from bone marrow, as well as other tissues, is reported to contain immature stem cells with considerable plasticity . Some cell lines also efflux Hoechst and generate SP profiles . Reverse transcription-polymerase chain reaction (RT-PCR) and efflux inhibition studies with the lung carcinoma cell line, A549, implicated the ABCG2 transporter as a Hoechst efflux pump . Furthermore, it is shown that transient expression of ABCG2 generates a robust SP phenotype in human embryonic kidney (HEK293) cells . The results allow the conclusion that ABCG2 is a potent Hoechst efflux pump . Semiquantitative RT-PCR was used to characterize the developmental pattern of expression of ABCG2 in hematopoiesis . It is expressed at relatively high levels in putative hematopoietic stem cells (isolated as SP, 34+/38- or 34+/KDR+ populations) and drops sharply in committed progenitors (34+/38+, 34+/33+, or 34+/10+) . Expression remains low in most maturing populations, but rises again in natural killer cells and erythroblasts . Comparison of messenger RNA (mRNA) levels for the 3 major multidrug-resistant efflux pumps, MDR1, MRP1, and ABCG2, in bone marrow SP cells reveals that ABCG2 is the predominant form in these cells . These data suggest that ABCG2 contributes significantly to the generation of the SP phenotype in hematopoietic stem cells . Furthermore, the sharp down-regulation of ABCG2 at the stage of lineage commitment suggests that this gene may play an important role in the unique physiology of the pluripotent stem cell.

Chin Med J (Engl), 2001 Sep, 114(9), 929 - 32
Reversing drug resistance in the ovarian carcinoma cell line SKOV3/mdr1 in vitro by antisense oligodeoxynucleotides; Pan L et al.; OBJECTIVE: To investigate the effect of multidrug resistance gene 1 (mdr1) antisense oligodeoxynucleotides (ODNs) on reversing multidrug resistance in the drug resistant ovarian carcinoma cell line SKOV3/mdr1 . METHODS: The ovarian carcinoma cell line SKOV3 transducted with a human multidrug resistance gene (mdr1) served as the drug resistant model (SKOV3/mdr1) . The mdr1 antisense ODNs was transfected into SKOV3/mdr1 cells while mediated by lipofectamine . Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression and the amount of the mdr1 mRNA in the cells . The positive rate and function of the mdr1 gene product P-glycoprotein (Pgp) in the mdr1 antisense ODNs treated SKOV3/mdr1 cells were determined by flow cytometry and rhodamine 123 efflux . Drug resistance in the SKOV3/mdr1 cell line was observed by MTT assay and cell colony culture . RESULTS: The mdr1 mRNA level was decreased to about 60% of that of beta-actin after mdr1 antisense ODNs treatment . The Pgp positive rate of mdr1 antisense ODNs treated SKOV3/mdr1 cells decreased from 100% to 52.6% (P < 0.01) . The intracellular rhodamine 123 retention was increased from 9.1% to 33.8% (P < 0.01) . The chemoresistance to taxol decreased to 58% of SKOV3/mdr1 with mdr1 antisense ODN treatment . Compared with SKOV3/mdr1 cells in the control group, under a certain range of drug concentrations, the number of drug resistance colonies in mdr1 antisense ODNs treated SKOV3/mdr1 cells for taxol and doxorubicin decreased by 8.6 +/- 0.8 fold and 3.1 +/- 0.6 fold, respectively . Some non-specific functions during oligodeoxyncleotide treatment was also detected . CONCLUSION: mdr1 expression in the SKOV1/mdr1 cell line was partially inhibited after mdr1 antisense ODNs treatment at the mRNA and protein level, increasing the chemotherapy sensitivity of this drug resistant ovarian carcinoma cell line.

Chin Med J (Engl), 2001 Jul, 114(7), 680 - 4
Human stem cell model to study signal transduction and molecular regulation mechanisms in CML; Fan E et al.; OBJECTIVE: To develop a primary human hematopoietic stem/progenitor cell model for chronic myeloid leukemia (CML) and study signal transduction and molecular regulation mechanisms in CML . METHODS: We developed a human model of p210BCR/ABL positive CML by transducing normal human umbilical cord blood CD34+ cells with a retroviral vector containing the b3a2 bcr/abl cDNA . We also examined whether this model recreated the cellular phenotype of CML by assessing cell adhesion, cell migration, cell proliferation and cell survival . RESULTS: We found that significantly more myeloid colony forming units grew from p210BCR/ABL expressing cells, adhesion of p210BCR/ABL expressing CD34+ cells to fibronectin was decreased but migration over fibronectin was enhanced compared with mock transduced CD34+ cells . In this model, we showed that the presence of p210BCR/ABL leads to elevated levels of p27kip in p210BCR/ABL expressing CD34+ cells . We also showed that multidrug resistance-1 (MDR-1) Pgp was upregulated in the p210BCR/ABL expressing cells which correlates with the expression of p210BCR/ABL . CONCLUSION: This primary human CML model recreates most of the features of CML and provides a useful tool to study signal transduction and downstream molecular regulation drived by the p210BCR/ABL oncogene in normal CD34+ cells.

Chin Med J (Engl), 2001 Jan, 114(1), 25 - 9
A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34+ cells to improve combination chemotherapy tolerance; Wang J et al.; OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs . METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml . Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors . RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis . Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently . The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells . CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

Zhonghua Zhong Liu Za Zhi, 2000 Jul, 22(4), 304 - 7
{Expression of LRP, MRP and MDR1 in non-small-cell lung cancer and its clinical significance}; Wang J et al.; OBJECTIVE: To detect expression of lung resistance protein (LRP), multidrug resistance-associated protein(MRP) and multidrug resistance 1(MDR1) mRNA, and its clinical significance in non-small-cell lung cancer (NSCLC) . METHODS: RT-PCR was used to investigate mRNA expression of the above mentioned genes . RESULTS: The frequency of expression of LRP, MRP and MDR1 was 74.2%, 80.3% and 37.9%, respectively . A significant positive correlation was observed between LRP and MRP(r = 0.47, P = 0.001), but this association was found neither between LRP and MDR1, nor between MRP and MDR1 . The expression of LRP, MRP and MDR1 did not vary with the grade of cell differentiation and TNM staging . In adenocarcinomas which responded to chemotherapy, there was lowered expression of both LRP and MRP than those which did not respond . In chemo-responsive squamous-cell carcinomas, however, this was true of LRP expression only . The median survival time of NSCLC patients with co-expressed 2 and 3 drug resistance related genes was significantly reduced . CONCLUSION: The intrinsic multidrug resistance of NSCLC involves the combined effects of LRP, MRP and MDR1.

Zhonghua Zhong Liu Za Zhi, 2000 Jul, 22(4), 273 - 5
{Establishment of MRP-overexpression subline of bladder carcinoma and its MDR phenotype}; Zhang X et al.; OBJECTIVE: To investigate MRP expression and multidrug resistance(MDR) phenotype of a bladder carcinoma subline transfected with the full length MRP cDNA . METHODS: After transfection, a stable MRP-overexpressed subline named EJ/MRP was established . Gene expression of MRP and mdr1 were detected by using RT-PCR and immunohistochemistry methods . Drug sensitivity testing of the EJ/MRP cells to 11 kinds of anti-cancer agents was examined . RESULTS: Compared with EJ/Vect which was mock transfected, MRP mRNA level of EJ/MRP increased 14.3 fold and MRP expression was mainly located in cytosol and plasma membrane . The relative resistance (RR) to VP-16, vincristine increased more than 10 fold, and that to doxorubicin, hydroxycamptothecin, thiotepa, mitomycin and cyclophosphamide increased 3 to 10 fold . CONCLUSION: Bladder carcinoma with stably over-expressed MRP presents typical MDR phenotype but without mdr1/P-gp expression . It provides a good model and positive control for the study of MRP-mediated MDR.

Zhonghua Zhong Liu Za Zhi, 2000 Sep, 22(5), 385 - 8
{Development of drug resistance and alteration of cell cycle in lung cancer: a flow cytometric study}; Xu M et al.; OBJECTIVE: To study the relationship between the expression of multidrug resistance-associated protein (MRP) and P-glycoprotein (P-gp) and alteration of cell cycle during the process of drug resistance in lung cancer . METHODS: Lung adenocarcinoma cell line GLC-82 was treated with low concentration of adriamycin . At 0, 24, 48, 72 and 96 hr following adriamycin treatment, the expression of MRP and P-gp, and cell cycle were examined by bi-parameter flow cytometry . RESULTS: Treatment of GLC-82 cells with adriamycin at a final concentration of 0.05 microgram/ml resulted in progressive decrease in cells in G1 phase and increase in cells in S phase of the cell cycle . The expression level of P-gp was very low and showed little change with time . In contrast, MRP expression was significantly increased in a time-dependent manner, and was positively correlated with changes in S phase of the cell cycle . CONCLUSION: MRP expression may play a major role in the development of resistance to adriamycin.

Zhonghua Zhong Liu Za Zhi, 2000 May, 22(3), 189 - 91
{Reversion of multidrug resistance in vitro of lung adenocarcinoma by mdr1 ribozyme}; Chen L et al.; OBJECTIVE: To investigate the possibility of reversion of multidrug resistant (MDR) in lung adenocarcinoma-resistant cell line A549/R by mdr1 ribozyme . METHODS: With GUC at 880 site of mdr1 mRNA selected as target point, plasmid expressing mdr1 ribozyme (pH beta Apr-1 neo/mdr1-Rb) was transduced to A549/R by lipofectin . The expression of mdr1 mRNA and Pgp, cellular rhodamine accumulation and sensitivity to doxorubicin were examined in ribozyme transduced and non-transduced cells . RESULTS: Transduction of mdr1 ribozyme to A549/R cells led to decrease in mdr1 mRNA and Pgp expression; increase in rhodamine accumulation . The sensitivity of the ribozyme transduced cells to doxorubicin was 200-fold as high as that of the parental drug-resistant cells . CONCLUSION: MDR of lung adenocarcinoma-resistant cell line A549/R can be effectively reversed with mdr1 ribozyme by cleavage of mdr1 mRNA and inhibition of Pgp expression.

Zhonghua Zhong Liu Za Zhi, 2000 May, 22(3), 184 - 8
{Synthesis of chimeric anti-MDR1 ribozymes (tRNA-Rzs) and their biological activities in cell-free system}; Wang F et al.; OBJECTIVE: Development of multidrug resistance(MDR) is the major obstacle to successful cancer chemotherapy . One strategy to block the P-glycoprotein(P-gp)-mediated MDR is to use a ribozyme (Rz) target against MDR1 mRNA . METHODS: Three kinds of anti-MDR1 chimeric hammerhead ribozymes, the first-one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second one, a stem-II base modified (U9-->G9, U13-->A13, G14-->A14, A18-->C18) Rz against codon 196 (196MDR1-sRz) and the third one, the stem-II base modified Rz directed against the -6(-)-4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized based on the cloned retroviral constructs: N2A + tRNAi(met)-196MDR1-Rz, N2A + tRNAi(met)-196MDR1-sRz, N2A + tRNAi(met)-iMDR1-sRz . RESULTS: In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules . The stem-II base modified Rz molecules were also more stable and efficient in catalytic activities than the unmodified ones . In control, the disabled tRNA-mut-iMDR1-sRz had no cleavage activity . CONCLUSION: Base modification in the Rz's stem-II of ribozyme structure and the development of chimeric tRNA-ribozyme molecules are able to enhance the cleavage efficacy.

Exp Cell Res, 2002 Jan 15, 272(2), 185 - 91
Retinoic acid inhibits telomerase activity and downregulates expression but does not affect splicing of hTERT: correlation with cell growth rate inhibition in an in vitro cervical carcinogenesis/multidrug-resistance model; Ding Z et al.; Telomerase, a ribonucleoprotein complex of hTERT, hTR, and TP1, has been reported to be associated with carcinogenesis and multidrug resistance (MDR) . This study used our in vitro human cervical multistep carcinogenesis/MDR model system in which normal human ectocervical and endocervical (HEN) cells were immortalized by HPV18 or 16, respectively, and subsequently transformed . The first evidence was found that immortalization and telomerase activation were correlated with increased expression specifically of two of the hTERT alternatively spliced mRNAs, one encoding wild-type protein containing the full-length functional reverse transcriptase (RT) region and one encoding a defective RT protein . Expression of neither hTERT mRNA containing full-length functional or defective RT motif was affected by transformation/MDR . All-trans-retinoic acid (ATRA) treatment of HPV-immortalized HEN-16-2 cells and transformed/MDR HEN-16-2/CDDP cells inhibited telomerase activity and downregulated expression of hTERT mRNAs containing full-length functional and a defective RT motif, but there were no changes in hTR and TP1 expression . Moreover, ATRA inhibited cell growth rate of HEN-16-2 and HEN-16-2/CDDP cells equally . These results provided the first evidence that ATRA equally in both immortalized and transformed/MDR cell lines inhibits telomerase activity and downregulates expression, but not splicing, of hTERT, and this is correlated with cell growth rate inhibition; the potential is implicated for applying ATRA to hTERT-targeted treatment of cervical cell carcinogenesis/MDR . (c)2001 Elsevier Science.

Zhonghua Kou Qiang Yi Xue Za Zhi, 1999 May, 34(3), 136 - 8
{Establishment and biological characteristics of vincristine: resistant cell line from human squamous cell carcinoma}; Zhang J et al.; OBJECTIVE: To establish a VCR-resistant cell line from human squamous cell carcinoma . METHODS: The cell line Tca8113 was utilized . The VCR concentration was increased gradually in consecutively passage of cultures . RESULTS: A VCR-resistant cell line Tca8113/V100 from Tca8113 was established, the cells grew steadily in 100 mumol/L medium and with 19.2 times of resistance to VCR . The cell line was also resistant to ADM, PYM, CDDP, MTX, 5-Fu at different degrees . The doubling time of the cells was 37.2 hours and showed microscopically and ultrastructurally typical squamous cell carcinoma when inoculated in nude mice . Blot hybridization showed the cells had increased espression of MDR-1mRNA . CONCLUSION: These results indicate that the cell line has multidrug resistant ability, and the reason for this may be over expression of MDR-1 gene . This cell line provides an important model for the study on drug-resistant cell both in cellular and molecular level, and on the mechanism of drug resistance.

Zhonghua Zhong Liu Za Zhi, 1999 Mar, 21(2), 112 - 3
{Clinical significance of multidrug resistance-associated protein(MRP) gene expression in non-small cell lung cancer}; Zhan M et al.; OBJECTIVE: To investigate expression of multidrug resistance-associated protein (MRP) in non-small cell lung cancer and its relation to histological type, TNM staging and prognosis . METHODS: In situ hybridization was used to examine mRNA expression of MRP . RESULTS: The overall positive rate of MRP expression was 74.1%, with 73.3% and 72.0% in adenocarcinoma and squamous-cell carcinoma, respectively . The expression of MRP was not related to histological subtypes, TNM staging and cell differentiation . In 47 patients who received chemotherapy, 35 patients with positive MRP expression(+(-)++) showed worse prognosis than in those with negative expression (P < 0.05) . The median survival time was 8.7 months and 21 months in patients with positive and negative MRP expession, respectively . In patients with squamous-cell carcinoma, the survival rate was significantly lower in patients (n = 12) with positive MRP expression than in those (n = 7) with negative MRP expression (P < 0.05) . Their median survival time was 6 months and 19.5 months, respectively (P < 0.05) . CONCLUSION: The expression of MRP gene is negatively correlated with survival of patients with squamous-cell carcinoma, but not adenocarcinoma, who received chemotherapy.

Zhonghua Zhong Liu Za Zhi, 1999 May, 21(3), 193 - 5
{Expression of multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in human rectal carcinomas and its clinical significance}; Peng X et al.; OBJECTIVE: To investigate the expression and clinical significance of multidrug resistance-associated protein(MRP) and lung resistance protein(LRP) in rectal carcinoma . METHODS: Fiftyseven paraffin embeded primary rectal carcinoma specimens were examined by immunohistochemical staining . RESULTS: Of the 57 tumour specimens examined, 28(49.1%) and 24(42.1%) were positively immunostaned for MRP and LRP, respectively . Co-expression of the two proteins was observed in 10(17.5%) specimens . Positive immunostaning of either of the two proteins was not correlated with cell differentiation and Dukes stage (P > 0.05) . Patients with MRP-positive tumours had shorter survival than MRP-negative patients (P < 0.05) . No such correlation was found for LRP expression, nor was found between MRP and LRP expression . CONCLUSION: Positive MRP expression in rectal carcinoma appears to be an independant factor of prognostic significance . To examine its expression may help guide rational comprehensive therapy of rectal carcinoma.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 Sep, 22(9), 562 - 3
{Cleaning cavity operation in treating relapse bacillary cavitary pulmonary tuberculosis}; Wei C et al.; OBJECTIVE: To study the effective surgical way for serious relapse multidrug resistant bacillary cavitary pulmonary tuberculosis . METHODS: 104 cases of serious relapse multidrug resistant bacillary cavitary pulmonary tuberculosis were cured by cleaning cavity operation from 1981 to 1998 . The operation contained resecting the back rib covering the cavity, separating the intercostal tissue, cutting the external wall of the cavity, cleaning the cavity thoroughly, scraping the cavity till exposing the fresh tissue, killing the tuberculosis germs with organic acid, filling the cavity with the intercostal muscle, fixing and suturing the muscles . RESULTS: Among the 104 cases, 103 cases (99.0%) were cured, 101 cases (98.1%) were cured after the first operation, one case (1.0%) became better . Among the 98 cases followed up, the rate of returning to work were 96%, no one died or relapsed . CONCLUSIONS: The design of cleaning cavity operation is reasonable and it is the effective method to treat the relapse bacillary cavitary pulmonary tuberculosis, the rate of cure and sputum conversion are promising.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 Sep, 22(9), 545 - 7
{The expression of MRP gene relates to the pathological features of lung cancer}; Wang Y et al.; OBJECTIVE: To evaluate the expression of MRP gene and its relation to pathological features in lung carcinoma . METHODS: A series of 35 resected primary lung cancer tissues from 9 post-chemotherapy and 26 untreated patients and adjacent normal lung tissues and 28 lymph node tissues were analyzed for MRP gene expression by RT-PCR method . RESULTS: Positive expression rate of MRP gene was 54% in 35 cases with primary lung cancer, 14% in tissues adjacent to the carcinoma site and the difference was significant (P < 0.005), the positive rate of MRP gene expression in metastatic lymph node tissues was 53% . In 9 cases of pre-operative chemotherapy, the positive expression rate of MRP gene was higher than that in 26 cases without chemotherapy . Nineteen of 35 tumors were found to show positive expression of MRP gene, comprising 11 of 18 adenocarcinoma, 9 of 15 squamous cell cancer . The relapse or metastasis rate with MRP-positive cases was higher(55%) than that with MRP-negative (15%) by follow-up of these patients . CONCLUSIONS: There are overexpression of MRP gene in primary lung cancer tissues . Preoperative chemotherapy may play inductive role in lung cancer multidrug resistance . The positive expression of MRP gene in lung cancer tissues is in accord with its lymph node tissues . Positive expression of MRP gene does not necessarily be associated with tumor size, clinical type and TNM stages, but it could be regarded as one poor prognostic factor.

Zhonghua Zhong Liu Za Zhi, 2000 Jan, 22(1), 27 - 9
{Expression and prognostic significance of multidrug resistance associated protein (MRP) gene in non-small cell lung cancer by in situ hybridization}; Shan G et al.; OBJECTIVE: To study the effect of MRP gene overexpression on prognosis of patients with non-small cell lung cancer (NSCLC) . METHODS: Paraffin-embedded tissues from 47 cases of NSCLC who had undergone radical tumor resection were examined for expression of MRP gene mRNA by in situ hybridization using labelled digoxigenin probes combined with immunohistochemistry . All the patients were retrospectively followed-up . RESULTS: All of the 47 lung cancer specimens were found to have overexpression of MRP mRNA . It was significantly correlated with patients' survival period, response to chemotherapy, recurrence and mestastases after surgery, but was not correlated with histology, tumor size, node status, TNM stage, degree of differentiation, age and sex . CONCLUSION: Overexpression of MRP gene is a marker of prognostic significance in patients with NSCLC.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 Nov, 22(11), 655 - 8
{Expression of multidrug resistance-associated protein in human non-small cell lung cancer}; Peng X et al.; OBJECTIVE: To investigate the expression of the multidrug resistance-associated protein(MRP) gene and MRP in human non-small-cell lung cancer tissues and to determine whether such expression was related to cell type, differentiation, tumour size, lymph node metastasis and prognosis . METHODS: 92 paraffin-embeded lung tumor samples (43 squamous cell carcinoma, 49 adenocarcinoma) and 16 fresh non-small-cell lung cancer samples were examined by using immunohistochemistry method and the reverse transcription-polymerase chain reaction (RT-PCR) technology respectively . RESULTS: The expression of MRP mRNA and MRP were detectable in 31% (5-16) and 54% (50/92) of non-small-cell lung cancer specimens respectively . Eighteen(42%) squamous cell carcinoma specimens and thirty-two(65%) adenocarcinoma specimens showed positive immunostaining for MRP(P < 0.05) . The expression of MRP was not related to tumour size, lymph node metastasis and cell differentiation significantly(P > 0.05) . The five-year survival rate after operation of patients with MRP-positive tumours and MRP-negative tumours were 16% (8/50), 52% (22/42) respectively . Patients with MRP-positive tumours had shorter survival time than the MRP-negative patients significantly(P < 0.01) . CONCLUSIONS: Adenocarcinoma had higher MRP expression than squamous carcinoma significantly, positive MRP immunostaining appears to be an independent indicator of poor prognosis in NSCLC.

Chin Med J (Engl), 2000 Nov, 113(11), 977 - 80
Upregulation of drug sensitivity of multidrug-resistant SGC7901/VCR human gastric cancer cells by bax gene transduction; Zhao Y et al.; OBJECTIVE: To investigate the role of bax in a vincristine (VCR)-induced multidrug-resistant (MDR) human gastric cancer cell line, SGC7901/VCR, in which the Bax protein expression level was significantly lower compared with that in parent cells . METHODS: A bax eukaryotic expression vector was constructed and transfected into SGC7901/VCR cells by lipofectamine, and resistant clones were selected by G418 . Western blotting detected Bax expression in transfectants . Tetrazolium blue (MTT) assay evaluated the differences in drug sensitivity and cell cycle changes of transfectants were analyzed using flowcytometry (FCM) . RESULTS: The bax eukaryotic expression vector was constructed and transfected into SGC7901/VCR cells . Through G418 selection, resistant clones were obtained . Western blotting demonstrated that the expression of Bax protein was markedly increased in bax transduced cells . These cells were more sensitive to adriamycin (ADR) and VCR than mock vector transducted cells . Moreover, bax transfection enhanced ADR-induced apoptosis and VCR-induced G2/M phase arrest of SGC7901/VCR cells . CONCLUSION: Bax was involved in the MDR of SGC7901/VCR cells.

Chin Med J (Engl), 2000 Sep, 113(9), 848 - 51
The role of c-myc in regulating mdr1 gene expression in tumor cell line KB; He Y et al.; OBJECTIVE: To investigate the relationship between c-myc expression and mdr1 regulation in multidrug resistance (MDR) tumor cell line KBv200 . METHODS: A DNA sequence encoding the ribozyme gene was incorporated into a eukaryotic expression vector (pH beta Apr-1 neo) and transfected into the cell line KB, which is resistant to vincristine and expresses the MDR phenotype . The changes of c-myc protein, P-glycoprotein (P-gp) and c-myc mRNA in the KB cell line were assessed before and after treatment with anti-mdr1-ribozyme using immunohisto-chemistry, flow cytometry and semi-quantitative RT-PCR methods . RESULTS: The results demonstrate elevated levels of c-myc mRNA and c-myc protein as well as P-gp in KBv200 cells which are resistant to vincristine (VCR), compared to these of the KB cell which is not resistant to VCR . The expression of c-myc protein, P-gp and c-myc mRNA in the multidrug resistant cell, KBv200, displayed higher than that in the sensitive cell, KB . However, after reversing the multidrug resistance phenotype by anti-mdr1-ribozyme, the level of c-myc protein, P-gp and c-myc mRNA expression showed significant down-regulation in KBv/5 mR3, without difference in KB/5 mR3 and KB . CONCLUSION: The results of our study suggest that there is a close relationship between c-myc gene and mdr1 gene . c-myc may be involved in regulating the expression of mdr1.

Chin Med J (Engl), 2000 Jun, 113(6), 536 - 9
{Chemoprotection of transfer of multidrug resistance gene into human hematopoietic progenitor cell}; Pan L et al.; OBJECTIVE: To observe the effect of the transfer of multidrug resistance gene (mdr1) into human hematopoietic progenitor cells (HPC) on the chemoprotection . METHODS: Human CD34+ cells served as a target of mdr1 gene transfer . Retroviral vector SF-mdr containing human total length mdr1cDNA was introduced into packing cells GP-envAM12 by liposome-mediated transfection . The mdr1 gene was transduced into human CD34+ cells by retroviral supernatants of packing cells . The integration and expression of the mdr1 gene and its protein (P170) in transduced cells were determined by PCR, RT-PCR, and flow cytometry . The drug resistance of chemotherapy in transduced HPC was determined by culturing colonies . RESULTS: The mdr1 gene was integrated and expressed in transduced CD34+ cells . The efficiency of mdr1 gene transfer was 10%-14% . Compared with untransduced controls, within a certain range of drug concentration, the number of drug-resistant colony in transduced HPC for taxol, doxorubicin, VCR and VP16 were increased by 3.6 +/- 2.1 fold, 2.9 +/- 0.3 fold, 1.9 +/- 0.4 fold, and 3.5 +/- 0.5 fold, respectively . CONCLUSION: The transfer of the mdr1 gene into human HPC can increase the drug resistance of the transduced cells to corresponding chemotherapeutic drugs that may provide some degree of chemoprotection for HPC.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 May, 22(5), 268 - 70
{Expression of multidrug resistance-associated protein gene in non-small cell lung cancer}; Xu M et al.; OBJECTIVE: To study the relationship between expression of multidrug resistance-associated protein (MRP) gene and occurrence of multidrug resistance (MDR) in non-small cell lung cancer (NSCLC) . METHODS: Thirty-two NSCLC samples were detected expression of MRP gene and chemosensitivity to 9 antitumor agents by RT-PCR, immunohistochemistry method and in vitro chemosensitivity assay . RESULTS: The positive expression rate of MRP mRNA in NSCLC was 72%, in lung tissue near carcinoma was 10%; the positive expression rate of MRP in NSCLC was 66%, positive staining of MRP located in membrane and cytoplasm of tumor cells, but no positive staining in lung tissue; MRP mRNA and MRP expression of moderate to high differentiation carcinoma was significantly higher than that of low differentiation carcinoma (P < 0.05) . There was significant correlation between expression of MRP mRNA and MRP in NSCLC . The expression of MRP in NSCLC, which was resistant to vincristine, etoposide and adriamycin, was higher than that being sensitive to these drugs (P < 0.05) . CONCLUSIONS: MRP gene may play an important role of intrinsic drug resistance in NSCLC, overexpression of MRP gene can be achieved through modulation at transcriptional and/or translation level . Overexpression of MRP is a major cause of resistance to vincristine, etoposide and adriamycin.

Chin Med J (Engl), 2000 Oct, 113(10), 957 - 60
Co-transfection of MRP and bcl-2 antisense S-oligodeoxynucleotides reduces drug resistance in cisplatin-resistant lung cancer cells; Wang J et al.; OBJECTIVE: To detect the influence of antisense s-oligodeoxynucleotides (S-ODNs) of bd-2 and multidrug resistance-associated protein (MRP) genes multidrug resistance-associated protein gene and bcl-2 antisense S-oligodeoxynucleotides on cisplatin-resistant lung adenocarcinoma cell line A549DDP which overexpresses both bcl-2 and MRP . METHODS: A549DDP cells were treated with sense and antisense S-ODN mediated by lipofection . Expression of MRP and bcl-2 mRNA and protein in the treated cells was measured by RT-PCR and flow cytometry (FCM), respectively . Apoptosis was identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling (TUNEL) . The degree of drug resistance of the treated cells was detected by a cell viability 3'-{4,5-dimethylthiazol-2-yl}-2, 5-diphenyl-tefrazolium bromide thiazolylblue (MTT) assay . RESULTS: Expression of bcl-2 and MRP significantly decreased in the cells treated with bcl-2 or/and MRP antisense S-ODN for 48 h as compared to the cells untreated and sense-treated (P < 0.05) . Resistance to cisplatin in the cells treated with bcl-2 or/and MRP antisense S-ODN decreased by 60.6% (6.5 times), 56.4% (7.2 times) and 71.0% (4.8 times), respectively, which paralleled the decrease of bcl-2 and MRP expression . Similarly, the resistance to etoposide and epirubicin in antisense-treated cells also reduced in parallel to decreases of the two gene expressions . The drug resistance in sense-treated cells was similar to that in untreated cells . Statistically significant dose- and concentration-dependent increases of apoptotic cells were observed in the groups exposed to 100 mumol/L cisplatin for 48 h after treatment by bcl-2 or/and MRP antisense . CONCLUSION: Bcl-2 and MRP were at least additive and possibly synergistic in conferring drug resistance in a cisplatin-resistant lung adenocarcinoma cell line . Antisense S-ODN could attenuate drug resistance by promoting cells apoptosis, which might lead to a new treatment for patients with non-small cell lung cancers (NSCLCs) who are refractory to conventional chemotherapy.

Chin Med J (Engl), 2000 Mar, 113(3), 228 - 31
Expression of the human multidrug resistance gene mdr1 in leukemic cells and its application in studying P-glycoprotein antagonists; Fu J et al.; OBJECTIVE: To investigate the retrovirus-mediated transfer and expression of multidrug resistance gene (mdr1) in hematopoietic cells and to develop a model for studying the possible reversal of the MDR-mediated phenotype . METHODS: A retroviral vector HaMDR expressing the human mdr1 gene was packaged by PA317 cells with a titer of up to 8.5 x 10(5) CFU/ml . K562 leukemia cells were infected with MDR retrovirus, and transfectant K562/MDR cells were generated . The integration and expression of the exogenous mdr1 gene in K562/MDR cells were determined by polymerase chain reaction and flow cytometry . The reversal ability of P-glycoprotein (P-gp) antagonists was analyzed by in vitro drug sensitivity, accumulation and efflux of rhodamine 123 (Rh123) in this model . RESULTS: Transduction with amphotropic MDR retrovirus resulted in integration and expression of the mdr1 gene in the resistant cells, where an aberrant splicing transcript of the mdr1 gene was found . The K562/MDR cells displayed a classic MDR phenotype with a 41-78 fold resistance to vincristine and colchicine in comparison with parental K562 cells . The drug sensitivity of K562/MDR cells to vincristine can be completely restored by cyclosporin A (CsA, 2 mg/L) and Cremophor EL (CRE 132 mg/L), either individually or in combination (P < 0.05) . CsA (3 mg/L) can block the efflux pump function of P-gp shown by the significantly increased accumulation and efflux reduction of Rh123 in K562/MDR cells . CONCLUSIONS: Retroviral vector HaMDR allows transfection with high-level expression of the mdr1 gene in human myeloid progenitor cells K562 . The transfected K562/MDR provides a simple, sensitive model for developing antagonists of P-gp and studying their mechanism of action.

Am J Gastroenterol, 2001 Dec, 96(12), 3368 - 78
The expression levels of plasma membrane transporters in the cholestatic liver of patients undergoing biliary drainage and their association with the impairment of biliary secretory function; Shoda J et al.; OBJECTIVES: Percutaneous transhepatic biliary drainage (PTBD) has been believed to reduce hyperbilirubinemia in patients with obstructive cholestasis and to lessen liver injury through bile acid retention . The efficacy may be closely related to the capability of cholestatic liver to produce and secrete bile, which in turn depends on the expressions and functional activities of plasma membrane transporters in the liver . The aim of the present study was to determine the expression levels of these transporters in the cholestatic liver of patients undergoing PTBD . METHODS: A total of 24 patients who had experienced obstructive cholestasis and had undergone preoperative PTBD were included in the study . Liver biopsy specimens were analyzed to determine the expression levels of the multidrug resistance-associated proteins (MRP) MRP2 and MRP3 and the canalicular bile salt export pump BSEP in the liver . RESULTS: The messenger RNA (mRNA) levels of MRP2, the canalicular bilirubin conjugate export pump, and bile salt export pump (BSEP) were unchanged in liver specimens from the 14 patients well drained by PTBD but were reduced in specimens from the 10 patients poorly drained, compared to the levels of control subjects . Immunostainings of MRP2 and BSEP outlined the canalicular membrane domain but seemed fuzzy to varying degrees in specimens obtained from cholestatic liver, especially in specimens from liver that had been poorly drained, in contrast to the linear and intense localization in the liver of control subjects, correlating with the impaired bilirubin conjugate and bile acid secretion . The mRNA of MRP3, functioning as an inducible export pump for bilirubin conjugate and bile acid, was expressed not only in the cholestatic liver but also in the liver of control subjects, and the mRNA level was increased in specimens from both the cholestatic liver that had been well drained and from the liver that had been poorly drained . Immunostaining of MRP3 was observed in the epithelia of intrahepatic bile ducts in the liver of both control subjects and cholestatic patients, and in the epithelia of proliferated bile ductules and the hepatocytes surrounding the portal tracts in the cholestatic liver . CONCLUSIONS: From the results of the present study, it is concluded that 1) the mRNA and immunohistochemical expression levels of MRP2 and BSEP may be altered in the cholestatic liver of patients undergoing PTBD; 2) both the decreased mRNA levels and the diminished canalicular membrane localization may be associated with the impairment of bile formation and secretion, i.e., the efficacy of PTBD; and 3) upregulated MRP3 in the cholangiocytes and hepatocytes may play a significant role in bile acid transport in the cholestatic hepatobiliary system.

Hum Cell, 2001 Sep, 14(3), 172 - 84
Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics; Kobayashi H et al.; One of the underlying mechanisms of multidrug resistance (MDR) is cellular overproduction of P-glycoprotein (P-gp), which acts as an efflux pump for various anti-cancer drugs . P-gp is encoded by a group of related genes termed MDR; only MDR1 is known to confer the drug resistance, and its overexpression in cancer cells has been a therapeutic target to circumvent the resistance . To overcome P-gp-mediated drug resistance, we have developed six anti-MDR1 hammerhead ribozymes and delivered them to P-gp-overproducing human leukemia cell line by a retroviral vector containing RNA polymerase III promoter . These ribozyme-transduced cells became vincristine-sensitive, concomitant with the decreases in MDR1 expression, P-gp amount and efflux pump function . Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the highest efficacy . The retrovirus-mediated transfer of this most potent anti-MDR1 ribozyme into a human lymphoma cell line, which was made resistant by infection of pHaMDR1/A retroviral vector and thus possessed a low degree of MDR due to P-gp expression relevant to clinical MDR, resulted in a complete reversal of MDR phenotype . In addition to retrovirus-mediated transfer of ribozymes, we evaluated the efficacy of cationic liposome-mediated transfer of ribozyme . Treatment of a P-gp-producing human breast cancer cell line with the liposome-ribozyme complex resulted in reversal of resistance, concomitant with the decreases in both MDR1 expression and P-gp amount . Confocal microscopic imaging of the cells after treatment with liposome/FITC-dextran showed cytoplasmic fluorescence that was abolished by cytochalasin B, indicating a high endocytotic activity in these cells . The endocytotic activity was well correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme . These distinct approaches using either retrovirus- or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells with different properties such as endocytotic activity as a specific means to reverse resistance.

Zhonghua Kou Qiang Yi Xue Za Zhi, 1998 May, 33(3), 137 - 9
{The correlation between P-glycoprotein and multidrug resistance of squamous carcinoma in oral and maxillofacial region}; Xie Z et al.; OBJECTIVE: To study the mechanism of drug resistance of oral carcinoma to chemotherapy . METHODS: 40 cases of squamous carcinoma in the oral and maxillofacial region were examined for the multidrug resistance gene product P-glycoprotein using a monoclonal antibody MRK16 . RESULTS: P-glycoprotein was detected in 62.5% of the sample . P-glycoprotein expression was related to the chemotherapy and the degree of differentiation . P-glycoprotein expression was higher in post-chemotherapy group than in unchemotherapy group (P < 0.05) . Well differentiated tumors expressed P-glycoprotein more frequently (P < 0.05) . P-glycoprotein expression was compared with clinic response to chemotherapy . The accuracy rate of prediction is 75% . CONCLUSION: P-glycoprotein plays an important role in mechanism of multidrug resistance of squamous carcinoma in the oral and maxillofacial region.

Int J Cancer, 2002 Jan 20, 97(3), 278 - 82
Y-box factor YB-1 predicts drug resistance and patient outcome in breast cancer independent of clinically relevant tumor biologic factors HER2, uPA and PAI-1; Janz M et al.; Intrinsic or acquired resistance to chemotherapy is responsible for failure of current treatment regimens in breast cancer patients . The Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug-resistant tumor phenotype . In human breast cancer, overexpression and nuclear localization of YB-1 is associated with upregulation of P-glycoprotein . In our pilot study, we analyzed the clinical relevance of YB-1 expression in breast cancer (n = 83) after a median follow-up of 61 months and compared it with tumor-biologic factors already used for clinical risk-group discrimination, i.e., HER2, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) . High YB-1 expression in tumor tissue and surrounding benign breast epithelial cells was significantly associated with poor patient outcome . In patients who received postoperative chemotherapy, the 5-year relapse rate was 66% in patients with high YB-1 expression . In contrast, in patients with low YB-1 expressions, no relapse has been observed so far . YB-1 expression thus indicates clinical drug resistance in breast cancer . Moreover, YB-1 correlates with breast cancer aggressiveness: in patients not treated with postoperative chemotherapy, those with low YB-1 expression are still free of disease, whereas the 5-year relapse rate in those with high YB-1 was 30% . There was no significant correlation between YB-1 expression and either HER2 expression or uPA and PAI-1 levels . Risk-group assessment achieved by YB-1 differed significantly from that by HER2 or uPA/PAI-1 . In conclusion, YB-1 demonstrated prognostic and predictive significance in breast cancer by identifying high-risk patients in both the presence and absence of postoperative chemotherapy, independent of tumor-biologic factors currently available for clinical decision making .

Int J Cancer, 2002 Jan 10, 97(2), 149 - 56
A very early induction of major vault protein accompanied by increased drug resistance in U-937 cells; Hu Y et al.; U-937 human leukemia cells were selected for resistance to doxorubicin in the presence or absence of a specific drug modulator that inhibits the activity of P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1) . Parental cells expressed low basal levels of the multidrug-resistance-associated gene (MRP1) and major vault protein (MVP) mRNAs and no MDR1 mRNA . Two doxorubicin-resistant cell lines were selected . Both drug-resistant cell lines upregulated the MVP mRNA level 1.5-fold within 1 cell passage . The MVP mRNA level continued to increase over time as the doxorubicin selection pressure was increased . MVP protein levels generally paralleled the mRNA levels . The 2 high molecular weight vault protein mRNAs were always expressed at constitutive levels . Fully formed vault particles consisting of the MVP, the 2 high molecular weight proteins and the vault RNA assembled and accumulated to increased levels in drug-selected cells . MVP induction is therefore the rate-limiting step for vault particle formation in U-937 cells . By passage 25 and thereafter, the selected cells were resistant to doxorubicin, etoposide, mitoxantrone and 5-fluorouracil by a pathway that was independent of MDR1, MRP1, MRP2 and breast cancer resistance protein . In summary, U-937 doxorubicin-selected cells are programmed to rapidly upregulate MVP mRNA levels, to accumulate vault particles and to become multidrug resistant .

Int J Cancer, 2002 Jan 1, 97(1), 21 - 7
Expression of heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) in human colorectal carcinoma; Tatebe S et al.; Gamma-glutamylcysteine synthetase (gamma-GCS) is a heterodimer consisting of heavy (gamma-GCSh) and light (gamma-GCSl) subunits . gamma-GCS catalyzes the rate-limiting de novo biosynthesis of glutathione (GSH), an abundant physiological antioxidant that plays important roles for regulating oxidative stress . Expression of gamma-GCSh and gamma-GCSl are sensitive to oxidative stress . To investigate whether expression of gamma-GCS is correlated with tumor progression, we used immunohistochemical approaches to examine 16 human colorectal adenomas and resected 57 carcinomas from untreated patients . In adjacent normal colorectal epithelium, levels of gamma-GCSh expression were low . Strong cytoplasmic staining for gamma-GCSh was detected in 3 (18.8%) adenoma and 48 (84.2%) carcinomas . The frequency of gamma-GCSh expression in carcinoma was significantly higher than in adenoma (p<0.0001) . We used RNase protation assay and Western blot to determine levels of gamma-GCSh mRNA and protein from 10 pairs of matched carcinomas with adjacent normal controls . Elevated expression of both gamma-GCSh mRNA and protein were found in 6 cases, suggesting that transcriptional and/or posttranscriptional regulation play an important role in the upregulation of gamma-GCS during colorectal carcinogenesis . We also examined the expression of another redox-regulated gene, multidrug resistance protein 1 (MRP1) . Strong staining for MRP1 was detected in 1 (6.3%) adenoma and 40 (70.2%) carcinomas . The frequency of MRP1 expression in carcinoma was significantly higher than in adenoma ( p<0.0001) . Nuclear p53 expression was detected in 30 (52.6%) of carcinomas . There is a significant correlation between gamma-GCSh and MRP1 expression (p=0.013) but not between gamma-GCSh and p53 . Since gamma-GCS is a sensor of oxidative stress, these results are consistent with the notion that oxidative stress is associated with colorectal tumor progression .

Clin Infect Dis, 2002 Feb 1, 34(3), 324 - 9 Epub 2001 Dec 26.
Trends in multidrug-resistant Mycobacterium tuberculosis in relation to sputum smear positivity in Hong Kong, 1989-1999; Kam KM et al.; We studied retrospectively the territory-wide occurrence and trends of multidrug-resistant tuberculosis (MDR-TB) in Hong Kong over an 11-year period during which a short-course directly observed therapy ("DOTS-Plus") strategy has been in operation . The overall MDR rate was 2.1% (primary, 1.4% and acquired, 9.5%) and declined at 0.08% per year: smear-positive primary MDR cases declined at yearly rate of -0.065% (R2=.23), and smear-negative primary MDR cases increased at 0.035% yearly . With declining rates of smear positivity, sputum culture has become the mainstay of detection of MDR-TB . Although the overall notification rates showed the elderly (age >65 years) age group to be most affected, the occurrence of MDR-TB has been consistently highest in the 35-65 year age group (60.4% of MDR caseload) . We demonstrated declining rates of sputum smear positivity of MDR-TB in a DOTS-plus environment and that a centralized laboratory database is essential in gathering epidemiological information for identifying particular risk groups and monitoring trends of MDR-TB in a community.

J Control Release, 2002 Jan 17, 78(1-3), 43 - 54
Role of transporters in the tissue-selective distribution and elimination of drugs: transporters in the liver, small intestine, brain and kidney; Kusuhara H et al.; Cumulative studies have revealed the importance of transporters in drug disposition in the body . Recently, organic anion transporters such as organic anion transporting polypeptides (OATPs), organic anion transporters (OATs) and multidrug resistance associated proteins (MRPs) have been identified . Their broad substrate specificity as well as the multiplicity of transporter gene products make these transporters suitable detoxification systems in the body . OATPs and OATs are responsible for the hepatic and renal uptake of organic anions, respectively, while MRP2 is a major transporter involved in the biliary excretion of organic anions . OATPs and MRP2 are involved in the hepatobiliary transport of pravastatin and temocaprilat . These are good examples of hepatobiliary transport maximizing their pharmacological effects, but minimizing their side-effects . Taking into consideration tissue-selective expression and substrate specificity, transporters are useful for delivering small molecules to target tissues . MRPs are also suggested to be involved in the barrier function in the small intestine, blood-brain barrier and blood-cerebrospinal fluid barriers by extruding their ligands into the luminal side . In this manuscript, we have summarized recent studies by others and ourselves on the role of these transporters in the tissue selective distribution and elimination of drugs.

Curr Pharm Des, 2001 Dec, 7(18), 1863 - 92
Radiolabelled tracers and anticancer drugs for assessment of therapeutic efficacy using PET; Brady F et al.; Positron Emission Tomography (PET) has the potential to improve efficacy of established and novel cancer therapies and to assist more rapid and rational progression of promising novel therapies into the clinic . This is due to PET's unrivalled sensitivity and ability to monitor the pharmacokinetics and pharmacodynamics of drugs and biochemicals radiolabelled with short -lived positron emitting radioisotopes . PET is a multidisciplinary science which employs chemists, biologists, mathematical modellers, pharmacologists as well as clinicians . Clinical research questions in oncology determine the methodological challenges faced by these other disciplines . Within this context we focus on the developments of the radiolabelled compounds that have underpinned the clinical work in oncology for monitoring tumour and normal tissue pharmacokinetics, assessment of tumour response, cell proliferation, gene expression, hypoxia, multidrug resistance and status of receptors on tumours.

Int J Tuberc Lung Dis, 2001 Dec, 5(12), 1137 - 42
Directly observed treatment for multidrug-resistant tuberculosis: an economic evaluation in the United States of America and South Africa; Wilton P et al.; OBJECTIVE: To estimate the cost-effectiveness of directly observed treatment compared to conventional therapy in reducing the spread of multidrug-resistant tuberculosis, for an industrialised country (represented by the United States of America) and a developing country (South Africa) . METHODS: Monte Carlo analysis using published data on probability, cost and health impact . RESULTS: In both countries, directly observed treatment is the dominant strategy, yielding cost savings and improved health outcomes . Cost savings for directly observed treatment relative to conventional therapy become more significant as more expensive second-line drugs are used in treatments . CONCLUSIONS: The cost-effectiveness of directly observed treatment relative to conventional therapy is demonstrated for both the USA and South Africa . Cost savings are more pronounced (especially for South Africa) as the likelihood of multidrug-resistant tuberculosis increases and more expensive second-line therapies are used . Given that health care resources are more severely constrained in developing countries, the data contained in this study are useful in guiding the design of policies for the effective management of multidrug-resistant tuberculosis in settings with limited resources.

Int J Tuberc Lung Dis, 2001 Dec, 5(12), 1129 - 36
Ambulatory treatment of multidrug-resistant pulmonary tuberculosis patients at a chest clinic; Kim HJ et al.; SETTING: Retrospective cohort analysis of multidrug-resistant tuberculosis (MDR-TB) patients treated at a Korean National Tuberculosis Association out-patient chest clinic . OBJECTIVE: To evaluate treatment outcomes and contributing factors . DESIGN: A review of clinical records of 1011 pulmonary MDR-TB patients retreated with individualised regimens selected on the basis of previous chemotherapy and drug susceptibility testing from 1988 to 1996 . RESULTS: The patients (mean age 38.6 years) had resistant organisms to an average of 3.7 drugs and were retreated with an average of 4.2 drugs which they had previously not taken and to which they were susceptible . Treatment outcomes were as follows: 487 cases (48.2%) cured, 82 (8.1%) failed, 394 (39.0%) defaulted, 45 (4.5%) transferred out, and three (0.3%) died . The treatment efficacy among those who completed chemotherapy was 85.6% . In a multivariate analysis favourable response was significantly associated with a greater number of newly prescribed drugs in the regimen to which they were susceptible (odds ratio {OR} 3.6; 95% confidence interval {CI} 1.3-9.5), younger age (OR 2.0; 95%CI 1.1-3.9), and a lower number of drugs to which they were resistant (OR 1.8; 95%CI 1.1-3.1) . The case fatality rate, including the follow-up period, was 1.7% (17 cases) . CONCLUSION: The cure rate of MDR-TB patients treated at an out-patient clinic was 48.2% due to a high defaulter rate (39.0%) . However, 85.6% of those who completed treatment were cured.

Int J Tuberc Lung Dis, 2001 Dec, 5(12), 1116 - 21
Pattern of mycobacterial resistance to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the state of Qatar after the implementation of DOTS and a limited expatriate screening programme; Al-Marri MR; OBJECTIVE: To determine the resistance pattern of Mycobacterium tuberculosis to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the State of Qatar after the implementation of DOTS and an expatriate screening programme on arrival . METHOD: A state-wide, population-based, retrospective analysis of all cases of pulmonary tuberculosis with positive M . tuberculosis culture reported to the Division of Public Health TB Unit from January 1996 to December 1998 . M . tuberculosis sensitivity testing was done by the Bactec method for isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB) . The results were interpreted as a daily change of the growth index of test vials (with drug) compared with controls . RESULTS: There were 406 isolates with positive M . tuberculosis culture . Sixty-one (15%) were resistant to one or more of the four anti-tuberculosis drugs, of which 58 (95%) were from newly diagnosed cases (primary) and three (5%) were from previously treated cases (acquired) . Primary resistance was as follows: any resistance 15%, INH 12.4%, RMP 2%, SM 5.2%, EMB 0.8% and multidrug resistance (MDR, resistance to INH and RMP at least) was found in 0.8% . Acquired resistance was as follows: any resistance 15%, INH 15%, RMP 5%, SM 5% and MDR 5% . CONCLUSION: The prevalence of resistance to four anti-tuberculosis drugs is strikingly low due to the limited expatriate screening programme (chest radiography) and implementation of DOTS . The four-drug regimen is recommended for the initial phase of therapy until the results of sensitivity testing are known.

Zhonghua Fu Chan Ke Za Zhi, 2001 Sep, 36(9), 549 - 53
{Establishment of the drug resistant cell line of choriocarcinoma and the reversal of drug resistance by transfection of human interleukin 2 gene}; Cui Z et al.; OBJECTIVE: To establish the drug-resistant cell line of choriocarcinoma and to study the transfection of the human interleukin 2 (hIL-2) gene into the established drug resistant cell line and investigate the reversal of the multidrug resistance . METHODS: The resistant cell line was established by pulse exposed choriocarcinoma cell line JEG-3 to etopside (VP-16) for ten months . The recombinant plasmid containing pcDNA3.1(+)-hIL-2 gene was constructed . The drug resistant cell line was transfected with the constructed plasmid by lipofectin, and the tumor cell colonies containing the IL-2 sequence were selected by genetin . The expression of hIL-2 and drug resistant-related genes was detected by reverse transcript polymerase chain reaction . The chemosensitivity of the gene-transfected tumor cells and the non transfected cell lines to methetraxate, VP-16, kengshengmycine, paclitaxol and 5-fluorouracil was determined by the methyl thiazolyl tetrazolium cytotoxicity assay . RESULTS: The transfected cells expressed human hIL-2 gene, and showed the reversal of multidrug resistance by methyl thiazolyl tetrazolium assay . The transfected cells expressed no multidrug resistance gene-1 (MDR1) on mRNA level . Drug resistance index to VP-16 decreased from 38.7 to 6.0 and 6.1, the index to methetraxate decreased from 14.5 to 2.6 and 2.5, to methetraxate from 13.0 to 2.0 . CONCLUSION: The transfection of hIL-2 gene into the drug resistance cell line of choriocarcinoma can modulate the MDR1 expression on the mRNA level, and reverse the drug resistance.

Drug Resist Updat, 2001 Jun, 4(3), 187 - 96
Drug resistant falciparum malaria: clinical consequences and strategies for prevention; Price RN et al.; The rising prevalence of multidrug resistant falciparum malaria is occurring at an alarming rate and has serious implications for the health of many of the world's poorest countries . The dangers of not changing treatment practices immediately are huge and irreversible, threatening to both exacerbate the scale and scope of the malaria pandemic, and deprive policymakers of future options against the disease . If a health care disaster is to be avoided then massive and long term funding is urgently required . Funds need to be applied in a cohesive manner, accountable to funding bodies and tailored to the specifics of each endemic region . The key elements of such an approach should be improving early diagnosis and treatment of infection and the deployment of combination regimens containing an artemisinin derivative.These short term measures will need to be accompanied by a longer term strategy to encourage antimalarial drug research and development.

Curr Drug Metab, 2001 Dec, 2(4), 367 - 77
The MRP family and anticancer drug metabolism; Suzuki T et al.; Acquirement of drug resistance by tumor cells is a major chemotherapeutic problem . It is well known that typical multidrug resistance is caused by P-glycoprotein and multidrug resistance related protein (MRP1) which belong to the ATP binding cassette (ABC) transporter family . Ishikawa proposed that the ATP-dependent glutathione-S-conjugate export pump (GS-X pump) and phase III detoxification system are essential to drug metabolism, and this constituted a new concept in drug metabolism and the detoxification of xenobiotics . The GS-X pump has been revealed to belong to the ABC transporter family and suggested to the contribution to anticancer drug resistance . The GS-X pump actively effluxes the glutathione S-platinum (GS-Pt) complex . We cloned novel ABC transporter cDNA from the PC-14/CDDP cell line, and the cloned cDNA was designated as a short-type MRP homologue, SMRP . Further investigation suggested that SMRP is a splicing variant of MRP5 . The MRP5 mRNA levels in tumors from lung cancer patients treated with platinum regimen were significantly higher than in tumors from patients treated with non-platinum regimens, and the MRP5 expression levels were correlate with the GCS expression levels that is the rate-limiting step enzyme in glutathione biosynthesis . These results suggested that MRP5 take part in the function of GS-X pump . Recently many transporter molecules belong to the ABC transporter family such as MRP family have been identified, and appear to express in various human tissues . It can be presumed that their molecules are affected by the disposition and metabolism of drugs, but their substrates are still unclear . If the substrate specificity is revealed in the future, it is expected that the anticancer agents transporter, moreover anti cancer drug resistance mechanisms, can be clarified . This review is cited in the cisplatin resistance and the GS-X pump, and finally describes an overview of the MRPs substrates recently clarified, mainly about anticancer drugs.

Kekkaku, 2001 Nov, 76(11), 699 - 706
{Anti-tuberculosis drug resistance in Japan and in the world}; Abe C; In 1994, the World Health Organization (WHO) and the International Union against Tuberculosis and Lung Disease (IUATLD) launched a global project on anti-tuberculosis drug resistance surveillance . The results from the first 4 years (1994-1997) and the second 4 years (1996-1999) of the projects were reported in 1998 and 2000, respectively . These surveillance results showed that resistance to anti-tuberculosis drugs is a global problem . The reports also showed that there were several hot spots around the world where prevalence of multidrug resistant tuberculosis (MDR-TB, defined as resistance to at least isoniazid and rifampin) was particularly high and could possibly threaten control programs . The Tuberculosis Research Committee of Japan (Ryoken) has conducted nationwide surveys for drug resistant tuberculosis at 2- or 5-yearly intervals since 1957 . The 1997 survey showed that among patients with no prior treatment, resistance to any of the four drugs was found in 10.3%, and the prevalence of primary MDR was 0.8% . The prevalence of drug resistance in the previously treated cases was 42.4% for any of the four drugs and 19.7% for MDR, indicating a high prevalence rate compared with those reported in the global project . Compared with the previous survey in 1992, the current survey shows increased prevalence of drug resistance in both new and re-treatment cases . No significant differences in resistance rates by sex, age group, nationality, district, and/or accompanying diseases were observed in any of the new or re-treatment cases . Other factors associated with the high prevalence in re-treatment cases remain to be determined . A total of 78 hospitals in various districts of Japan participated the cooperative study . Each collaborating laboratory sent all the isolated mycobacterial cultures to the Research Institute of Tuberculosis (RIT) . In the local laboratories, the absolute concentration method using 1% Ogawa egg slant, its modified methods using a 48-well plate and a 16-well plate, combination of above 2 or 3 methods, and other method were used for drugsusceptibility testing, and the proportion method using 1% Ogawa egg slant was used in the RIT . The results in the local laboratories were compared with those in the RIT . There was no significant difference in the concordance rates according to the test drugs among methods for drug susceptibility testing used in the local laboratories . Relatively lower concordance rates were seen in the laboratories using the Microtiter method related to high overestimation rates, compared with those in the laboratories using the standard method and Well-pack method . However, relatively lower concordance rates (less than 90%) were seen in the laboratories using any of the three methods, indicating that there are variations among facilities.

Gen Physiol Biophys, 2001 Sep, 20(3), 215 - 37
Drug transporters and their role in multidrug resistance of neoplastic cells; Kvackajova-Kisucka J et al.; Multidrug resistance (MDR) of neoplastic cells, i.e . resistance towards large groups of unrelated drugs, represents the phenomenon that dramatically depresses the effectiveness of cancer chemotherapy . Membrane transport of ATPases from ABC superfamily plays an important role in MDR . In the present paper we are aiming to compare two members of this family: P-glycoprotein (PGP products of mdr genes) and multidrug resistance-associated protein (MRP, products of mrp genes) and their impact for MDR of neoplastic cells.

Probl Tuberk, 2001, (7), 60 - 2
{Genotyping mycobacteria, isolated from incarcerated tuberculosis patients}; Chernousova LN et al.; The aim of the investigation was to genotype clinical Mycobacterium tuberculosis isolates circulating at the penitentiaries of the Ivanovo Region . Mycobacterial strains were genotyped by the polymorphism of the lengths of restrictive fragments containing the insertion sequence 6110 by the routine procedure . The genotypes of Mycobacterium strains were classified in accordance with the PHRI database (New York) . The strains were characterized by their sensitivity to antituberculous drugs by the absolute concentration method . The investigations indicated that in terms of the IS6110 genotype there were prevalent Mycobacteria strains belonging to 2 families: a W family (62.5% of the strains) and an AI family (25%) . In addition to the monocultures, genotyping also revealed 3 mixed cultures consisting of 2 strains . Determining the drug sensitivity of the genotyped strains demonstrated both drug-sensitive and drug-resistant strains, including multidrug resistance among the strains of the same genotyping family (W or AI).

Probl Tuberk, 2001, (7), 13 - 8
{Effectiveness of the standard mode of chemotherapy for newly-diagnosed patients with destructive pulmonary tuberculosis with bacterial isolation}; Mishin VIu et al.; The authors evaluated the efficiency of a routine drug therapy regimen by the WHO category 1 in treating 149 new cases of destructive pulmonary tuberculosis and bacterial isolation . They used not only the WHO sputum smear negativization criterion, but the data of cultural studies and on lung cavernous closure . The specific features of the approach applied were compulsory cultural studies determining Mycobacterium sensitivities before treatment and compulsory correction of chemotherapy after there was evidence for the sensitivity . Retrospective analysis of 6-month chemotherapy has ascertained that the efficiency of the routine drug therapy regimen largely depends on the baseline extent of infiltrative and destructive changes in the lung and on the baseline resistance of Mycobacteria tuberculosis . They showed a high baseline resistance to streptomycin (20.6%) and streptomycin and isoniazid (33.1%) and a low baseline resistance to ethambutol (5.1%) . In these cases, the more optimum regimen was a combination of rifampicin, pyrazanamide, and ethambutol . When Mycobacteria showed multidrug resistance, the routine regimen was ineffective and caused amplification to a larger number of drugs . A modified treatment course using the routine regimen in the intensive phase was developed . If Mycobacterial resistance was present, compulsory correction was made by using reserve agents, pathogenetic treatments, artificial pneumothorax or surgical interventions, which made it possible to abacillate 94.1% of patients by their smears and culture by months 4-5 and to close caverns in 91.3% of cases by months 8-10.

Anticancer Drug Des, 2001 Feb, 16(1), 27 - 36
A novel B-ring modified homocamptothecin, 12-Cl-hCPT, showing antiproliferative and topoisomerase I inhibitory activities superior to SN-38; Bailly C et al.; We report the synthesis and pharmacological evaluation of a novel homocamptothecin (hCPT) derivative, 12-Cl-hCPT, which contains a seven-membered beta-hydroxylactone in place of the conventional six-membered alpha-hydroxylactone found in camptothecin (CPT) and bears a chloro substituent at position 12 . The capacity of 12-Cl-hCPT to inhibit DNA topoisomerase I was compared with that of SN-38, the active metabolite of the clinically used antitumour prodrug CPT-11 . In the DNA relaxation assay, 12-Cl-hCPT proved to be slightly more potent than SN-38 at stimulating the formation of nicked plasmid DNA molecules . A series of radiolabelled DNA restriction fragments were employed to identify and compare the position of the DNA cleavage sites induced by topoisomerase I in the presence of 12-Cl-hCPT and SN-38 . These sequencing studies confirm that both 12-Cl-hCPT and SN-38 strongly promote DNA cleavage by topoisomerase I and reveal that the majority of the cleavage sites are located at the same nucleotide positions for the two drugs . However, a certain number of DNA cleavage sites were found to be specific to 12-Cl-hCPT . These sites, previously characterized with unsubstituted hCPT, generally correspond to 5'-CG sites whereas the sites common to the 12-Cl-hCPT and SN-38 essentially correspond to 5'-TG sites . We also quantified the formation of drug-induced protein-DNA complexes formed in HT29 human colon carcinoma cells . Trapping of endogenous proteins onto DNA was found to be much more efficient with 12-Cl-hCPT than with SN-38 . These data provide a molecular basis to account for the enhanced antiproliferative activity of 12-Cl-hCPT compared with that of SN-38 . Biological evaluation on a panel of sensitive and drug-resistant cell lines revealed 12-Cl-hCPT to be more cytotoxic to tumour cells than SN-38 . 12-Cl-hCPT proved 14- and 23-fold more active than SN-38 toward the K562adr and T24anp multidrug-resistant cell lines, respectively . The marked topoisomerase I inhibitory properties of 12-Cl-hCPT coupled with its interesting antiproliferative activity, in particular against cancer cells presenting multidrug resistance phenotype with overexpression of P-glycoprotein, makes 12-Cl-hCPT a valid candidate for subsequent preclinical evaluation . Collectively, the data strengthen homocamptothecin as an extremely promising template to generate novel and potent antitumour agents.

J Clin Pharmacol, 2001 Dec, 41(12), 1271 - 9
P-glycoprotein (P-gp) is upregulated in peripheral T-cell subsets from solid organ transplant recipients; Donnenberg VS et al.; Immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and corticosteroids are substrates for the transmembrane multidrug resistance pump P-glycoprotein (P-gp) . Experience in oncologyhas suggested that chronic exposure to P-gp substrates induces upregulation of P-gp activity, which could result in resistance to immunosuppressive drugs . The authors investigated P-gp function in CD4+ and CD8+ T cells from the peripheral blood of solid organ transplant recipients (SOTX) . Subjects included 14 stable SOTX (10 liver, 4 lung) and 16 healthy controls . Four-color flow cytometry was used to simultaneously measure intracellular concentration of the fluorescent P-gp substrate Rhodamine 123 (Rh123) and surface expression of CD45RO (nominal memory/effector), CD45RA (naive), and either CD4 or CD8 . P-glycoprotein function was measured by a dye efflux assay in which activity was inferred from a decrease in Rh123 fluorescence . CD4+ and CD8+ T cells from patients and control subjects eliminated Rh123, and this activity was inhibited by verapamil, a known P-gp substrate . CD8+ T cells had greater P-gp activity than CD4+ cells, and naive and transitional T cells displayed greater activity than memory T cells . Activity was bimodal in CD8+ CD45RO+ T cells, with a subset of these cells expressing the greatest P-gp activity . Patient CD8+ naive and transitional T cells had upregulated P-gp activity compared to control subjects . We conclude that (1) P-gp activityis significantly upregulated in specific T-cell subsets (CD8+/CD45RA+) in the peripheral blood of SOTX, and (2) the bimodal nature of P-gp response in CD8+ T cells complicates analysis of the effect of chronic administration of P-gp substrates to SOTX.

Pharm Res, 2001 Nov, 18(11), 1542 - 9
Effect of probenecid on fluorescein transport in the central nervous system using in vitro and in vivo models; Sun H et al.; PURPOSE: The purpose of this study was to characterize the function of multidrug resistance-associated proteins (MRPs) (or MRP-like organic anion transport systems) in the blood-brain harrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) using both an in vitro BBB model and an in vivo microdialysis model . METHODS: In vitro functional studies were performed using bovine brain microvessel endothelial cells (BBMEC) . The accumulation of fluorescein, an anionic fluorescent dye, in BBMEC was determined with and without the presence of inhibitors of various efflux transport proteins . In vivo microdialysis simultaneously monitored fluorescein concentrations in cortical extracellular fluid and cerebrospinal fluid . The effect of probenecid on the in vivo distribution of fluorescein was studied using a balanced crossover design in the rat . RESULTS: In vitro experiments showed that probenecid, indomethacin, LY-329146, and all MRP inhibitors significantly increased (two- to threefold) the accumulation of fluorescein in BBMEC, whereas LY-335979, a P-gp inhibitor, had no effect on the accumulation of fluorescein . Probenecid significantly increased fluorescein plasma concentration and the plasma free fraction in vivo . The distribution of fluorescein across the BBB and BCSFB was enhanced by 2.2- and 1.9-fold, respectively, when probenecid was coadministered, even after correction for increased fluorescein plasma concentrations and free fraction . CONCLUSIONS: These results demonstrate that MRPs or MRP-like transport system(s) may play an important role in fluorescein distribution across both BBB and BCSFB . This study showed that microdialysis proved to be a powerful in vivo technique for the study of transport systems in the central nervous system, and in vitro/in vivo correlations are possible using these model systems.

Pharm Res, 2001 Nov, 18(11), 1535 - 41
Influence of P-glycoprotein modulators on cardiac uptake, metabolism, and effects of idarubicin; Kang W et al.; PURPOSE: The clinical utility of anthracyclines like idarubicin (IDA) is limited by the occurrence of multidrug resistance and cardiotoxicity . Previous studies have demonstrated that the multidrug transporter P-glycoprotein (P-gp) is present in the heart and have suggested that it exerts a protective function . We sought to determine the influence of P-gp inhibitors verapamil and PSC 833 on myocardial uptake, metabolism, and actions of IDA . METHODS: In Langendorff-perfused rat hearts, the outflow concentration-time curve and the residual amount in cardiac tissue of IDA and its active metabolite idarubicinol (IDOL) were measured after 0.5 mg dose of IDA in the absence and presence of the P-gp inhibitors verapamil and PSC 833 . RESULTS: During perfusion (80 min), 2% of the IDA dose was converted to IDOL in the heart . Myocardial uptake of IDA was significantly increased by verapamil but not by PSC 833, which increased the recovery of IDA and IDOL . IDA significantly decreased left ventricular developed pressure to approximately 40% and increased coronary vascular resistance to 140% of baseline level, respectively . The vasoconstrictive effect was markedly potentiated by PSC 833 . CONCLUSIONS: The enhancement of myocardial IDA uptake by verapamil could be due to a decrease in P-gp-mediated efflux . PSC 833 inhibits cardiac metabolism (non-IDOL pathways) and increases the acute cardiotoxicity of IDA.

Zhonghua Fu Chan Ke Za Zhi, 2001 Aug, 36(8), 493 - 5
{Modulation of multidrug resistance by 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol in SKOV3-adriamycin-resistant cell line}; Yuan Y et al.; OBJECTIVE: To study the modulation of mdr1 and P-glycoprotein (P-gp) by 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in SKOV3-adriamycin-resistant (SKOV3/AdrR) cell line . METHODS: SKOV3/AdrR cells were treated with PPMP, mRNA expression of multidrug resistant (mdr1) gene was analyzed by reverse transcriptase-polymerase chain reaction . Intracellular rhodamine (Rh123) concentration was measured by flow cytometry . RESULTS: PPMP was found to inhibit mdr1 expression of SKOV3/AdrR at the mRNA level . This modulation of gene expression was content dependent and complete inhibition appeared at 25 mumol/L PPMP treatment . PPMP could increase intracellular Rh123 accumulation in SKOV3/AdrR cells . After 15, 25 mumol/L PPMP treatment, Rh123 accumulation in SKOV3/AdrR was markedly enhanced Rh123 fluorescence intensity were 389.98, 426.08 respectively (P < 0.05) . CONCLUSIONS: PPMP could modulate mdr1 expression at the mRNA level in a content dependent manner . P-gp mediated outward efflux of cytotoxic agents can be blocked by PPMP in SKOV3/AdrR cell . PPMP possesses multi-drug resistance activity in ovarian cancer.

Blood, 2002 Jan 1, 99(1), 326 - 35
Novel triterpenoid CDDO-Me is a potent inducer of apoptosis and differentiation in acute myelogenous leukemia; Konopleva M et al.; It has been shown that the novel synthetic triterpenoid CDDO inhibits proliferation and induces differentiation and apoptosis in myeloid leukemia cells . In the current study the effects of the C-28 methyl ester of CDDO, CDDO-Me, were analyzed on cell growth and apoptosis of leukemic cell lines and primary acute myelogenous leukemia (AML) . CDDO-Me decreased the viability of leukemic cell lines, including multidrug resistant (MDR)-1-overexpressing, p53(null) HL-60-Dox and of primary AML cells, and it was 3- to 5-fold more active than CDDO . CDDO-Me induced a loss of mitochondrial membrane potential, induction of caspase-3 cleavage, increase in annexin V binding and DNA fragmentation, suggesting the induction of apoptosis . CDDO-Me induced pro-apoptotic Bax protein that preceded caspase activation . Furthermore, CDDO-Me inhibited the activation of ERK1/2, as determined by the inhibition of mitochondrial ERK1/2 phosphorylation, and it blocked Bcl-2 phosphorylation, rendering Bcl-2 less anti-apoptotic . CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and monocytic differentiation in primary cells . Of significance, colony formation of AML progenitors was significantly inhibited in a dose-dependent fashion, whereas normal CD34(+) progenitor cells were less affected . Combinations with ATRA or the RXR-specific ligand LG100268 enhanced the effects of CDDO-Me on cell viability and terminal differentiation of myeloid leukemic cell lines . In conclusion, CDDO-Me is an MDR-1- and a p53-independent compound that exerts strong antiproliferative, apoptotic, and differentiating effects in myeloid leukemic cell lines and in primary AML samples when given in submicromolar concentrations . Differential effects of CDDO-Me on leukemic and normal progenitor cells suggest that CDDO-Me has potential as a novel compound in the treatment of hematologic malignancies.

Leuk Res, 2002 Feb, 26(2), 143 - 54
Evaluation of the clinical relevance of the expression and function of P-glycoprotein, multidrug resistance protein and lung resistance protein in patients with primary acute myelogenous leukemia; Tsimberidou AM et al.; The multidrug resistance (MDR) transporter-proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP) and lung resistance protein (LRP) have been associated with treatment failure . The aim of this study was to investigate prospectively the clinical significance of expression and function of the MDR proteins, considering other prognostic factors, such as age, immunophenotype, and cytogenetics . Mononuclear cells of peripheral blood or bone marrow from 61 patients with de novo acute myelogenous leukemia (AML) were analyzed . The monoclonal antibodies JSB1, MRPm6 and LRP56 were used for expression studies . Accumulation and retention studies were performed using the substrates Daunorubicin, Calcein-AM, Rhodamine-123 and DiOC(2) in the presence or absence of the modifiers Verapamil, Genistein, Probenecid, BIBW22S and PSC833 . Induction treatment consisted of a 3+7 combination of Ida/Ara-C for patients < or = 60 years of age and a 3+5 Ida/VP-16 combination per OS for patients >60 . MDR function was expressed as the ratio of mean fluorescence intensity substrate in the presence of modifier over the substrate alone (resistance index, RI) . Patients with advanced age, low CD15 expression and high RI for accumulation of DiOC(2) in the presence of BIBW22S had significantly lower complete remission (CR) rates . No factor was prognostic for event-free survival analysis, which was limited to remitters only . Overall survival was shorter in patients with advanced age, poor prognosis cytogenetics, high CD7 expression, and high RI for Daunorubicin efflux modulated by Verapamil . These results suggest that MDR transporter-proteins have a limited role in the treatment failure of patients treated with Idarubicin-based regimens.

Leuk Res, 2002 Feb, 26(2), 121 - 7
Multidrug resistance (MDR-1) expression in AIDS-related lymphomas; Tulpule A et al.; P-glycoprotein is a product of the multidrug resistance (MDR-1) gene . In non-Hodgkin's lymphoma, less than 20% of untreated de novo lymphomas express MDR-1 compared with approximately 50% after failure of chemotherapy . We wished to study the expression of MDR-1 in AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) . Tissue biopsies from 50 patients with newly diagnosed AIDS-NHL were studied by immunohistochemical analysis using C494, a monoclonal antibody specific for the MDR-1 isoform of P-gp . MDR-1 expression was correlated with patient demographics, lymphoma characteristics, response to chemotherapy, and survival . Forty-six males and four females with a median age of 38 years (range 26-63) were studied . A prior AIDS-defining opportunistic infection was reported in 35 patients (70%) . The median CD4+ lymphocyte count was 69/mm(3) (range 0-920) . Thirty-two patients (63%) had received prior anti-HIV therapy, including a protease inhibitor in five (10%) . Pathologic types consisted of diffuse large cell in 13 (26%), immunoblastic in 13 (26%), small non-cleaved in 22 (44%), and high grade not otherwise specified in two (4%) . The majority of patients (76%) had stage III/IV disease . Pre-treatment lymphoma tissues from 33 patients (66%) stained positively for MDR-1 . MDR-1 positive patients had a significantly lower complete remission rate compared to MDR-1 negative patients (33 versus 65%, P=0.042) . Duration of complete response was significantly longer in MDR-1 negative patients compared with MDR-1 positive patients (not reached versus 9.9 months, P=0.003) . Strategies to overcome MDR-1 expression may be important for initial treatment in patients with AIDS-NHL.

J Nat Prod, 2001 Dec, 64(12), 1514 - 20
Modulation of the multidrug-resistance phenotype by new tropane alkaloid aromatic esters from Erythroxylum pervillei; Silva GL et al.; Nine tropane alkaloid aromatic esters (1-9) were isolated from the roots of Erythroxylum pervillei by following their potential to reverse multidrug-resistance with vinblastine-resistant oral epidermoid carcinoma (KB-V1) cells . All isolates, including seven new structures (3-9), were evaluated against a panel of human cancer cell lines, and it was found that alkaloids 3 and 5-9 showed the greatest activity with KB-V1 cells assessed in the presence of vinblastine, suggesting that these new compounds are potent modulators of P-glycoprotein . Confirmatory results were obtained with human ovarian adenocarcinoma (SKVLB) cells evaluated in the presence of adriamycin and synergistic studies performed with several cell lines from the NCI tumor panel . The structures of the new compounds were determined using spectroscopic techniques . Single-crystal X-ray analysis was performed on the monoester, tropane-3 alpha,6 beta,7 beta-triol 3-phenylacetate (1).

Cancer, 2001 Dec 15, 92(12), 3085 - 92
A novel taxane active against an orthotopically growing human glioma xenograft; Laccabue D et al.; BACKGROUND: The development of effective chemotherapy for central nervous system tumors is hampered by the blood-brain barrier and by limited drug diffusion in the brain tissue . BAY 59-8862 is a new taxane analog that was selected and developed for its activity against tumors with a P-glycoprotein-mediated, multidrug-resistant phenotype . Because P-glycoprotein is implicated in limiting the access of drugs to central nervous system tumor targets, the objective of this study was to evaluate the ability of intravenously administered BAY 59-8862 to affect the growth of central nervous system tumors . METHODS: The U-87 MG human glioma cell line was xenografted orthotopically (intracranially) in nude mice . Paclitaxel or BAY 59-8862 was delivered intravenously four times every fourth day, and antitumor efficacy was assessed by examining the effects on mouse survival time and by histologic examination of mouse brain . Drug levels in plasma and brain were determined according to a high-performance liquid chromatography method . RESULTS: The analog was as potent as paclitaxel in inhibiting the proliferation of three human glioma cell lines (U-87 MG, SW1783, and GBM) and was as effective as paclitaxel in inhibiting the heterotopic (subcutaneous) tumor growth in nude mice of U-87 MG cells (tumor weight inhibition, approximately 60%) . In contrast, BAY 59-8862 was more active than paclitaxel (P < 0.05 in two of three experiments) in increasing the survival time of mice that were injected orthotopically with U-87 MG cells . The results were supported by the pharmacokinetic data, which indicated a much higher (about 15-fold) brain:plasma level ratio in BAY 59-8862-treated animals compared with paclitaxel-treated animals . CONCLUSIONS: The study provides evidence of an additional pharmacologic advantage of BAY 59-8862, i.e., the ability to affect the growth of intracranial tumors, probably due to the lack of recognition by the P-glycoprotein-mediated transport systems . The favorable behavior of BAY 59-8862 supports the potential interest in the analog for clinical studies in patients with brain tumors or metastases .

J Pharmacol Exp Ther, 2002 Jan, 300(1), 97 - 104
Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats; Cherrington NJ et al.; Many phase I and II microsomal enzyme inducers share common mechanisms of transcriptional activation and thus share a similar battery of genes that are coordinately regulated . Many phase II metabolites are thought to be transported out of cells by multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) . The purpose of this study was to determine the organ distribution of these three transporters in rat, and whether they are coordinately regulated with phase I and II drug-metabolizing enzymes . Therefore, Mrp1, 2, and 3 mRNAs were quantified using branched DNA signal amplification in multiple tissues and in tissues from rats that were treated with 18 chemicals thought to induce drug-metabolizing enzymes by six different transcription activation mechanisms {aryl-hydrocarbon receptor ligands, constitutive androstane receptor (CAR) activators, pregnane-X-receptor ligands, peroxisome proliferator activator receptor ligands, electrophile response element (EpRE) activators, and CYP2E1 inducers} . It was found that Mrp1 was expressed at a high level in kidney, lung, intestine, and brain, with low expression in liver . Mrp2 was highly expressed in liver and duodenum, and Mrp3 was highly expressed throughout the intestine but very low in liver . Microsomal enzyme inducers did not markedly increase the expression of Mrp1 or Mrp2 . However, Mrp3 expression was significantly increased by each of the CAR activators and an EpRE activator in liver . Mrp3 was not similarly induced in kidney and large intestine, demonstrating that the coordinate inducibility of Mrp3 is specific to the liver . We conclude that rat hepatic Mrp3 is induced by CAR activators, thus enhancing the vectoral excretion of some phase II metabolites from the liver to the blood.

Carcinogenesis, 2001 Dec, 22(12), 1931 - 7
Mechanisms of tolerance to DNA damaging therapeutic drugs; Karran P; The cytotoxic effect of many anticancer drugs relies on their ability to damage DNA . Drug resistance can be associated with the ability to remove potentially lethal DNA lesions . DNA damage tolerance offers an alternative route to resistance . In a drug-tolerant cell, persistent DNA damage has become uncoupled from cell death . Tolerance to some DNA damaging drugs is accompanied by inactivation of the cell's DNA mismatch repair pathway . This is widely acknowledged as the mechanism underlying resistance to methylating agents and to 6-thioguanine which produce structurally similar types of DNA damage . Defects in mismatch repair are also associated with resistance to numerous drugs that produce a wide variety of structurally diverse DNA lesions . Here I consider possible mechanisms by which mismatch repair might influence drug resistance and the extent to which loss of mismatch repair might be considered to confer a multidrug resistance phenotype.

Cancer Res, 2001 Dec 15, 61(24), 8851 - 8
Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells; Conze D et al.; It has been shown that serum levels of interleukin (IL)-6 are elevated in patients with various types of cancer . However, the exact source of IL-6 in these patients and its role in tumor progression remain unclear . Here we demonstrate that the autocrine production of IL-6 by tumor cells promotes resistance of the cells to chemotherapy, a novel function of IL-6 in cancer biology . Breast cancer cells that are sensitive to drug treatment do not express IL-6, whereas high levels of IL-6 are produced by multidrug-resistant breast cancer cells . Expression of the IL-6 gene in drug-sensitive breast cancer cells increases their resistance to drug treatment by activating the CCAAT enhancer-binding protein family of transcription factors and inducing mdr1 gene expression . Thus, the autocrine production of IL-6 by tumor cells is an important factor in determining the susceptibility or resistance of these cells to drug treatment . Because tumors from some breast cancer patients contain IL-6-producing cells, it is possible that IL-6 could potentially be used as a prognostic factor for chemotherapy resistance.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 166 - 70
Pfmdr1 alleles and response to ultralow-dose mefloquine treatment in Gabonese patients; Mawili-Mboumba DP et al.; The identification of parasite molecular markers involved in resistance to antimalarial compounds is of great interest for monitoring the development and spread of resistance in the field . Polymorphisms in Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) have been associated with chloroquine resistance and mefloquine susceptibility . In the present study, carried out in Lambarene, Gabon, we investigated the relationship between the presence of mutations at codons 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene and the success of ultralow-dose mefloquine treatment (1.1 mg/kg of body weight) . Sixty-nine patients were included in the study, and depending on the level of in vivo resistance to mefloquine, they were classified as sensitive responders (S), patients with low-grade resistance (RI), and nonresponders (NR) . We found that the prevalences of the Tyr-86 mutation among isolates from patients in groups S, RI, and NR were 100, 96, and 90%, respectively, and that the prevalence of the Phe-184 mutation among the isolates was 80% in each group . A prevalence of about 10% point mutations at codons 1042 and 1246 was detected only in isolates from patients in groups RI and NR . There was no statistically significant association between the presence of the Tyr-86 mutation and the in vivo response (P = 0.79) . Among the parasite isolates from patients with drug-resistant infections, 83% had the wild-type pfmdr1 genotype (S(1034)-N(1042)-D(1246)) . No link between the presence of this genotype and parasite resistance was detected (P = 0.42) . Among the isolates analyzed, 85 had double mutations (Y(86)-F(184) or Y(86)-Y(1246)) and 11 had triple mutations (Y(86)-D(1042)-Y(1246), Y(86)-F(184)-Y(1246), or Y(86)-F(184)-D(1042)) . These findings are not consistent with those of previous in vitro studies and suggest that further evaluation of pfmdr1 gene polymorphism and in vivo mefloquine sensitivity are needed.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 144 - 50
Optimization of xanthones for antimalarial activity: the 3,6-bis-omega-diethylaminoalkoxyxanthone series; Kelly JX et al.; Hydroxyxanthones have been identified as novel antimalarial agents . The compounds are believed to exert their activity by complexation to heme and inhibition of hemozoin formation . Modification of the xanthone structure was pursued to improve their antimalarial activity . Attachment of R-groups bearing protonatable nitrogen atoms was conducted to enhance heme affinity through ionic interactions with the propionate side chains of the metalloporphyrin and to facilitate drug accumulation in the parasite food vacuole . A series of 3,6-bis-omega-diethylaminoalkoxyxanthones with side chains ranging from 2 to 8 carbon atoms were prepared and evaluated . Measurement of heme affinity for each of the derivatives revealed a strong correlation (R(2) = 0.97) between affinity and antimalarial potency . The two most active compounds in the series contained 5- and 6-carbon side chains and exhibited low nanomolar 50% inhibitory concentration (IC(50)) values against strains of chloroquine-susceptible and multidrug-resistant Plasmodium falciparum in vitro . Both of these xanthones exhibit stronger heme affinity (8.26 x 10(5) and 9.02 x 10(5) M(-1), respectively) than either chloroquine or quinine under similar conditions and appear to complex heme in a unique manner.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 89 - 94
Prediction of abacavir resistance from genotypic data: impact of zidovudine and lamivudine resistance in vitro and in vivo; Walter H et al.; Abacavir is frequently used in antiretroviral combination therapies as a potent nucleoside reverse transcriptase inhibitor (NRTI) . Four mutations are selected for by abacavir in vitro and in vivo: K65R, L74V, Y115F, and M184V . Abacavir resistance has also been observed in NRTI multidrug-resistant samples . Furthermore, abacavir resistance has been described in the context of zidovudine resistance . To evaluate the genetic basis of abacavir resistance, the viral genotype and phenotypic resistance were analyzed for 307 patient samples . Low- and high-level resistances were defined as 2.5- to 5.5-fold- and >5.5-fold-reduced susceptibility, respectively . If all samples with abacavir-selected and NRTI multidrug resistance-associated mutations were scored as resistant, 27.6% of the samples were misclassified, mainly due to samples falsely scored as susceptible . Therefore, the relative frequencies of other mutations were evaluated . Mutations at codons 44 and 118 were rarely detected in abacavir-susceptible samples but were overrepresented in resistant samples . Site-directed mutagenesis of E44D, V118I, and M184V resulted in low-level resistance for the double mutant 44/184 and the triple mutant . Low-level abacavir resistance was also detected for a viral clone carrying zidovudine mutations only . Additional insertion of M184V into the zidovudine background doubled the resistance, whereas 44/118 did not lead to a further increase . Incorporating combinations of zidovudine mutations and M184V into the scoring system markedly reduced the number of misclassified samples, whereas 44/118 did not improve the prediction . In conclusion, the combination of M184V with zidovudine mutations gives rise to high-level abacavir resistance, which may be clinically relevant . Thus, options for useful sequential combinations of NRTI are limited.

Toxicology, 2002 Jan 15, 170(1-2), 55 - 62
Multidrug-resistance mdr1a/1b double knockout mice are more sensitive than wild type mice to acute arsenic toxicity, with higher arsenic accumulation in tissues; Liu J et al.; Arsenic is an environmental toxicant . Active extrusion via the ArsAB pump is a mechanism for arsenic detoxication in bacteria . However, how arsenic is effluxed from mammalian cells is not completely known . Our recent work shows that acquired arsenic resistance is associated with overexpression of P-glycoprotein and can be reversed by PSC833, an inhibitor for P-glycoprotein . This study utilized the mdr1a/1b(-/-) mice, which lack mdr1-type P-glycoproteins, to examine whether these mice are sensitive to arsenic toxicity and have higher arsenic accumulation in their tissues . The mdr1a/1b(-/-) and wild-type FVB mice were given arsenic as sodium arsenite (12-19 mg/kg, sc) and toxicity was examined 24 h later . The mdr1a/1b(-/-) mice were more sensitive than wild-type mice to arsenite-induced lethality, with LD(50) of 14.5 and 17 mg/kg, respectively . Histologically, arsenite produced more frequent and more severe lesions in the liver and kidney of the mdr1a/1b(-/-) mice than in wild-type mice . Serum alanine aminotransferase activity and blood urea nitrogen levels, indicative of hepatic and renal damage respectively, were increased 4 to 6-fold in the mdr1a/1b(-/-) mice as compared with 1-2-fold increases in wild-type mice . The mdr1a/1b(-/-) mice accumulated more arsenic in the liver (15.3 vs . 5.2 microg/g), kidney (7.23 vs . 3.22 microg/g), small intestine (3.98 vs . 1.57 microg/g) and brain (0.45 vs . 0.17 microg/g), as compared with wild-type mice 24 h after sodium arsenite (14 mg/kg, s.c.) administration . In summary, this study demonstrated that the mdr1a/1b(-/-) mice were more sensitive to acute arsenic toxicity and accumulated more arsenic than wild-type mice, suggesting that P-glycoproteins are involved, at least in part, in arsenic efflux in mammalians.

Acta Pharmacol Sin, 2001 Aug, 22(8), 731 - 5
Multiple drug resistance phenotype of human endothelial cells induced by vascular endothelial growth factor 165; Zhang XS et al.; AIM: To investigate the effect of vascular endothelial growth factor 165 (VEGF165) on sensitivity of endothelial cells to anticancer drugs . METHODS: Human dermal microvessel endothelial cells (HDMEC) were incubated with anticancer drugs in the presence of VEGF165 . Survival of endothelial cells was assayed by MTT method . DNA fragments of apoptosis were detected by agarose electrophoresis . Potential mechanisms underlying the effect of VEGF165 on endothelial cells were investigated with RT-PCR and Western blot analysis . RESULTS: VEGF165 induced the multidrug resistance phenotype of HDMEC to a wide variety of anticancer drugs such as epirubicin, cisplatin, etoposide, mytomycin C, vincristine, CPT-11, and taxol in vitro . This protective effect was partly due to the up-regulation of lung drug resistance protein (LRP) and multidrug resistance-associated protein (MRP), as well as the down-regulation of Bax protein induced by VEGF165 . CONCLUSION: VEGF165 induced multidrug resistance phenotype of endothelial cells, which implicated the anti-angiogenic effect of anticancer drugs might depend on microenvironment of tumors in vivo.

Acta Pharmacol Sin, 2001 Nov, 22(11), 1023 - 7
Reversal effects of droloxifene on multidrug resistance in adriamycin-resistant K562 cell line; Li J et al.; AIM: To study the reversal effects of droloxifene (DRO) on multidrug resistance (MDR) in K562 cell line resistant to adriamycin (ADR) . METHODS: K562 cell line resistant to ADR (K562/A02) and K562 cell line sensitive to ADR (K562) were treated with DRO . Using MTT assay, chemosensitivity to ADR in DRO-treated K562 cell lines was studied . Before and after the treatment with DRO 10 micromol/L, MDR1 and GSTpi gene expression were assayed by reverse transcription-polymerase chain reaction and immunocytochemistry assay . Flow cytometry was used to determine intracellular ADR concentration . RESULTS: DRO significantly reversed MDR in K562/A02 (P < 0.01) . After treatment of DRO 20, 10, and 5 micromol/L, the chemosensitivity to ADR was increased to 14, 13, and 4 folds, respectively . The reversal activity of DRO was similar to that of verapamil (VRP) . After treated with DRO 10 micromol/L, both MDR1 and GSTpi mRNA expression began to decline on the 2nd day, and significantly decreased on the 5th day (P<0.01) . The changes in P-gp and GSTpi protein expression were similar to that of their mRNA expression . Two hours after treatment of DRO 20, 10, and 5 micromol/L, intracellular ADR concentration in K562/A02 was increased to 2.9, 2.3, and 1.5 folds, respectively . However, DRO did not markedly increase ADR accumulation in K562 . CONCLUSION: DRO had strong reversal effect on MDR in K562/A02, which was comparable to that of VRP, but the reversal effect was via different pathways.

Acta Pharmacol Sin, 2001 Oct, 22(10), 949 - 55
Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin; Wang JS et al.; AIM: To investigate whether human peripheral blood hematopoietic progenitor cells (PBPC) modified with human aldehyde dehydrogenase class-3 gene (ALDH-3) and multidrug resistance gene 1 (MDR1) would increase chemotherapy resistance to 4-hydroperoxycyclophosphamide (4-HC) and -glycoprotein effluxed drugs . METHODS: A bicistronic retroviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and used to transfect the packaging cell lines PA317 by electroporation . CD34+ PBPC were isolated with a high-gradient magnetic cell sorting system (MACS), and then were transfected with supernatant of retrovirus containing human ALDH-3 and MDR1 cDNA . PCR, RT-PCR, Southern blot, Northern blot, FACS, and MTT assay were used to evaluate the transfection and expression of the transgene in target cells . RESULTS: The bicistronic retroviral vector construction was verified by PCR and restriction endonuclease analysis . Dual drug resistance genes were integrated into the genomic DNA of CD34+ PBPC and expressed efficiently . The efficiency of gene transfection in CD34+ PBPC was tested to be 18 % on colonies . Nested PCR and Neor rescue assay indicated that no helper virus was present in this system . Compared with the untransduced cells, transgene recipient cells conferred 4.5-fold resistance to 4-HC, 6.6-fold and 7.8-fold resistance to P-glycoprotein effluxed drug, vincristine and daunorubicin, respectively . CONCLUSION: Efficient transduction of two different types of drug resistance genes into human peripheral blood hematopoietic progenitor cells and the co-expression may decrease cumulative myelosuppression of combination chemotherapy.

J Infect Dis, 2001 Dec 1, 184(11), 1480 - 4 Epub 2001 Nov 13.
The white morphotype of Mycobacterium avium-intracellulare is common in infected humans and virulent in infection models; Mukherjee S et al.; Isolates of Mycobacterium avium-intracellulare (MAI) form multiple colony types named red-opaque, white-opaque, red-transparent (RT), and white-transparent (WT) . The newly discovered WT morphotype is multidrug resistant relative to other variants in vitro . To determine whether the WT morphotype occurs in humans, 32 MAI-positive clinical samples from 2 sites were plated directly onto indicator agar without prior passage in vitro . WT was the predominant morphotype in 26 (81%) of these samples and was absent in only 2 samples . WT variants grew better than isogenic RT variants in mouse and human macrophage models of infection, and RT clones that passed through such systems underwent rapid shifts to the WT morphotype . The RT morphotype was heterogeneous with regard to infectivity . In summary, the white morphotype was common in humans and was favored in disease models . It may play an important role in the establishment and persistence of MAI infection.

J Biol Chem, 2002 Feb 22, 277(8), 6497 - 503 Epub 2001 Dec 17.
Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2); Sasaki M et al.; Human organic anion transporting polypeptide 2 (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2) play important roles in the vectorial transport of organic anions across hepatocytes . In the present study, we have established a double-transfected Madin-Darby canine kidney (MDCK II) cell monolayer, which expresses both OATP2 and MRP2 on basal and apical membranes, respectively . The basal-to-apical transport of 17 beta estradiol 17 beta-d-glucuronide (E(2)17 beta G), pravastatin, and leukotriene C(4) (LTC(4)), which are substrates of OATP2 and MRP2, was significantly higher than that in the opposite direction in the double-transfected cells . Such vectorial transport was also observed for taurolithocholate sulfate, which is transported by rat oatp1 and Mrp2 . The K(m) values of E(2)17 beta G and pravastatin for the basal-to-apical flux were 27.9 and 24.3 microm, respectively, which were comparable with those reported for OATP2 . Moreover, the MRP2-mediated export of E(2)17 beta G across the apical membrane was not saturated . In contrast, basal-to-apical transport of estrone-3-sulfate and dehydroepiandrosterone sulfate, which are significantly transported by OATP2, but not by MRP2, was not stimulated by MRP2 expression . The double-transfected MDCK II monolayer expressing both OATP2 and MRP2 may be used to analyze the hepatic vectorial transport of organic anions and to screen the transport profiles of new drug candidates.

Emerg Infect Dis, 2001 Sep-Oct, 7(5), 855 - 61
Multidrug-resistant tuberculosis in prison inmates, Azerbaijan; Pfyffer GE et al.; In a tuberculosis (TB) program in the Central Penitentiary Hospital of Azerbaijan, we analyzed 65 isolates of Mycobacterium tuberculosis by IS6110-based restriction fragment-length polymorphism (RFLP) and spoligotyping . From 11 clusters associated with 33 patients, 31 isolates had an IS6110-based banding pattern characteristic of the Beijing genotype of M . tuberculosis . In addition, 15 M . tuberculosis isolates with similar RFLP patterns constituted a single group by spoligotyping, matching the Beijing genotype . Multidrug resistance, always involving isoniazid and rifampin, was seen in 34 (52.3%) of 65 isolates, with 28 belonging to the Beijing genotype.

Emerg Infect Dis, 2001 Sep-Oct, 7(5), 842 - 8
Molecular identification of streptomycin monoresistant Mycobacterium tuberculosis related to multidrug-resistant W strain; Bifani P et al.; A distinct branch of the Mycobacterium tuberculosis W phylogenetic lineage (W14 group) has been identified and characterized by various genotyping techniques . The W14 group comprises three strain variants: W14, W23, and W26, which accounted for 26 clinical isolates from the New York City metropolitan area . The W14 group shares a unique IS6110 hybridizing banding motif as well as distinct polymorphic GC-rich repetitive sequence and variable number tandem repeat patterns . All W14 group members have high levels of streptomycin resistance . When the streptomycin resistance rpsL target gene was sequenced, all members of this strain family had an identical mutation in codon 43 . Patients infected with the W14 group were primarily of non- Hispanic black origin (77%); all were US-born . Including HIV positivity, 84% of the patients had at least one known risk factor for tuberculosis.

Biochemistry, 2001 Dec 25, 40(51), 15752 - 61
Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: use of a permeant phosphoinositide-binding peptide; Bucki R et al.; During platelet activation, phosphatidylserine (PS) exposure on the extracellular face of the plasma membrane is associated with increased procoagulant activity . PS externalization is generally attributed to an increase in intracellular Ca(2+) . Various phospholipid transporters, such as specific scramblases or proteins from the family of multidrug resistance proteins, and cofactors such as phosphatidylinositol 4,5-bisphosphate (PIP2) have been proposed to participate in this process . In this study, we used a membrane-permeant polycationic peptide (RhB-QRLFQVKGRR), derived from the PIP2-binding site of gelsolin (GS 160-169) and linked to rhodamine B, to investigate the role of PIP2 in PS externalization in whole platelets . The peptide penetrated rapidly into the platelets, specifically bound to PIP2, and induced PS exposure to a similar extent as thrombin or collagen, but independently of changes in intracellular Ca(2+) or phosphoinositide 3-kinase activity . A pretreatment of platelets with quercetin, an inhibitor of phosphoinositide metabolism, drastically decreased PS exposure induced by agonists or peptide . In large unilamellar vesicles (LUVs), the presence of PIP2 was strictly required for the induction of scrambling of NBD-labeled phospholipids (PC and PS) by the peptide . In inside-out vesicles from erythrocytes (IOVs), the peptide also induced redistribution of PC and PS . Our data suggest that, in intact platelets, PIP2 acts as a target of polycationic effectors, including Ca(2+), to promote PS exposure . The use of a membrane-permeant and fluorescent peptide which binds to PIP2 is a promising tool to investigate the role of PIP2 in various cellular processes.

Br J Cancer, 2001 Dec 14, 85(12), 1998 - 2003
Selective modulation of P-glycoprotein-mediated drug resistance; Bebawy M et al.; Multidrug resistance associated with the overexpression of the multidrug transporter P-glycoprotein is a serious impediment to successful cancer treatment . We found that verapamil reversed resistance of CEM/VLB(100) cells to vinblastine and fluorescein-colchicine, but not to colchicine . Chlorpromazine reversed resistance to vinblastine but not to fluorescein-colchicine, and it increased resistance to colchicine . Initial influx rates of fluorescein-colchicine were similar in resistant and parental cells, whereas vinblastine uptake was about 10-fold lower in the resistant cells . These results provide indirect evidence that fluorescein-colchicine is transported from the inner leaflet of the membrane and vinblastine from the outer membrane leaflet . Verapamil inhibited fluorescein-colchicine transport in inside-out vesicles made from resistant cells, whilst chlorpromazine was found to activate the transport of fluorescein-colchicine . The chlorpromazine-induced activation of fluorescein-colchicine transport was temperature-dependent and may reflect its interaction with phospholipids localised in the same bilayer leaflet . Conversely, chlorpromazine localisation in this leaflet may be responsible for its allosteric inhibition of vinblastine transport from the opposing membrane leaflet . The proposed relationship between the selectivity of modulation of P-glycoprotein and the membrane localisation of the cytotoxic drug substrates and modulators may have important implications in the rational design of regimes for the circumvention of multidrug resistance clinically.

J Pharm Sci, 2001 Nov, 90(11), 1829 - 37
Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line; Perloff MD et al.; The present study characterized the response of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) to chronic ritonavir (RIT) exposure by assessing increases in P-gp and MRP1 protein expression and activity . LS-180V intestinal carcinoma cells were exposed for 3 days to 1-100 microM RIT concurrently with controls . P-gp and MRP1 protein was quantified by Western blot analysis . Cell accumulation assays, using the P-gp substrate rhodamine 123 (RH123), the P-gp/MRP1 substrate doxorubicin (DOX), and the MRP substrate carboxyfluorescein (CBF), were performed as a measure of transporter activity . RIT strongly induced P-gp and MRP1 expression (maximum 6-fold and 3-fold increases, respectively) in a concentration-dependent fashion . Following extended exposure to RIT (> 10 microM), cells accumulated < 50% of the RH123 and DOX compared with controls, whereas accumulation of CBF was decreased by 30% at 30 microM . Differences in cell accumulation of RH123 could be eliminated with verapamil (100 microM; a P-gp inhibitor), whereas decreased DOX cell accumulation was only partially reversed by verapamil . Indomethacin (100 microM; an MRP1 inhibitor) had no significant effect on RH123 or DOX accumulation, suggesting limited MRP1-mediated activity . Thus, RIT induced protein expression of P-gp and MRP1 and increased cellular drug exclusion of RH123, DOX, and CBF . Similar in vivo phenomena may occur during anti-HIV drug therapy, explaining potential decrements in therapeutic efficacy due to decreases in bioavailability or alterations in drug distribution .

J Pharm Sci, 2001 Oct, 90(10), 1593 - 8
Evaluation of creatine transport using Caco-2 monolayers as an in vitro model for intestinal absorption; Dash AK et al.; Creatine is a nutraceutical that has gained popularity in both well-trained and casual athletes for its performance-enhancing or ergogenic properties . The major disadvantages of creatine monohydrate formulations are poor solubility and oral bioavailability . In the present study, creatine transport was examined using Caco-2 monolayers as an in vitro model for intestinal absorption . Confluent monolayers of Caco-2 cells (passage 25-35) were used for the permeability studies . Monolayers were placed in side-by-side diffusion chambers . (14)C-Creatine (0.1-0.5 microCi/mL) was added to either the apical or basolateral side, and the transport of the creatine across the Caco-2 monolayer was measured over a 90-min period . The apical to basolateral transport of (14)C-creatine was small, ranging from 0.2-3% of the original amount appearing on the receiver side in a 90-min period . Interestingly, the basolateral to apical permeability of radiolabeled creatine was substantially greater than that observed in the apical to basolateral direction . Studies with drug efflux transport inhibitors indicate that neither the P-glycoprotein nor multidrug resistance-associated protein is involved in the enhanced basolateral to apical transport of creatine .

J Pharm Sci, 2001 Oct, 90(10), 1540 - 52
Influence of multidrug resistance (MDR) proteins at the blood-brain barrier on the transport and brain distribution of enaminone anticonvulsants; Cox DS et al.; Previous in vitro studies evaluating the permeability of enaminones suggested that their blood-brain barrier (BBB) transport might be influenced by the presence of an efflux mechanism . Therefore, transport mechanisms responsible for these anticonvulsants across the BBB were examined . The transport of enaminones (1 x 10(-4) M) were evaluated over 120 min with verapamil (50 microM) and probenecid (100 microM) using bovine brain microvessel endothelial cells (BBMECs) to assess the role of multidrug resistant (MDR) transport proteins {i.e., P-glycoprotein (Pgp) and MDR protein 1 (MRP1)} on efflux, respectively . Uptake studies in the presence and absence of rhodamine 123 (R123; 3.2 and 5.0 microM) were also performed in a Pgp overexpressing cell line, MCF-7/Adr . Select enaminone esters (12.5 mg/kg) were administered intravenously to mdr 1 a/b (+/+), mdr 1 a/b (-/-) knockout and probenecid pretreated mice (20 +/- 5g) . Enaminones and R123 were assayed with validated ultraviolet and fluorescence high-performance liquid chromatography methods, respectively . Verapamil and probenecid significantly ( p>0.05) inhibited the transport of select enaminone esters across BBMECs . Two enaminones caused a statistically significant increase in the uptake of R123 in MCF-7/Adr cells . Concentrations of select enaminones in mdr 1 a/b (-/-) mice brains were significantly higher ( p<0.05) compared with those in mdr 1 a/b (+/+) mice brains; however, no differences were observed in probenecid pretreated animals . Taken together, these results strongly suggest that Pgp may influence enaminone transport at the BBB and hence affect epilepsy treatment with these agents .

Int J Cancer, 2001 Dec 15, 94(6), 864 - 72
Targeting multidrug resistant tumor cells with a recombinant single-chain FV fragment directed to P-glycoprotein; Niv R et al.; The MDR1 gene product P-glycoprotein (Pgp) plays a key role in multidrug resistance of cancer cells . Pgp is an ATP-driven efflux pump that extrudes a variety of dissimilar hydrophobic cytotoxic compounds . P-glycoprotein overexpression results in multidrug resistance (MDR) of tumor cell lines in vitro as well as in cancer patients . To selectively target and eliminate MDR tumor cells, we have isolated a monoclonal antibody that specifically reacts with the first extracellular loop of the human Pgp . We have cloned the variable domain genes of this antibody and assembled a functional single-chain Fv fragment capable of specifically targeting various Pgp-expressing MDR carcinoma cells lines . Targeting and specific elimination of Pgp-dependent MDR human cancer cells was achieved by constructing a single-chain immunotoxin in which the scFv fragment was fused to a truncated form of Pseudomonas exotoxin (PE38) . We conclude that recombinant Fv-immunotoxins or other Fv-based molecules armed with potent cytotoxins represent an effective tool in targeted cancer therapy aimed at specific elimination of MDR tumor cell sub-populations . Recombinant antibody fragments targeting MDR proteins such as Pgp may be also used for intracellular expression and consequent phenotypic knockout of MDR .

Int J Cancer, 2001 Nov, 94(4), 492 - 9
Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma; Nies AT et al.; Treatment of hepatocellular carcinoma (HCC) by chemotherapy is often impeded by the intrinsic multidrug resistance (MDR) of this frequent primary cancer of the liver . The MDR phenotype can be caused by ATP-dependent export of chemotherapeutic drugs across the plasma membrane being mediated by transporters of the MDR P-glycoprotein family or of the multidrug resistance protein (MRP) family . To elucidate the role of MRP family members in HCC, we analyzed the expression and subcellular localization of MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3); all 3 isoforms have been shown to confer resistance to chemotherapeutic drugs . Semiquantitative RT-PCR demonstrated that MRP2 and MRP3 mRNA expression in HCC was at least 10-fold higher than MRP1 mRNA expression . MRP2 immunostaining was observed in 87% (33/38) of HCC samples . MRP2 was localized in the plasma membrane in a polarized fashion, either in trabecular structures resembling the canalicular membrane or in the luminal membrane when cells had a pseudoglandular arrangement . MRP3 was detected in all samples examined (9/9) by RT-PCR and by immunofluorescence microscopy . MRP3 was localized to the basolateral membrane of carcinoma cells . Double-label immunofluorescence microscopy with antibodies specific for MRP2 or MRP3 indicated that carcinoma cells expressed both MRP isoforms simultaneously . When MRP1 was detected by immunofluorescence microscopy, it was localized on the intracellular membranes of carcinoma cells . Thus, plasma membrane expression of MRP2 and MRP3, but not of MRP1, can contribute to the MDR phenotype of HCC .

Int J Cancer, 2001 Nov 1, 94(3), 377 - 82
Overexpression of the human major vault protein in astrocytic brain tumor cells; Berger W et al.; Evidence has shown that the major human vault protein (MVP), which is identical to lung resistance-related protein (LRP), may be causally involved in a special type of multidrug resistance (MDR) . The purpose of this study was to investigate the expression and cellular localization of MVP in cells derived from brain tumors and other tumors of neuroectodermal origin . Using both established cell lines (n = 22) and primary explants (n = 30), we show that a distinct overexpression of the MVP gene at the mRNA (RT-PCR) and protein (Western blot) levels is a characteristic feature of cells derived from astrocytic brain tumors . Primary cultures obtained from meningioma specimens also expressed high MVP levels, in contrast to neuroblastoma and medulloblastoma cells, which rarely contained detectable amounts of MVP . Normal human astrocytes cultured in vitro expressed MVP, although at low amounts compared with most malignant cell types . Basal MVP expression correlated with resistance against diverse antineoplastic drugs including anthracyclins, cisplatin and etoposide . By Western blot, MVP was also detected in all tumor samples taken from 7 glioma and 3 meningioma patients . Taken together, these data suggest overexpression of MVP as one explanation for the low efficacy of chemotherapeutic treatment of astrocytic brain tumors .

Cancer, 2001 Oct 1, 92(7), 1753 - 8
Suppression of proline-directed protein kinase F(A) potentiates apoptotic induction and greatly enhances chemosensitivity in human acute lymphoblastic leukemia cells; Hsu CP et al.; BACKGROUND: Previously, the authors reported that specific antisense suppression of overexpressed proline-directed protein kinase (PDPK) F(A) enhances the chemosensitivity of various clinical anticancer drugs up to > 100-fold in human prostate carcinoma cells, suggesting an association of PDPK F(A) with drug resistance in human malignancies . METHODS: In this report, by using a similar approach, the authors demonstrate further that the suppression of PDPK F(A) enhances even more dramatically the chemosensitivity of clinically used anticancer drugs in various types of human acute lymphoblastic leukemia (ALL) cells . RESULTS: Compared with parental and control transfected cells, transduced ALL cells (both Jurkat and CCRF-CEM cells) with low levels of PDPK F(A) displayed an enhanced sensitivity to vincristine, vinblastine, paclitaxel, methotrexate, doxorubicin, and daunorubicin . Estimation of the 50% inhibitory concentration (IC(50)) index further revealed that the transduced cells displayed up to > 3000-fold drug sensitivity, and there was a correlation between suppressed levels of PDPK F(A) and drug sensitivity . A mechanistic study further revealed that the enhanced chemosensitivity in transduced ALL cells was due mainly to the potentiation of apoptotic induction . CONCLUSIONS: Taken together, the results demonstrate that the suppression of overexpressed PDPK F(A) greatly enhances the chemosensitivity of various clinical anticancer drugs in both types of human ALL cells, providing initial evidence for an important role of this PDPK in controlling multidrug resistance of ALL .

Cancer, 2001 Sep 15, 92(6), 1591 - 605
Homoharringtonine: history, current research, and future direction; Kantarjian HM et al.; BACKGROUND: Cephalotoxine esters, including homoharringtonine (HHT), have shown encouraging activity in leukemia in initial studies in China and in later studies in the U.S . METHODS: The authors conducted a review of the literature to examine the studies pertinent to HHT in relation to preclinical studies and Phase I-II trials in patients with hematologic malignancies and solid tumors . RESULTS: HHT and analogues appear to induce differentiation and apoptosis . Studies from China reported high response rates in patients with leukemia . Trials in the U.S . using short HHT infusions (3-4 mg/m(2) daily for 5 days) resulted in a high incidence of cardiovascular complications that were reduced using continuous infusion schedules of 3-7 mg/m(2) daily for 5-7 days initially, and later lower dose schedules of 2.5 mg/m(2) daily for 7-14 days . Results in solid tumors were negative . However encouraging results were reported in patients with acute myeloid leukemia, myelodysplastic syndrome, acute promyelocytic leukemia, and, most important, chronic myeloid leukemia (CML) . In CML patients, HHT has been investigated alone and in combination with interferon-alpha and low-dose cytarabine in late and early chronic phases, with positive results . Additional areas of interest include the potential use of HHT for the treatment of central nervous system leukemia, polycythemia vera, and other nonmalignant conditions such as malaria . New semisynthetic preparations and HHT derivatives that bypass multidrug resistance may improve the efficacy and toxicity profiles, and broaden the range of antitumor efficacy . CONCLUSIONS: HHT and its derivatives appear to have promising activity in hematologic malignancies, a finding that needs to be pursued .

Cancer, 2001 Sep 15, 92(6), 1577 - 90
A Phase I study of infusional vinblastine in combination with the P-glycoprotein antagonist PSC 833 (valspodar); Bates S et al.; BACKGROUND: PSC 833 is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance (MDR) . The authors conducted a Phase I study of orally administered PSC 833 in combination with vinblastine administered as a 5-day continuous infusion . METHODS: Seventy-nine patients with advanced malignant disease were enrolled in the trial and treated with escalating doses of PSC 833 . Pharmacokinetic interactions between PSC 833 and vinblastine were anticipated . Accordingly, when dose limiting toxicities were observed, the dose of vinblastine was reduced as PSC 833 was escalated . Three schedules and two formulations of PSC 833 were used in the study . RESULTS: The maximum tolerated doses of PSC 833 were 12.5 mg/kg orally every 12 hours for 8 days for the liquid formulation in combination with 0.9 mg/m(2) per day vinblastine as a continuous intravenous infusion (CIV) for 5 days; and 4 mg/kg orally every 6 hours for 8 days for the microemulsion formulation in combination with 0.6 mg/m(2) per day vinblastine CIV for 5 days . The principal toxicities for PSC 833 were ataxia and paresthesias and for the combination, constipation, fever . and neutropenia . Increased oral bioavailability and increased peak and trough concentrations were observed with the microemulsion formulation . Significant interpatient variability in pharmacokinetic parameters was observed . Ten patients studied at the MTD for PSC 833 (4 mg/kg orally every 6 hours for 8 days) had inhibition of rhodamine efflux from CD56 positive peripheral lymphocytes as a surrogate for Pgp antagonism . Among 43 evaluable patients with clear cell carcinoma of the kidney, 3 patients had complete responses, and 1 patient had a partial response . CONCLUSIONS: PSC 833 in combination with vinblastine can be administered safely to patients provided the vinblastine dose is adjusted for pharmacokinetic interactions . The high interpatient variability is a significant confounding factor . Surrogate studies with CD56 positive cells suggest that Pgp inhibition in the clinical setting is achievable . Improved methods for predicting pharmacokinetic interactions should improve future studies .

Cancer, 2001 Sep 15, 92(6), 1556 - 66
Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression; Zhan M et al.; BACKGROUND: p53 mutations occur in almost half of all soft tissue sarcomas (STS) and may contribute to multidrug resistance (MDR) in patients with STS . Doxorubicin (Dox) is one of the most active single agents in STS but is less effective in STS with p53 mutations . The effect of reintroducing wild type (wt) p53 into STS cells harboring p53 mutations on the cytotoxicity of DOX in vitro and in vivo was studied . METHODS: The following cell lines were used in this study: SKLMS-1 STS cells, which do not express wt p53; two wt p53 stable transfectant cells derived from SKLMS-1 cells; and SKLMS-1 transfectant cells from a p53 temperature-sensitive mutant that expresses wt p53 at 32 degrees C and mutant p53 at 38 degrees C . The cytotoxicity of Dox was examined by {3-(4,5-dimethylthiazzol-2-yl)-2,5-diphenltetrazolium} (MTT) and clonogenetic assay, and the effect of reintroducing wt p53 on tumor suppression by Dox was evaluated with a tumorigenicity assay . DNA fragmentation was used to detect apoptosis . MDR-1 P-glycoprotein (P-gp) expression was detected by Western blot and immunohistochemical analyses of protein levels and by Northern blot analysis of mRNA levels, respectively . The intracellular accumulation of Dox was detected by flow cytometric analysis . RESULTS: The 50% inhibitory concentration (IC(50)) of Dox for the SKLMS-1 wt p53 transfectants decreased 16-fold compared with SKLMS-1 parental cells expressing mutant p53 . Colony formation of SKLMS-1 cells after Dox treatment also was inhibited by wt p53 reintroduction . The tumorigenicity of SKLMS-1 cells was inhibited by wt p53 reintroduction alone or by Dox treatment alone and was inhibited further when p53 introduction was combined with Dox treatment in severe combined immunodeficient mice . Although no difference in DNA fragmentation, Bax expression, or Bcl-2 expression was detected among wt p53 transfectants and parental SKLMS-1 cells after Dox treatment, MDR-1 P-gp expression was decreased in wt p53 transfectants compared with parental SKLMS-1 cells . Furthermore, higher intracellular accumulations of Dox were found in wt p53 transfectants than that in SKLMS-1 cells . CONCLUSIONS: Reintroduction of wt p53 into STS cells harboring p53 mutations can enhance their chemosensitivity to Dox through the inhibition of MDR-1 P-gp expression . Thus, the combination of p53 gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients with STS .

Semin Liver Dis, 2001 Nov, 21(4), 551 - 62
Role of multidrug resistance 3 deficiency in pediatric and adult liver disease: one gene for three diseases; Jacquemin E; Class III multidrug resistance P-glycoproteins, mdr2 in mice and MDR3 in humans, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion . The role of an MDR3 gene defect in liver disease was initially suspected in a subtype of progressive familial intrahepatic cholestasis called PFIC3 . Several MDR3 mutations have been identified in children with PFIC3 and are associated with a low level of phospholipids in bile, leading to a high biliary cholesterol saturation index . Mutations leading to a truncated protein are associated with an absence of canalicular MDR3 protein . The phenotypic spectrum of PFIC3 ranges from neonatal cholestasis to cirrhosis in young adults . There is now strong evidence that in addition to PFIC3, an MDR3 defect can be involved in intrahepatic cholestasis of pregnancy and in cholesterol gallstone disease . Therefore, at least three human liver diseases are due to a single gene deficiency . Patients with PFIC3 due to MDR3 deficiency may benefit from ursodeoxycholic acid therapy and could be good candidates for cell therapy in the future.

Drug Metab Dispos, 2002 Jan, 30(1), 96 - 9
A pharmacophore for human pregnane X receptor ligands; Ekins S et al.; The pregnane X receptor (PXR) is involved in transcriptional regulation of multiple cytochromes P450 and multidrug resistance-associated protein (MDR1), which encodes for the drug transporter P-glycoprotein . Crystal structure analyses suggest that the ligand binding domain is highly hydrophobic and flexible, allowing molecules of differing sizes to bind in multiple orientations . Using literature data for EC(50) (half-maximal inhibitory concentration) values for PXR activation derived for 12 human PXR ligands, a pharmacophore was developed . This pharmacophore supports the hydrophobic nature of the ligand binding domain recently deduced from the X-ray crystal structure because it contains four hydrophobic regions and one hydrogen bond acceptor . These features are consistent with at least one of the three experimentally determined orientations in which SR12813 binds to PXR, as determined by overlay studies . SR12813 fulfills all of the five pharmacophore features, as does the potent ligand hyperforin . The pharmacophore was also used to predict the binding affinity for 28 molecules not in the model but known to be PXR ligands of differing potencies . The pharmacophore distinguished the most potent activators of PXR (that display >5-fold activation/deactivation), like ecteinascidin, troglitazone, nifedipine, and dexamethasone-t-butylacetate, from poor activators, such as scopoletin and kaempferol . The model could be useful in drug development, potentially acting as a high-throughput filter for identifying compounds that may bind to PXR before in vitro determination . Ultimately, this will aid in the selection of molecules with a lesser capacity to be potent PXR ligands and thus avoid induction of numerous drug-metabolizing enzymes and MDR1.

Drug Metab Dispos, 2002 Jan, 30(1), 20 - 6
Induction of multidrug resistance-1 and cytochrome P450 mRNAs in human mononuclear cells by rifampin; Asghar A et al.; Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative, competitive RT-PCR were used to examine the capability of rifampin to induce the expression of mRNA derived from multidrug resistance-1 (MDR1) and drug-metabolizing cytochrome P450 (P450) genes in the mononuclear fraction (lymphocytes) of human blood . A total of 50 healthy volunteers (age, 18-74) participated in two studies in which 600 mg of rifampin was administered orally once daily in the evening for 7 days . Twenty of these individuals also received fexofenadine before and after rifampin dosing . MDR1 and CYP2C8 mRNAs were expressed in 100% (50 of 50) and 95% (35 of 37) of individuals, respectively, at baseline . A significant (P < 0.05; n = 37) increase in the expression of MDR1 mRNA from 176,900 +/- 122,000 to 248,500 +/- 162,300 molecules/microg of RNA was observed following rifampin administration in the human lymphocytes . There was no significant (P > 0.05) difference in MDR1 mRNA expression between males and females at baseline . Interestingly, 58% of the individuals (n = 29) demonstrated a 120% increase {95% confidence interval (CI); 120%; range, 81-153%; responders} in MDR1 mRNA expression . In contrast, the remaining 42% of individuals (n = 21) exhibited a mean decrease of -5.2% (95% CI; -5.2%; range, -15 to +4%; nonresponders) . Rifampin steady-state trough serum concentrations were not significantly different (P > 0.05) between responders and nonresponders . Likewise, there was no relationship between the observed induction in MDR1 mRNA expression in lymphocytes and the observed increase in fexofenadine oral clearance in twenty volunteers . The mRNA of CYP2E1, CYP3A5, CYP3A7, CYP4A11, and CYP4B1 genes were variably expressed at baseline and following rifampin treatment . In contrast, CYP2C9 and CYP3A4 mRNAs were undetectable in lymphocytes both before and after rifampin dosing . Interindividual variability in baseline expression and inducibility of MDR1 and P450 mRNA in human lymphocytes appeared to be substantial and may not reflect the expression of these enzymes in other tissues.

Drug Metab Dispos, 2002 Jan, 30(1), 4 - 6
Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas; Nakamura T et al.; The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), and cytochrome P450 3A (CYP3A) in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas . Caco-2 cells (passages 36-88) were kindly supplied by several laboratories in Japan . Human duodenal enterocytes were obtained from five healthy male volunteers . Normal colorectal tissues and colorectal adenocarcinomas were simultaneously obtained from seven patients with primary colorectal adenocarcinoma . MDR1, MRP1, MRP2, and CYP3A mRNA levels were determined by real-time quantitative polymerase chain reactions (PCR) . Relative concentrations of mRNAs for target proteins (MDR1, MRP1, MRP2, and CYP3A) and glyceraldehyde-3-phosphate dehydrogenase in Caco-2 cells were 1.00 +/- 0.15, 1.02 +/- 0.06, 0.94 +/- 0.10, and 0.68 +/-0.60, respectively, and those in human enterocytes were about 12-, 3-, 7-, and 8000-fold higher than in the Caco-2 cells, respectively . In contrast, MDR1, MRP1, and CYP3A mRNA levels in Caco-2 cells were comparable to those in normal colorectal tissue and colorectal adenocarcinoma.

J Clin Virol, 2002 Feb, 24(1-2), 93 - 8
Reappearance of HIV multidrug-resistance in plasma and circulating lymphocytes after reintroduction of antiretroviral therapy; Albrecht D et al.; BACKGROUND: After the discontinuation of antiretroviral therapy in HIV-infected patients with highly resistant virus, the detectability of viral resistance mutations quickly decreases . To which extent this represents a true loss of resistance or rather a detectability phenomenon remains unclear . OBJECTIVES: To monitor virologic response and resistance pattern during a non-strategic treatment interruption in the presence of highly drug-resistant viral strains . STUDY DESIGN: We performed serial genotypic resistance analyses on viral DNA isolated from a patient with a multidrug-resistant human immunodeficiency virus infection who discontinued and later on reintroduced antiretroviral therapy . Sequencing was performed on viral DNA from plasma as well as DNA from circulating leukocytes . RESULTS: While under combination antiretroviral therapy with two nucleosidic reverse transcriptase inhibitors, a non-nucleosidic reverse transcriptase inhibitor and a protease inhibitor, the viral load of the patient was around five logs . Genotypic resistance to all available agents was detected during this time . Antiretroviral therapy was then interrupted, and 14 weeks later an almost complete reversion of the virus to wild type was observed . After introduction of a new antiretroviral therapy regimen, the reappearance of nearly all of the formerly present resistance mutations had to be noted within 6 weeks, including mutations without known relation to any of the drugs in the new regimen . CONCLUSIONS: We obviously observed not the de novo appearance of a complex resistance pattern under just 6 weeks of potent antiretroviral therapy, but a reappearing archival strain of the virus . This finding provides evidence for subdetectable persistence of resistant variants during treatment interruptions . Therefore, resistance analyses from peripheral blood performed in times of treatment interruptions should be interpreted with caution as they may provide incomplete information about the resistance profile soon after reintroduction of therapy.

Brain Res Mol Brain Res, 2001 Dec 16, 97(1), 43 - 50
Transendothelial permeability of chlorpyrifos in RBE4 monolayers is modulated by astrocyte-conditioned medium; Yang J et al.; The immortalized rat brain endothelium 4 (RBE4) cell line preserves many features of the in vivo brain endothelium . It has been used as an in vitro model of the blood-brain barrier (BBB) . Astrocyte-endothelial cell interactions are crucial for maintenance of BBB characteristics . The present study investigated morphological and permeability properties of the RBE4 cell line . Immunohistochemical studies showed positive staining in RBE4 cells for E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule . Western blot immunoassay showed that RBE4 cells consistently express E-cadherin and that its expression significantly increased (P<0.001) in the presence of astrocyte-conditioned medium (ACM) . The transendothelial permeability of chlorpyrifos, an organophosphorus insecticide, was significantly decreased (P<0.001) when the RBE4 cells were grown in ACM compared with control medium . Additional studies were carried out to determine whether chlorpyrifos is a substrate for the multidrug resistance protein, P-glycoprotein (P-gp) . No significant change in chlorpyrifos transendothelial permeability was noted in the presence of verapamil, a P-gp blocker . Thus, in this system, chlorpyrifos is not a substrate for P-gp . This work demonstrates that with additional refinements the RBE4 monolayers might serve as a useful in vitro model for the study of BBB permeability and modulation by astrocyte-derived soluble factors.

Am J Physiol Regul Integr Comp Physiol, 2002 Jan, 282(1), R191 - 8
Xenobiotic efflux pumps in isolated fish brain capillaries; Miller DS et al.; To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from killifish and dogfish shark brain using confocal microscopy and quantitative image analysis . In killifish brain capillaries, luminal accumulation of fluorescent derivatives of cyclosporin A and verapamil was concentrative, specific, and energy dependent (inhibition by KCN) . Transport was reduced by PSC-833, but not by leukotriene C4, indicating the involvement of P-glycoprotein . The ability of capillaries to transport the cyclosporin A derivative was unchanged over 20 h, demonstrating the long-term viability of the preparation . Luminal accumulation of the fluorescent organic anions sulforhodamine 101 and fluorescein-methotrexate was also concentrative, specific, and energy dependent . Transport of these compounds was reduced by leukotriene C4, but not by PSC-833, indicating the involvement of a multidrug resistance-associated protein (Mrp) . Similar results were obtained for isolated capillaries from dogfish shark . Immunostaining localized P-glycoprotein and Mrp2 to the luminal surface of the killifish brain capillary endothelium . These findings validate a new and long-lived comparative model for studying drug transport across the blood-brain barrier and, as in mammals, implicate P-glycoprotein and Mrp2 in transport from the central nervous system to blood in fish.

Br J Cancer, 2001 Nov 30, 85(11), 1746 - 52
High levels of the MDM2 oncogene in paediatric rhabdomyosarcoma cell lines may confer multidrug resistance; Cocker HA et al.; The MDM2 protein is known to be overexpressed in some sarcomas including rhabdomyosarcoma . However, the extent to which the MDM2 protein influences sensitivity to chemotherapeutic drugs is unclear . We have analysed this further using stable transfection of the mdm2 gene into 4 well-characterised human paediatric rhabdomyosarcoma cell lines . Transfection with the mdm2 gene resulted in increased levels of the MDM2 protein in all the cell lines . In 2 of the lines, SCMC and RD, the mdm2 gene caused between 2-fold and 61-fold increase in resistance to vincristine, etoposide and doxorubicin but not to cisplatin . In these lines there was an increase in expression of the mdr-1 gene which encodes P-glycoprotein, but not the mrp1 gene which encodes the multidrug resistance protein (MRP) . The resistance was reversible using the MDR modulator PSC833, confirming the presence of P-glycoprotein . We conclude that MDM2 overexpression may be a mechanism by which multidrug resistance is regulated in some rhabdomyosarcomas . (c) 2001 Cancer Research Campaign

J Biol Chem, 2002 Feb 15, 277(7), 5110 - 9 Epub 2001 Dec 07.
ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain; Hou YX et al.; Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP . ATP hydrolysis by either one or both NBDs is essential to drive transport of solute . Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely . How events at these two domains are coordinated during the transport cycle have not been fully elucidated . Earlier reports (Gao, M., Cui, H . R., Loe, D . W., Grant, C . E., Almquist, K . C., Cole, S . P., and Deeley, R . G . (2000) J . Biol . Chem . 275, 13098-13108; Hou, Y., Cui, L., Riordan, J . R., and Chang, X . (2000) J . Biol . Chem . 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold . This suggested transmission of a positive allosteric interaction from NBD2 to NBD1 . To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with {alpha-(32)P}8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs . However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2 . Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold . This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP . Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by {alpha-(32)P}8-N(3)ADP as well as by either {alpha-(32)P}- or {gamma-(32)P}8-N(3)ATP (Hou et al . (2000), see above) . These mutations also strongly diminished the enhancement by ATP of {alpha-(32)P}8-N(3)ADP labeling and the transport activity of the protein . Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2 . The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.

Acta Pharmacol Sin, 2001 Feb, 22(2), 111 - 6
P glycoprotein regulated transport of glutamate at blood brain barrier; Liu XD et al.; AIM: To study whether efflux of glutamate (Glu) at blood brain barrier (BBB) was regulated by P-glycoprotein (P-gp) . METHODS: 1) After intracerebral microinjection {3H}Glu 5 min, recoveries were determined in injected cerebrums in presence of multidrug-resistant(MDR) reversing agents verapamil (Ver), vincristine(VCR), an d cyclosporin A(CsA); 2) apparent transfer constants (Kin) of {3H}Glu from plasma to brain were determined after the in situ rat brain perfusion 2 min us ing solution containing MDR-reversing agents; 3) uptake amount of {3H}Glu by prima ry cultured bovine brain capillary endothelial cells(BCEC) was analyzed; and 4) In presence of MDR-reversing agents and antibody of P-gp, C(219), uptake amount of {3H}Glu by luminal membrane vesicles derived from BCEC was also determined . RESULTS: In control rats, remaining percentage of {3H}Glu in inject ed cerebrums was 25 %+/-16 % at 5 min after intracerebral injection . After pre-treating with CsA 10, 100 micromol/L, VCR 20 micromol/L and Ver 100 micromol/L, the remaining percentages of {3H}Glu were increased to about 2.2, 2.5, 2.3, a nd 2.7 folds of control, respectively . In the in situ rat brain per fusion experiment, VCR and CsA in perfusion medium concentration-dependently increased {3H}Glu BBB permeability to brain . Co-administration o f CsA 40 micromol/L mad e BBB permeability of {3H}Glu in cerebral cortex, hippocampus and striatum increase to about 9, 3, 7, and 4.6 folds of control, respectively . Steady-state uptake of {3H}Glu by BCEC was also increased up to 2.5 folds in presence of 100 micromol/L CsA . MDR-reversing agents and antibody of P-gp, C219, level-dependently inhibited the uptake of {3H}Glu by luminal membrane vesicles of BCEC . And this process is ATP-dependent . CONCLUSION: Efflux of Glu at BBB may be regulated by P-gp.

J Med Chem, 2001 Dec 20, 44(26), 4668 - 76
Chemosensitization of a multidrug-resistant Leishmania tropica line by new sesquiterpenes from Maytenus magellanica and Maytenus chubutensis; Kennedy ML et al.; Parasite resistance to drugs has emerged as a major problem in current medicine, and therefore, there is great clinical interest in developing compounds that overcome these resistances . In an intensive study of South American medicinal plants, herein we report the isolation, structure elucidation, and biological activity of dihydro-beta-agarofuran sesquiterpenes from the roots of Maytenus magellanica (1-14) and M . chubutensis (14-17) . This type of natural products may be considered as privileged structures . The structures of 10 new compounds, 1, 3, 6-9, and12-15, were determined by means of (1)H and (13)C NMR spectroscopic studies, including homonuclear (COSY and ROESY) and heteronuclear correlation experiments (HMQC and HMBC) . The absolute configurations of eight hetero- and homochromophoric compounds, 1, 3,6-9, 12, and 13, were determined by means of CD studies . Fourteen compounds, 1-3 and 6-16, have been tested on a multidrug-resistant Leishmania tropica line overexpressing a P-glycoprotein-like transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation . From this series, 1, 2, 3, 14, and 15 showed potent activity, 1 being the most active compound . The structure-activity relationships of the different compounds are discussed.

AAPS PharmSci . 2000;2(3):E26.
No evidence for the involvement of the multidrug resistance-associated protein and/or the monocarboxylic acid transporter in the intestinal transport of fluvastatin in the rat; Lindahl A et al.; Fluvastatin, an amphiphilic anion, shows a nonlinear increase in effective intestinal permeability (P(eff)) with increasing lumenal concentrations in rats . The main objective of this study was to investigate whether or not this observation could be attributed to an efflux-mediated transport by the multidrug resistance-associated protein (MRP) . In parallel, we investigated the possible involvement of the monocarboxylic acid transporter (MCT) in the rapid intestinal absorption of fluvastatin . Single-pass perfusions were performed in the ileum and colon of the rat, with and without the presence of well-established inhibitors/substrates for the MRP (probenecid) and the MCT (nicotinic acid) . The results suggest that neither the MRP nor the MCT are involved to any significant extent in the absorption process of fluvastatin in the rat intestine . Thus, the previously reported concentration-dependent P(eff) of fluvastatin in these intestinal regions of the rat is probably not attributable to saturation of any efflux mediated by MRP.

J Biol Chem, 2002 Feb 15, 277(7), 5360 - 8 Epub 2001 Dec 05.
Functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxification; Li L et al.; We have identified a detoxifying efflux carrier from Arabidopsis using a functional cloning strategy . A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin . After transformation of KAM3 cells with an Arabidopsis cDNA library, transformants were selected for restored growth on the toxic medium . One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria . We named this Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1) . A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis genome . Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds . Interestingly, AtDTX1 was also capable of detoxifying Cd(2+), a heavy metal . Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.

Br J Pharmacol, 2001 Dec, 134(8), 1609 - 18
Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein; Taylor AM et al.; 1 . Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein . Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies . 2 . No significant differences were observed in substrate ({(3)H}-azidopine) binding to wt or cys-less P-gp . Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace {(3)H}-azidopine from the wt or cys-less P-gp . These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions . 3 . The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical . The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control . However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine . 4 . Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways . The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.

Br J Pharmacol, 2001 Dec, 134(8), 1601 - 8
Saint John's wort: an in vitro analysis of P-glycoprotein induction due to extended exposure; Perloff MD et al.; 1 . Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin . Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability . 2 . The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system . Experiments were performed with 3 to 300 microg ml(-1) of methanol-extracted SJW and 0.03 to 3 microM HYP, representing low to high estimates of intestinal concentrations . 3 . In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir) . P-gp was quantified using Western blot analysis . P-gp expression was strongly induced by SJW (400% increase at 300 microg ml(-1)) and by HYP (700% at 3 microM) in a dose-dependent fashion . Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor . Fluorescence microscopy of intact cells validated these findings . In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested . 4 . SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations . Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW.

Bioorg Med Chem, 2002 Jan, 10(1), 195 - 205
Peptidomimetic glutathione analogues as novel gammaGT stable GST inhibitors; Burg D et al.; Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a role in the onset of multidrug resistance (MDR) . To evaluate the contribution of GST to this process, inhibitors are needed . Glutathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly . In this paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase mediated breakdown . The peptide bonds in GSH were replaced by isosteres, such as the 'reduced' amide (which was prepared using a novel method), N-methylamide, urethane, and methylene linkages . The in vitro evaluation of the compounds focuses on GST inhibition and stability towards gamma-glutamyl-transpeptidase (gammaGT), the main enzyme involved in GSH breakdown . The compounds were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor . All novel GSH-analogues were shown to inhibit rat liver cytosolic GSTs . Furthermore, peptidomimetic changes of the gamma-glutamyl-cysteine amide bond greatly improved stability towards gammaGT . These compounds may therefore be useful in the design of novel in vivo applicable GST inhibitors.

Eur J Biochem, 2001 Dec, 268(24), 6569 - 77
Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells; Klokouzas A et al.; Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself . Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter . This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1 . The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1 . In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells . It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells . These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action.

Pharmacol Rev, 2001 Dec, 53(4), 569 - 96
Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations; Lee G et al.; Drug transport in the central nervous system is highly regulated not only by the blood-brain and the blood-cerebrospinal fluid barriers but also in brain parenchyma . The novel localization of drug transporters in brain parenchyma cells, such as microglia and astrocytes, suggest a reconsideration of the present conceptualization of brain barriers as it relates to drug transport . That is, the cellular membranes of parenchyma cells act as a second "barrier" to drug permeability and express transporters whose properties appear similar to those localized at the conventional brain barriers . This review will focus on the molecular characteristics, localization, and substrate specificities of several classes of well known membrane drug transporters (i.e., the organic cation, organic anion, nucleoside, P-glycoprotein, and multidrug resistance proteins) in the brain . Comparisons to similar transporters localized within the peripheral system and clinical implications of the functional expression of specific drug transport families will be discussed when appropriate . Nutrient and neurotransmitter transporters, whose characteristics have been reviewed extensively elsewhere, will not be considered in this review.

Cancer Lett, 2002 Jan 10, 175(1), 39 - 44
The preferential induction of apoptosis in multidrug-resistant KB cells by 5-fluorouracil; Warr JR et al.; It has been previously been shown that multidrug resistance may be associated with biochemical changes which increase the sensitivity of resistant cells to the induction of apoptosis by certain agents . We have shown here that 48 h exposure to 5-fluorouracil (5-FU) induces both a significantly greater proportion of apoptotic cells and much greater cleavage of the apoptosis-related protein poly-(ADP-ribose)-polymerase in the multidrug-resistant (MDR) carcinoma cell line, KB-A1, than in corresponding drug-sensitive control KB-3.1 cells . Exposure to 5-FU also reduced the level of the anti-apoptotic protein, protein kinase B, in the MDR cells, but not in the control cells.

Gynecol Oncol, 2001 Dec, 83(3), 523 - 32
Characterization of a human carcinosarcoma cell line of the ovary established after in vivo change of histologic differentiation; Mobus VJ et al.; OBJECTIVES: Cell lines are valuable in vitro models for clinical and basic research . Most ovarian cancer cell lines described are serous cystadenocarcinomas or poorly differentiated adenocarcinomas . The establishment of ovarian cancer cell lines with rare histologic differentiation is especially of interest . We describe the establishment of a carcinosarcoma cell line of the ovary after in vivo selection . METHODS: The cell line OV-MZ-22 was established from a solid tumor mass in the upper abdomen . At the time of establishment, the patient underwent secondary debulking and was pretreated with six cycles of cis-platinum/epirubicin/cyclophosphamide . Features of the cell line studied included morphology, ultrastructure, heterotransplantation, chromosome analysis, and analysis of intermediate filament proteins and actins by immunocytochemistry . RESULTS: The first histologic report of the patient described a papillary cystadenocarcinoma, which changed to a carcinosarcoma with predominantly sarcomatous differentiation at secondary debulking . This cell line is aneuploid and shows no expression of the tumor-associated antigens CA-125 and CEA, but an overexpression of MDR-1, lung resistance protein, p53, and topoisomerase I and II, but not of multidrug-resistance-associated protein . The cell line did not give rise to transplant tumors in nude mice . The histologic and immunocytochemical comparison of the primary and the relapsed tumor proved evidence of an in vivo change of differentiation from predominantly papillary cystadenocarcinoma to carcinosarcoma . Morphological characteristics and intermediate filament pattern underlined the sarcomatous differentiation and origin of this cell line . The differentiation phenotype of OV-MZ-22 cells is that of smooth-muscle cells . CONCLUSION: The change of histologic differentiation was apparently due to a selection process caused by platinum-containing chemotherapy . The origin of the cell line and its rarity make this new line an appropriate tool for further investigation.

Neuroreport, 2001 Nov 16, 12(16), 3557 - 60
P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain; Potschka H et al.; Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs) . In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms . The mechanisms that lead to drug resistance in epilepsy are not known . Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy . However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs . In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid . Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine . The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

Int J Parasitol, 2001 Dec, 31(14), 1681 - 5
Identification of two putative ATP-cassette genes in Encephalitozoon intestinalis; Bonafonte MT et al.; Currently existing chemotherapeutic compounds are limited and few are effective for treating microsporidiosis . It is possible that resistance of Encephalitozoon to some drugs occurs by efflux mechanisms similar to those previously described for mammalian tumour cells, bacteria or protozoal parasites such as Plasmodium, Leishmania and Entamoeba histolytica . The data in the present study suggest that Encephalitozoon intestinalis contains at least one multidrug resistance gene . We report here two complete sequences EiABC1 and EiABC2, encoding different ATP-binding cassette genes from E . intestinalis, including a P-gp.

Ital J Anat Embryol, 2001, 106(2 Suppl 1), 59 - 68
Intracellular P-glycoprotein in multidrug resistant tumor cells; Arancia G et al.; The overexpression of the drug-efflux molecular pump P-glycoprotein (P-gp) may confer to tumor cells the multidrug resistant (MDR) phenotype, which is one of the causes of cancer chemotherapy failure . By investigating several in vitro models of human tumor cells, we observed that P-gp, in addition to its localization on the plasma membrane, can also be found intracellularly . In particular, by using immunocytochemical and cytofluorimetric methods, we revealed that in MDR breast cancer cells (MCF-7) a significant level of P-gp was expressed in the Golgi apparatus, which is the major site of accumulation of the antitumoral compound doxorubicin . Moreover, we demonstrated the intracellular location of P-gp in three stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane . Double immunofluorescence labelling and immunoelectron microscopy revealed, also in this tumor cell type, the location of P-gp in the Golgi apparatus where it seems to play a pivotal role in intracellular drug transport . Finally, we analyzed the expression, localization and function of drug transport proteins in human colon carcinoma lines (LoVo) exhibiting different degrees of intrinsic or drug-induced resistance . We found that only MDR LoVo cells expressed P-gp on the plasma membrane while both low-level drug resistant clonal LoVo cells and MDR LoVo cells appeared to be positive for intracellular P-gp . Our findings suggest a functional role of the intracytoplasmic P-gp in the transport and sequestration of drugs . This represents a complementary protective mechanism of tumor cells against cytotoxic agents.

Novartis Found Symp, 2001, 240, 269 - 81; discussion 282-9
The multiple mechanisms of multidrug resistance and cellular pH; Simon S; Multidrug resistance (MDR) describes a variety of strategies that tumour cells develop to evade the cytotoxic effects of anti-cancer drugs . Tumour cells that have become MDR show decreased cellular sensitivity to the drug employed in chemotherapy as well as a broad spectrum of drugs without obvious common targets or structural homology . MDR is a major obstacle to the successful treatment of tumours . Our understanding of multidrug resistance is limited, in part, by our limited knowledge of what makes tumour cells more sensitive to chemotherapeutics . Drug resistance may subvert the same mechanisms that make tumour cells hypersensitive or use molecular modifications unrelated to the mechanisms that lead to hypersensitivity in tumour cells . Indeed, multidrug resistance is likely to be the consequence of a multitude of mechanisms . Some drug-resistant and drug-sensitive mammalian cell lines have shown aberrations in regulation of cellular pH . These changes can contribute to some of the physiological and cell biological changes that are observed both in drug-sensitive and MDR tumour cell lines . As such, at least in some tumour cell lines, the changes of pH contribute both to the physiology of transformation and the physiology of drug-resistance . The observation that these variations from normal pH are not universally observed in either tumour or MDR cells suggests that these mechanisms cannot be generalized to all transformed or MDR cells . It remains to be resolved to what extent these changes are a consequence of studying cell lines grown in vitro or if they reflect the in situ physiology of human tumours . There are no detectable changes in cytosolic or organellar pH in cells transfected with P-glycoprotein, a plasma membrane-based protein implicated in drug resistance . Thus, there are likely to be multiple mechanisms contributing to multiple drug-resistance.

Novartis Found Symp, 2001, 240, 232 - 47; discussion 247-50, 265-8
pH and multidrug resistance; Roepe PD; Most chemotherapeutic drugs in use today are hydrophobic small molecules that are also typically either weakly basic, weakly acidic or charged . Thus, changes in the electrochemical parameters of tumour cell membranes have important effects on their transmembranous diffusion and cellular retention . Changes in these parameters can also modulate the function of immunological agents, and affect the signal transduction associated with induction of apoptosis . For these reasons, it is logical to propose that many (if not most) of the characteristics of multidrug-resistant (MDR) tumour cells could be due to perturbations in cellular ion transport . Indeed, many reports of altered ion transport in MDR cells can be found in the literature . Moreover, many studies suggest that P glycoprotein (Pgp) overexpression confers this altered ion transport, however, detailed physical-chemical analysis of this phenomenon has been confused by the complexity of the model systems devised to study Pgp . To help resolve this confusion, our laboratory has focused on a detailed characterization of 'pure' and stable Pgp transfectants unadulterated by the complications of chemotherapeutic drug exposure, various yeast strains and yeast vesicle preparations, and purified, reconstituted Pgp preparations . Recent data obtained with these model systems are summarized in this paper.

Hum Pathol, 2001 Nov, 32(11), 1240 - 4
Expression of multidrug resistance 1 and glutathione-S-transferase-Pi protein in nasopharyngeal carcinoma; Chen CL et al.; Radiotherapy is the modality of choice for the treatment of nasopharyngeal carcinoma (NPC) . However, systemic chemotherapy has recently been found to play an increasing role in the treatment of advanced or metastatic disease . The status of drug resistance gene expression that has crucial impact on chemotherapy has not been fully addressed for patients with NPC . In this study, we examined the expression of multidrug resistance 1 (MDR-1) and glutathione-S-transferase-Pi (GST-Pi) in primary, recurrent, and metastatic NPC using results of immunohistochemical examinations . The results were correlated with the expression of Epstein-Barr virus (EBV) latent protein, latent membrane protein 1 (LMP1), and clinicopathologic features, including stage, histopathologic types, and survival rates . MDR-1 protein expression was detected in 18 (12.6%) of 143 patients with primary NPC, 14 (32.6%) of 43 with recurrent NPC, and O (0%) of 20 with metastatic NPC, whereas 83 (58%) of 143 patients with primary NPC, 30 (69.8%) of 43 with recurrent NPC, and 13 (65%) of 20 with metastatic NPC expressed GST-Pi . EBV-LMP1 was expressed in 59 (41.3%) of 143 patients with primary NPC, 23 (53.5%) of 43 with recurrent NPC, and 9 (45%) of 20 with metastatic NPC . Simultaneous expression of MDR1 and GST-Pi was observed in 13 (72.2%) of 18 patients with primary NPC and 12 (85.7%) of 14 with recurrent NPC . The expression of LMP1 was detected in only 6 of the 13 patients with primary NPC and 6 of the 12 with recurrent NPC . We concluded that the expression of GST-Pi was more frequent in NPC tumor tissues than the expression of MDR-1 . The expression of MDR-1 correlated with clinicopathologic features of primary NPC, including the histopathologic types and survival rates, but not with disease stage . The expression of GST-Pi did not correlate with clinicopathologic features . The expression of MDR-1 and GST-Pi did not correlate with expression of EBV-LMP1 for patients with NPC .

Haematologica, 2001 Dec, 86(12), 1287 - 95
Multiparametric analysis of apoptotic and multi-drug resistance phenotypes according to the blast cell maturation stage in elderly patients with acute myeloid leukemia; Suarez L et al.; BACKGROUND AND OBJECTIVES: Acute myeloid leukemia (AML) is a heterogeneous group of malignant diseases, often characterized by coexistence of more than one subpopulation of blast cells . Multiparametric flow cytometry immunophenotyping has proven to be a reliable and sensitive approach for the discrimination of myeloid blast cells from residual normal cells present in bone marrow samples from AML patients and, at the same time, allows the identification of different maturation compartments among myeloid blasts . Therefore, it provides a unique tool for assessing apoptotic and multidrug resistance (MDR)-associated phenotypes in individual subsets of leukemic cells . DESIGN AND METHODS: The aim of the present study was to explore the simultaneous expression of proteins related to both apoptosis (APO2.7, bcl-2, bax) and multidrug resistance (MDR1, MRP, LRP) in the different blast cell subpopulations detected at diagnosis in a group of 72 elderly patients with AML . In addition, we included 5 bone marrow samples from healthy adult donors in the analysis . RESULTS: Immature blast cells (CD34+: subset I) showed a significantly higher level of bcl-2 expression (p <0.0001) together with a lower reactivity for APO 2.7 (p=0.02) as compared to the other more mature CD34- cell subsets . The expression of Bax parallelled that of APO 2.7, although the difference between immature CD34+ blast cells and the mature blast cell subsets did not reach statistical significance (p=0.18) . These results translated into a significantly (p<0.0001) higher bcl-2/bax ratio for the CD34+ blast cells as compared to that of the two CD34- blast cell subpopulations . Regarding the expression of the multidrug resistance-associated proteins Pgp and MRP, CD34+ blast cells displayed a greater expression of both proteins as compared to the more mature CD34- AML blast cells, but differences according to maturation stage of AML blast cells did not reach statistical significance . In contrast, LRP expression was significantly lower in the more immature CD34+ blast cell subset than in the more mature ones (p=0.01) . INTERPRETATIONS AND CONCLUSIONS: As far as normal bone marrow is concerned our results suggest that all blast cell subpopulations are more protected from apoptosis than their normal counterparts . We conclude that in elderly patients with AML the more immature blast cells are more resistant to apoptotic processes, which could explain why, when AML relapses, the blast cells frequently display a more immature phenotype than that observed at diagnosis . Contradictory results in multidrug resistance profile support the hypothesis that failure to respond to chemotherapeutic drugs in AML is a multifactorial phenomenon.

Biochem Biophys Res Commun, 2001 Dec 7, 289(3), 733 - 7
Analysis of chromatin-immunopurified MeCP2-associated fragments; El-Osta A et al.; As molecular biologists, we are continuing to unravel the interactions by which DNA binding proteins mediate the expression of genes . The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between chromatin structure and its role in regulating transcription, replication, and recombination in vivo . We describe a robust assay used to identify the molecular determinants associated with chromatin . In this article we illustrate the ChIP technique and use the transcriptionally silent-hypermethylated multidrug resistance (MDR1) gene as the platform for methyl-CpG binding protein 2 (MeCP2) localization on chromatin . Driven by the hypothesis that repression is strongly dependent on the methylation profile of the endogenous promoter, we demonstrate that MDR1 is targeted by MeCP2 . Methylated MDR1 chromatin is highly enriched with MeCP2 and is in striking contrast to localization observed in cells in which MDR1 is transcriptionally active . In a distinct model system we discuss experimental methods used to immunopurify the MeCP2 repressor complex on chromatin and quantify protein-DNA association by competitive PCR approach . (c) 2001 Elsevier Science.

J Clin Microbiol, 2001 Dec, 39(12), 4420 - 5
Multidrug-resistant Trichosporon asahii infection of nongranulocytopenic patients in three intensive care units; Wolf DG et al.; Trichosporon asahii (Trichosporon beigelii) infections are rare but have been associated with a wide spectrum of clinical manifestations, ranging from superficial involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients . We report on the recent recovery of T . asahii isolates with reduced susceptibility in vitro to amphotericin B (AMB), flucytosine, and azoles from six nongranulocytopenic patients who exhibited risk factors and who developed either superficial infections (four individuals) or invasive infections (two individuals) while in intensive care units . The latter two patients responded clinically and microbiologically to AMB treatment . All six isolates were closely related according to random amplified polymorphic DNA studies and showed 71% similarity by amplified fragment length polymorphism analysis, suggesting a common nosocomial origin . We also review the literature pertaining to T . asahii infections and discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus.

Int J Infect Dis, 2001, 5(3), 126 - 32
Molecular epidemiology of tuberculosis among eight hospitals in New York City, 1996-1997; Magnani J et al.; OBJECTIVE: To determine the molecular epidemiology of tuberculosis isolated from patients cared for at eight hospitals scattered throughout New York City . MATERIALS AND METHODS: Cases of tuberculosis occurring in 1996 and 1997 at collaborating hospitals were identified, and demographic data were extracted from patient charts . All available isolates were analyzed by IS6110 for genetic relatedness . The molecular fingerprints were compared both to each other and to the larger repository of strains from New York City developed and maintained at the Public Health Research Institute . RESULTS: One hundred and eighty cases were fully characterized . Compared with New York City cases, study patients were more likely to be Asian and less likely to be non-Hispanic blacks . Overall, 97 (54%) of the cases were clustered with respect to other study strains or with respect to the other New York City isolates . Clustered strains were significantly more likely to be from non-Hispanic blacks or patients born in the United States . The largest cluster (n = 17) was the "W" strain previously associated with an outbreak of multidrug-resistant tuberculosis in New York City . In the current study, the majority of W strain isolates were fully drug-susceptible . CONCLUSIONS: High rates of genetically related tuberculosis continue to occur among patients in New York City, in spite of improved control of nosocomial outbreaks and dramatic decreases in the overall case rates . The use of molecular techniques to suggest patterns of transmission has become essential in developing and assessing routine tuberculosis control strategies.

Anticancer Res, 2001 Jul-Aug, 21(4A), 2745 - 51
Influence of hypotonic stress on the biliary excretion of doxorubicin in Wistar and TR- rats; Gaugg M et al.; In tumor cells, doxorubicin (DOX) is excreted by P-glycoprotein (P-gp) and the multidrug resistance-associated protein (mrp) . Both transporters might also be involved in cellular regulatory volume decrease (RVD) . To study the hepatobiliary excretion of DOX during RVD, isolated livers of Wistar and multidrug resistance-associated protein 2 (mrp2)-deficient TR- rats were first perfused with an isotonic and later with a hypotonic medium . In both rat strains, DOX is effectively excreted into the bile . Within 30 minutes, the biliary excretion of DOX-derived fluorescence steadily increased to 1.1+/-0.11 and 0.84+/-0.05 nmoles/min . g liver in Wistar and TR- rats, respectively . Under hypotonic conditions, DOX excretion followed the biphasic-increase in bile flow . Excretion was increased to 136+/-19% and 176+/-9% (first peak) and to 141+/-11% and 157+/-14% (second peak) in Wistar and TR- rats, respectively . Our data show that in the liver of both strains, exposure to a hypotonic medium stimulates DOX excretion, presumably by activation of P-gp and mrp2 during RVD.

Anticancer Res, 2001 Jul-Aug, 21(4A), 2709 - 12
Effects of butaclamol, clopenthixol, mepromazine and cannabinol stereoisomers on apoptosis induction; Varga A et al.; The efflux pump of multidrug resistant mdr cells have different sensitivities to some stereoisomeric forms of CNS-active compounds . The ABC transporters of mdr cells were more sensitive to (-)butaclamol than to its stereoisomeric counterpart (8), which may function to alter the membrane structure . We suppose that the drug-accessible membrane structure possesses an important role in the induction (or prevention) of apoptosis . Therefore the apoptosis-inducing effect of three stereoisomeric pairs was studied on mouse lymphoma cells . In these experiments levo- and dextromepromazine had similiar effects . The cis- and trans-clopenthixol were less effective in apoptosis induction than the 12H-benzo(a)-phenothiazine used as a positive control . The effect of stereoisomeric pairs on induced apoptosis was studied when the cells were exposed to the stereoisomers for 60 minutes before subjection apoptosis induction by benzo(a)phenothiazine, a well-known apoptosis inducer . Then the pretreated cells were exposed to 12H-benzo(a)-phenothiazine for 60 minutes . The samples were washed and incubated for 24 hours . The cells were stained with annexin-V-FITC and propidium iodine and investigated by flow cytometry . The mdr cells with increased membrane integrity may result in the preferential killing of multidrug resistant cancer cells in the presence of some stereoisomers.

Anticancer Res, 2001 Jul-Aug, 21(4A), 2357 - 62
Expression of Y box-binding protein-1 correlates with DNA topoisomerase IIalpha and proliferating cell nuclear antigen expression in lung cancer; Gu C et al.; Y box-binding protein-1 (YB-1), a member of the DNA binding protein family, interacts with inverted CCAAT boxes (Y-boxes) . Y-boxes are located on the promoter of numerous genes, such as DNA topoisomerase IIalpha (Topo IIalpha), proliferating cell nuclear antigen (PCNA) and multidrug resistance 1 (MDR1) . In this study, we used immunohistochemical (IHC) staining to detect YB-1 expression in 59 lung cancer tissues and to evaluate whether YB-1 expression was associated with the expression of YB-1 target genes such as Topo IIalpha, PCNA and MDR1 in human lung carcinoma . Twenty-eight out of 59 cases (47.5%) were stained positive for YB-1 in the cytoplasm, while 30 out of 59 cases (50.8%) were positive for PCNA in the nuclei . Topo IIalpha-positive cells were detected in 16 out of 59 cases (27.1%) . Eight out of 59 cases (13.6%) had greater than 5% P-gp positive cells expression . There was a significant correlation between the YB-1 and Topo IIalpha expression in small cell lung cancer (SCLC) (p=0.0242) . YB-1 expression also correlated with PCNA expression in non-small cell lung cancer (NSCLC) (p=0.0001) . Higher levels of YB-1 expression were associated with T3-4 and Stage III-IV tumors in adenocarcinomas (p=0.0072; p=0.0168) . In contrast, no relationship was found between YB-1 expression and P-gp expression . Our study suggests that YB-1 expression correlates with Topo IIalpha and PCNA expression in lung cancer.

Anticancer Res, 2001 Jul-Aug, 21(4A), 2273 - 80
Screening and discovery of novel MDR modifiers from naturally occurring bisbenzylisoquinoline alkaloids; Fu LW et al.; BACKGROUND: The failure of conventional cancer chemotherapy has been linked to overexpression of a membrane associated P-glycoprotein (P-gp) that acts as an energy-dependent drug efflux pump . A promising strategy to conquer multidrug resistance (MDR) is to develop functional MDR modifiers that can inhibit the activity of P-gp . Materials and METHODS: We used MTT in combination with other in vitro drug evaluation assays to screen potential MDR modifiers from a series of naturally occurring Bisbenzylisoquinoline Alkaloids (BBIs) that were isolated from natural plants . RESULTS: Our in vitro screening assays indicated that at least six of these natural compounds (FF0019, FF0018, FF0015, FF0014, FF0011 and FF0012) showed potent activities to restore sensitivity of resistant tumor cells, such as MCF-7/adr and KBv200 cells, to many antitumor drugs including doxorubicin and vincristine . Further analyses by measurement of radioactive {3H}-Vincristine indicated that these BBIs increased intracellular drug accumulation in MDR cells, but had little effect on drug-sensitive cells . CONCLUSIONS: These results suggested that the mechanism of these compounds to reverse MDR was associated with the increase in the intracellular drug accumulation through inhibiting the activity of P-gp . Another important feature is that the in vitro cytotoxic effect of these naturally occurring BBIs themselves on tumor cells was very low . Thus, these compounds may possess great promise in being developed into novel MDR modifiers.

J Hum Genet, 2001, 46(11), 656 - 63
Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution; Conrad S et al.; The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells . In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs . Using polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences . Among several mutations found, two are expected to cause amino acid substitutions . One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain . To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined . Transport of the MRP1 substrates leukotriene C4, 17beta-estradiol 17beta-(D)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1.

Proteomics, 2001 Oct, 1(10), 1233 - 48
Proteomics for studying cancer cells and the development of chemoresistance; Hutter G et al.; Extensive studies during the last decades have identified several mechanisms through which cells escape the cytotoxic effects of a variety of chemotherapeutic drugs . One type of drug resistance is called multidrug resistance (MDR), because selection with one anticancer drug leads to cross-resistance with a wide range of other drugs . These MDR cells express frequently plasma transport proteins like p-glycoprotein . But cellular resistance to chemotherapy is multifactorial and may be affected by the cell cycle stage and proliferation status, biochemical mechanisms such as detoxification, cellular drug transport, or DNA replication and repair mechanisms . Several laboratory techniques, such as polymerase chain reaction, immunocytochemistry, flow cytometry, blotting, and fluorescent microscopy have been used for the identification of MDR markers and mechanisms . We review the possibilities in studying cancer biology and development of chemoresistance in cancer treatment using the proteomic approach.

Zhonghua Xue Ye Xue Za Zhi, 1999 Jun, 20(6), 288 - 91
{Modulation of multiple drug resistance by nomegestrol acetate and droloxifene in K562/A02}; Li J et al.; OBJECTIVE: To study the modulation of mdr1, glutathione S-transferase Pi (GST pi), topoisomerase II alpha (Topo II alpha) and multidrug resistance-associated protein (MRP) by nomegestrol acetate (NOM) and droloxifene (DRO) in K562 cell line . METHODS: Adriamycin (ADM)-resistant K562 (K562/A02) and parental K562 cells were treated with NOM and DRO . The alterations of chemosensitivity to ADM were evaluated by MTT assay, mRNA and protein expression of drug-resistant gene by RT-PCR and immunohistochemistry and accumulation of ADM by flow cytometry (FCM) . RESULTS: Both NOM and DRO markedly enhanced chemosensitivity to ADM of K562/A02 cells . After 20, 10 and 5 mumol/L of NOM or DRO treatment, the chemosensitivities to ADM were increased by 17.7, 15.1 and 5.1 times, respectively in NOM treated cells and by 12.3, 12.9 and 4.0 times, respectively in DRO treated cells . In both the drugs treated cells the accumulation of ADM was increased by 2-3 times . The MDR reversal activity of NOM was similar to that of VRP, but the modulatory action of DRO on K562/A02 cells was weaker than that of VRP at concentration of 20 mumol/L . After 20 mumol/L of NOM treatment, chemosensitivity to ADM of K562 cells was markedly enhanced (P < 0.05) . Both NOM and DRO were found to significantly inhibit mdr1 and GST pi expression and increase Topo II alpha expression at the mRNA and protein level . This modulation of gene expression was time dependent and the maximal effects appeared 5 days after drugs treatment . Both NOM and DRO failed to modulate the MRP expression . In K562 cells, NOM and DRO also inhibited GST pi expression (P < 0.05) . CONCLUSION: Both NOM and DRO could markedly reverse the MDR of K562/A02 cells . The reversal activity of NOM was comparable to that of VRP . Both the drugs could modulate mdr1, GST pi and Topo II alpha expression in a time dependent manner.

Lung Cancer, 2001 Dec, 34 Suppl 2, S91 - 4
Drug resistance in non-small cell lung cancer; Monzo M et al.; There is an underlying feeling in the oncology community that chemotherapy has reached a therapeutic glass ceiling in non-small cell lung cancer (NSCLC) and that we will never attain the acceptable survival rates that are just beyond our reach . Multiple clinical trials have tested doublets, triplets and sequential chemotherapy in what is often regarded as a futile attempt to break through this ceiling . Recent large randomized trials have not demonstrated that any of these combinations is superior to any of the others . Nevertheless, the analysis of DNA or RNA from patients can permit us to assess genes that may be specific targets of certain cytotoxic agents and others that may be markers of multidrug resistance . With this in mind, the Spanish Lung Cancer Group has designed a trial to test the involvement of various genes in resistance to gemcitabine and cisplatin.

J Nat Prod, 2001 Nov, 64(11), 1398 - 403
Leishmanicidal, antiplasmodial, and cytotoxic activity of novel diterpenoid 1,2-quinones from Perovskia abrotanoides: new source of tanshinones; Sairafianpour M et al.; Cryptotanshinone (1), a quinoid diterpene with a nor-abietane skeleton, and three new natural products, 1beta-hydroxycryptotanshinone (2), 1-oxocryptotanshinone (3), and 1-oxomiltirone (4), were isolated from roots of the Iranian medicinal plant Perovskia abrotanoides . Their structures were established using homo- and heteronuclear two-dimensional NMR experiments, supported by HRMS . The total amount of tanshinones isolated from dry roots of Perovskia abrotanoides was about 1.5% . The compounds exhibited leishmanicidal activity in vitro (IC(50) values in the range 18-47 microM) . These findings provide a rationale for traditional use of the roots in Iran as a constituent of poultices for treatment of cutaneous leishmaniasis . The isolated tanshinones also inhibited growth of cultured malaria parasites (3D7 strain of Plasmodium falciparum), drug-sensitive KB-3-1 human carcinoma cell line, multidrug-resistant KB-V1 cell line, and human lymphocytes activated with phytohaemagglutinin A (IC(50) values in the range 5-45 microM) . The toxicity of tanshinones toward the drug-sensitive KB-3-1 and the multidrug-resistant KB-V1 cells was the same, indicating that the compounds are not substrates for the P-glycoprotein drug efflux pump.

Br J Cancer, 2001 Nov 16, 85(10), 1564 - 71
MRP1 gene expression level regulates the death and differentiation response of neuroblastoma cells; Peaston AE et al.; We have previously reported a strong correlation between poor prognosis in childhood neuroblastoma (NB) patients and high-level expression of the transmembrane efflux pump, Multidrug Resistance-associated Protein (MRP1), in NB tumour tissue . In this study, we inhibited the endogenous expression of MRP1 in 2 different NB tumour cell lines by stably transfecting an MRP1 antisense expression vector (MRP-AS) . Compared with control cells, MRP-AS transfectant cells demonstrated a higher proportion of dead and morphologically apoptotic cells, spontaneous neuritogenesis, and, increased synaptophysin and neurofilament expression . Bcl-2 protein expression was markedly reduced in MRP-AS cells compared to controls . Conversely, we found that the same NB tumour cell line overexpressing the full-length MRP1 cDNA in sense orientation (MRP-S) demonstrated resistance to the neuritogenic effect of the differentiating agent, all-trans-retinoic acid . Taken together, the results suggest that the level of MRP1 expression in NB tumour cells may influence the capacity of NB cells for spontaneous regression in vivo through cell differentiation and death.

Eur Biophys J, 2001 Oct, 30(6), 430 - 42
The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence; Rothnie A et al.; In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity . We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers . Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads . P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers . For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents . Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment . Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein . Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization . The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes . In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes . However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers . In summary, P-gp is able to cause perturbation in the organization of bilayer constituents . Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.

J Exp Clin Cancer Res, 2001 Sep, 20(3), 393 - 400
Interferon-alpha 2b modulation of doxorubicin sensitivity in a multidrug resistant cell line; Lucero Gritti MF et al.; We evaluated the effect of interferon-alpha2b as a chemosensitiser on HCT-15 cell line in treatment with doxorubicin . Chemosensitivity was determined by {3H}-thymidine incorporation and tetrazolium assays . The levels of expression of P-glycoprotein, Bcl-2 oncoprotein and HLA-ABC complex, and cell cycle/apoptosis analysis were determined by flow cytometry . Dox 50 ng/ml - IFN alpha 2b 500 IU/ml treatment inhibited cell proliferation (47.2 +/- 1.4%, p < 0.0001; MTT assay: 40.6 +/- 1.2%, p < 0.0001) and augmented the expression of P-170, Bcl-2 and HLA-ABC, while it didn't exert apoptosis, producing a slight G2/M arrest . A concentration of IFN-alpha2b, that by itself is not cytotoxic, can potentiate the efficacy of the anticancer drug . This effect is not due to a down-modulation of P-170 . The absence of apoptosis and augmented levels of Bcl-2 expression suggests that this could be one of the mechanisms of drug resistance exerted by these cells.

Zhonghua Jie He He Hu Xi Za Zhi, 2001 Aug, 24(8), 458 - 60
{Clinical significance of the expression of lung resistance protein in non-small cell lung carcinomas}; Lu M et al.; OBJECTIVE: To investigate the expression of the lung resistance-related protein (LRP) in non-small cell lung carcinomas (NSCLC) and evaluate its clinical significance . METHODS: Using immunohistochemistry, LRP was examined in 69 NSCLC specimens obtained by either transbronchial biopsy via fibrobronchoscopy or transcutaneous needle biopsy or surgical resection . Of the 69 patients from whom tumor specimens were obtained, 52 were male and 17 were female . RESULTS: LRP expression was detected in 39/69 (57%) tumor specimens . There was no correlation between LRP expression and NSCLC histological classification (P > 0.05) . No significant differences were found in gender and among age groups . The positive rates of LRP were 67% (18/27), 55% (17/31), 52% (23/44), 64% (16/25), 83% (5/6), 57% (36/63) and 50% (3/6) in adenocarcinoma, squamous, staging T1-2, T3-4, N3, M0 and M1 respectively . It was shown that LRP expression was not associated with primary tumor size and metastasis status (N,M) . NSCLC patients with positive LRP expression responded more poorly to chemotherapy than those with negative LRP expression (P < 0.05) . CONCLUSIONS: The frequencies of multidrug resistance caused by LRP are similar between adenocarcinoma and squamous . Expression of LRP is related to multidrug resistance of NSCLC, which in turn related to the efficacy and prognosis of chemotherapy . The examination of LRP expression may be valuable for choice of chemotherapy regimen.

Chin Med Sci J, 1998 Mar, 13(1), 24 - 8
Reversion of multidrug resistance in the P-glycoprotein positive breast cancer cell line (MCF-7/ADR) by introduction of hammerhead ribozyme; Yuan Y et al.; A hammerhead ribozyme which site-specifically cleaved the GUC position in codon 880 of the mdr1 mRNA was designed . The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump . A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH beta Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype . The ribozyme was stably expressed in the cell line by the RNA dot blotting assay . The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdr1 mRNA expression by 83.5%; and the expressed ribozyme could inhibit the formation of P-glycoprotein detected by immuno-cytochemistry assay and could reduce the cell's resistance to adriamycin; this means that the resistant cells were 1,000-fold more resistant than the parental cell line (MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line . These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

Curr Opin Investig Drugs, 2001 Sep, 2(9), 1294 - 301
Chimeric fusion proteins--diphtheria toxin-based; Frankel AE et al.; Most cancer patients receive chemotherapy drugs that target DNA or the cell division apparatus . Many of these patients develop multidrug-resistant tumor cells, thus, novel methods to overcome drug resistance are needed . One approach is to target tumor cell protein synthesis . Peptide toxins, which catalytically inactivate protein synthesis, have been re-engineered to selectively bind and intoxicate tumor cells . Diphtheria toxin (DT), a member of the class of peptide toxins, has been subjected to structural and genetic analysis and protein engineering for several decades . In this review, we will examine the structure, function, synthesis and pharmacology of anticancer DT conjugates.

Am J Trop Med Hyg, 2001 Nov, 65(5), 450 - 5
In vitro susceptibility of Plasmodium falciparum isolates from Myanmar to antimalarial drugs; Wongsrichanalai C et al.; In vitro drug susceptibility profiles were assessed in 75 Plasmodium falciparum isolates from 4 sites in Myanmar . Except at Mawlamyine, the site closest to the Thai border, prevalence and degree of resistance to mefloquine were lower among the Myanmar isolates as compared with those from Thailand . Geometric mean concentration that inhibits 50% (IC50) and 90% (IC90) of Mawlamyine isolates were 51 nM (95% confidence interval {CI}, 40-65) and 124 nM (95% CI, 104-149), respectively . At the nearest Thai site, Maesod, known for high-level multidrug resistance, the corresponding values for mefloquine IC50 and IC90 were 92 nM (95% CI, 71-121) and 172 nM (95% CI, 140-211) . Mefloquine susceptibility of P . falciparum in Myanmar, except for Mawlamyine, was consistent with clinical-parasitological efficacy in semi-immune people . High sensitivity to artemisinin compounds was observed in this geographical region . The data suggest that highly mefloquine-resistant P . falciparum is concentrated in a part of the Thai-Myanmar border region.

Jpn J Cancer Res, 2001 Nov, 92(11), 1242 - 50
Paclitaxel and SN-38 overcome cisplatin resistance of ovarian cancer cell lines by down-regulating the influx and efflux system of cisplatin; Komuro Y et al.; Cisplatin (DDP) is one of the key drugs used to treat patients with ovarian cancer, although resistance to DDP can occur . Paclitaxel and SN-38 (an active metabolite of irinotecan (CPT-11)) are two drugs that are effective in patients with DDP-resistant ovarian cancer . To study how these drugs may overcome the intrinsic and / or acquired resistance of cancer cells to DDP, we investigated the effect of a combination of DDP with paclitaxel and a combination of DDP with SN-38 on three ovarian cancer cell lines, RTSG (intrinsically resistant cell line), KF (DDP-sensitive cell line), and KFra (acquired resistant cell line obtained from KF) . We found that these combinations showed additive to synergistic antitumor activity . A time-dependent platinum (Pt) accumulation was observed in the DDP-sensitive KF cell line, while a decrease occurred in the KFra cell line . Little accumulation was observed in RTSG . Intracellular Pt accumulation was increased in all three cell lines by exposure to paclitaxel or SN-38 . Ouabain, a Na(+),K(+)-ATPase inhibitor, decreased Pt accumulation in KF and KFra cell lines and inhibited the paclitaxel- and SN-38-induced increases in Pt accumulation in these cell lines . When we assessed the mRNA levels of the multidrug resistance-associated protein (MRP), which may be an efflux pump for DDP, the combination of paclitaxel or SN-38 with DDP down-regulated these levels, which are up-regulated by DDP alone . These results suggest that paclitaxel and SN-38 overcome DDP resistance of ovarian cell lines by controlling intracellular accumulation of DDP via both the influx and efflux systems.

Jpn J Cancer Res, 2001 Nov, 92(11), 1235 - 41
Reversal of multidrug resistance by novel nitrophenyl pyrones, SNF4435C and D; Kurosawa K et al.; SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein . These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells {vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM}, human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR . Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete . SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells . Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice . When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained . These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.

Int J Oncol, 2001 Dec, 19(6), 1169 - 78
A synthetic triptycene bisquinone, which blocks nucleoside transport and induces DNA fragmentation, retains its cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines; Wang B et al.; In contrast to the parent triptycene (code name TT0), triptycene bisquinone (code name TT2) is cytostatic (IC50: 300 nM) and cytotoxic (IC50: 230 nM) in wild-type (WT), drug-sensitive HL-60 cells (HL-60-S) at day 4 . Therefore, the effects of this new quinone antitumor drug were assessed and compared to those of daunorubicin (DAU, daunomycin) in the multidrug-resistant (MDR) HL-60-RV and HL-60-R8 sublines, which respectively overexpress P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP) . In contrast to DAU, which loses its cytostatic {resistance factors (RFs): 22.9-35.7} and cytotoxic (RFs: 23.8-31.3) activities in MDR sublines, TT2 decreases tumor cell proliferation (RFs: 0.9-1.3) and viability (RFs: 0.9-1.5) as effectively in HL-60-S as in HL-60-RV and HL-60-R8 cells at days 2 and 4 . Similarly, DAU inhibits the rate of DNA synthesis less effectively in MDR than in parental HL-60 cells (RFs: 8.1-11.9) but TT2 decreases the incorporation of 3{H}-thymidine into DNA to the same degree in HL-60-S, HL-60-RV and HL-60-R8 cells (RFs: 1.2) . In contrast to DAU, which is inactive, the advantage of TT2 is its ability to block the cellular transport of purine and pyrimidine nucleosides in WT tumor cells, an effect which persists in both MDR sublines (RFs: 1.0-1.2) . Moreover, the concentrations of DAU which induce maximal DNA cleavage in HL-60-S cells at 24 h lose all or most of their DNA-damaging activity in HL-60-RV and HL-60-R8 cells, whereas treatments with 4 microM TT2 produce similar peaks of DNA fragmentation in all WT and MDR cell lines . Since TT2 not only mimics the antitumor effects of DAU but also blocks nucleoside transport and retains its effectiveness in MDR cells that have already developed different mechanisms of resistance to DAU, this new quinone antitumor drug might be valuable to develop new means of polychemotherapy.

Chest, 2001 Nov, 120(5), 1514 - 9
Increased incidence of multidrug-resistant tuberculosis in diabetic patients on the Bellevue Chest Service, 1987 to 1997; Bashar M et al.; STUDY OBJECTIVES: To investigate the characteristics of tuberculosis infection in diabetic patients at Bellevue Hospital . DESIGN: We conducted a case-control study retrospectively reviewing the records of patients at Bellevue Hospital Center from 1987 to 1997 with a discharge diagnosis of tuberculosis and diabetes mellitus . SETTING: Bellevue Hospital Center is a 1,200-bed, inner-city municipal hospital located in the Lower East Side of New York City . PATIENTS: Fifty-three identified patients had verified tuberculosis infection and diabetes; of these, 50 charts were available for review . One hundred five control cases were selected from nondiabetic patients with a discharge diagnosis of tuberculosis during the same time period . MEASUREMENTS AND RESULTS: Thirty-six percent (18 cases) of the patients with diabetes and tuberculosis had multidrug-resistant tuberculosis (MDR-TB) compared to only 10% (10 cases) in the control group (p < 0.01) Controlling for homelessness, HIV status, and directly observed therapy, the relative risk of MDR-TB was calculated to be 8.6 (confidence interval, 3.1 to 23.6) in the diabetic group compared to the control group . CONCLUSIONS: There was a significant association between diabetes and MDR-TB . Diabetes continues to be a risk factor for tuberculosis and was associated with MDR-TB in our patients.

Anticancer Res, 2001 Jul-Aug, 21(4B), 2925 - 32
Immunohistochemical expression of topoisomerase IIalpha (Topo IIalpha) and multidrug resistance-associated protein (MRP), plus chemosensitivity testing, as chemotherapeutic indices of ovarian and endometrial carcinomas; Koshiyama M et al.; OBJECTIVE: The purpose of this study was to investigate the chemosensitive and chemoresistant indices of gynecologic malignancies . METHODS: We studied the expression of topoisomerase II alpha(Topo II alpha) and multidrug resistance-associated protein (MRP), and then correlated them with the in vitro chemosensitivities of gynecologic tumor cells using immunohistochemistry and a tetrazolium dye (MTT) assay . RESULTS: In the 19 ovarian carcinomas examined, the mean Topo II alpha index (%) and the tumor cell growth inhibition rate (I.R.: %) for doxorubicin and etoposide in the clear cell adenocarcinomas {15.8, 21.4, 32.3} were all lower than in the endometrioid {33.9; p<0.001, 58.3, 61.9; p<0.05, respectively} and serous adenocarcinomas {43.6; p<0.001, 75.0, 79.8; p<0.01, respectively} . Comparing these markers with the clinical response to chemotherapy, the overall predictive accuracy of the Topo II alpha index and the MTT assay was 87.5% (14/16) and 81.3% (13/16), respectively . In the 24 endometrial carcinomas examined, the mean Topo II a index and the I.R for doxorubicin and etoposide in the G1 carcinomas {22.2, 26.8, 21.5} were significantly lower than in the G2/G3 carcinomas {38.4, 54.0; p<0.001, 40.5; p<0.05} . Furthermore, strong MRP expression (> or = 50%) was detected in 13 (93%) of the 14 G1 carcinomas, but in only 4 (44%) of the 9 G2/G3 carcinomas (p<0.05) . CONCLUSIONS: The Topo II alpha index and the results of in vitro chemosensitivity testing may be of assistance in selecting the appropriate chemotherapeutic drugs against gynecologic malignancies based on their histological type and differentiation, along with MRP expression.

Clin Infect Dis, 2001 Dec 15, 33(12), 2009 - 16 Epub 2001 Nov 09.
Artemisinin antimalarials in pregnancy: a prospective treatment study of 539 episodes of multidrug-resistant Plasmodium falciparum; McGready R et al.; The emergence and spread of multidrug-resistant Plasmodium falciparum compromises the treatment of malaria, especially during pregnancy, where the choice of antimalarials is already limited . Artesunate (n=528) or artemether (n=11) was used to treat 539 episodes of acute P . falciparum malaria in 461 pregnant women, including 44 first-trimester episodes . Most patients (310 {57.5%}) received re-treatments after earlier treatment with quinine or mefloquine . By use of survival analysis, the cumulative artemisinin failure rate for primary infections was 6.6% (95% confidence interval, 1.0-12.3), compared with the re-treatment failure rate of 21.7% (95% confidence interval, 15.4-28.0; P=.004) . The artemisinins were well tolerated with no evidence of adverse effects . Birth outcomes did not differ significantly to community rates for abortion, stillbirth, congenital abnormality, and mean gestation at delivery . These results are reassuring, but further information about the safety of these valuable antimalarials in pregnancy is needed.

Br J Cancer, 2001 Oct 19, 85(8), 1175 - 84
Cross-talk between signalling pathways and the multidrug resistant protein MDR-1; Ding S et al.; The multidrug resistant protein MDR-1 has been associated with the resistance to a wide range of anti-cancer drugs . Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer . We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification . We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged . Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold . Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated . This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself . Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells . This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol .

Am J Clin Pathol, 2001 Nov, 116(5), 729 - 37
Neoadjuvant chemotherapy in cervical carcinoma: regulators of cell cycle, apoptosis, and proliferation as determinants of response to therapy and disease outcome; Costa S et al.; To evaluate whether cellular markers predict the responsiveness to neoadjuvant chemotherapy (NAC) in cervical cancer, 21 patients with stages I and II cervical carcinomas treated by NAC before surgery were followed up for a mean of 52.3 months . Pre-NAC biopsy and operative specimens were subjected to counting of apoptotic (AI/V) and mitotic (MI/V) indices, detection of human papillomavirus (HPV) DNA, and immunohistochemical analysis of cell cycle and proliferation markers (p21, p53, pRb, proliferating cell nuclear antigen {PCNA}, Ki-67) and multidrug resistance gene (MDR1), as related to NAC response (RAC), recurrence-free (RFS), and overall (OS) survival . Adenosquamous histology and lymph node involvement were significant determinants of nonsurvival . All carcinomas contained HPV DNA . In univariate analysis, p21, pRb, and MDRI in the biopsy specimen and PCNA, Ki-67, and pRb in the surgical sample significantly predicted RAC, while age, AI/V number of lymph nodes removed, and MI/V predicted RFS . Highly significant predictors of OS were AI/V number of lymph nodes removed, post-NAC MDR1 expression, MI/V and recurrence . Multivariate analysis confirmed the strong post-NAC effects of histologic type, AI/V, and MDR1 expression for RFS, and recurrence, age, and Ki-67 expression for OS . NAC responders with slightly decreased AI/V and increased MI/V had a poor prognosis.

Cancer Chemother Pharmacol, 2001 Oct, 48(4), 319 - 26
Antitumor efficacy of 26-fluoroepothilone B against human prostate cancer xenografts; Newman RA et al.; BACKGROUND: Epothilone compounds (e.g . epothilones A and B) represent a new structural class of microtubule inhibitors with the remarkable ability to inhibit tumor growth of multidrug-resistant cell lines at low nanomolar or even subnanomolar concentrations . Unfortunately, this therapeutic efficacy has only been achieved to date with a narrow therapeutic window . Hence, other structural analogs of compounds such as epothilone B are currently being synthesized in the hope that they will demonstrate equivalent antitumor efficacy with reduced systemic toxicity . PURPOSE: To evaluate the relative efficacy and toxicity of selectively modified epothilone compounds . METHODS: Compounds were initially screened for relative cytotoxicity against the human prostate cancer cell lines PC3, LNCaP, MDA PCa 2a and MDA PCa 2b . Growth inhibitory IC50 values of 0.5 to 4 nM were obtained . From this initial screen, one epothilone compound, 26-fluoroepothilone B, was chosen for further evaluation against the growth of s.c.-implanted MDA PCa 2b- and PC3-derived prostate tumors in athymic nude mice . The compound was administered intravenously at 2, 5 and 10 mg/kg after the tumors had reached 300 mm3 . Two control groups were used: paclitaxel (40 mg/kg) and saline . RESULTS: Following treatment with 10 mg 26-fluoroepothilone B/kg, there was a sustained decrease in tumor size for 30 days reaching a maximal reduction of 80% when compared with tumor growth in the saline control group . Sustained suppression (> 20 days) of tumor growth was observed following the second drug injection . Although a maximal body weight loss of 30% occurred after the second injection, all mice completely regained their initial body weight in 20 days . A lower dose (2 mg/kg) produced a 58% maximal reduction in tumor size and a 20% body weight loss . Minimal inhibition of tumor growth, however, was obtained with paclitaxel at a maximally tolerated dose (40 mg/kg) . Other epothilones tested were either less effective and/or more toxic than 26-fluoroepothilone B . This new fluorinated epothilone compound supports the growth of paclitaxel-dependent Tax-18 mutant CHO cells and produces microtubule bundles similar to those produced by paclitaxel, indicating that the two drugs share a similar mechanism of action . CONCLUSION: A new fluorinated epothilone compound, 26-fluoroepothilone B, has been described that stabilizes microtubule structures based on its support of growth of a mutant paclitaxel-dependent CHO cell line . Its antitumor activity against human prostate cancer in nude mice is superior to that of paclitaxel at equivalent toxic doses . Further research is required to determine optimal dosing strategies and to fully assess the compound's activity against other malignant diseases.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3635 - 9
Multidrug resistant Mycobacterium leprae from patients with leprosy; Maeda S et al.; Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines . Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M . leprae.

Microbes Infect, 2001 Nov, 3(13), 1111 - 3
Drug resistance of strains of Mycobacterium tuberculosis isolated in Brazil; Almeida da Silva PE et al.; Tuberculosis remains a serious public health problem, worsened by an increased frequency of multidrug-resistant Mycobacterium tuberculosis . We report here a retrospective study of resistance to antituberculosis drugs of 170 strains of M . tuberculosis isolated from the state of Rio Grande do Sul, Brazil . The frequency of resistance to at least one drug was 34%, while 22% were resistant to more than one drug . Among the strains isolated from patients without a history of previous treatment for tuberculosis, patients with positive serology for HIV and patients with previous treatment for tuberculosis, the resistance to at least one drug was 14, 27 and 73%, respectively . Multidrug-resistant tuberculosis, defined as resistant to at least rifampicin (RMP) and isoniazid (INH), was found in the groups of patients without previous treatment, HIV co-infected and with previous treatment for tuberculosis at 10, 17 and 44%, respectively . With the purpose of evaluating whether the sensitivity test to INH and RMP would be a good marker to indicate resistance to other antituberculosis drugs, sensitivity tests were performed with four more drugs in 32 strains, initially classified as resistant to INH, RMP or both . Of 18 strains resistant to INH and RMP simultaneously, 89% showed resistance to four more drugs.

Biochem Pharmacol, 2001 Nov 15, 62(10), 1337 - 43
Impact of the basic amine on the biological activity and intracellular distribution of an aza-anthrapyrazole: BBR 3422; Chou KM et al.; The anthrapyrazoles have entered clinical trials and show significant activity against breast cancer . However, these drugs are cardiotoxic and ineffective in multidrug-resistant (MDR) tumor cells . We have reported previously on the synthesis and antitumor characteristics of the 9-aza-anthrapyrazoles and their lack of cardiotoxicity; unfortunately, the leading candidates are cross-resistant in MDR-expressing cells . The results also indicated that the side arm structures of 9-aza-anthrapyrazole play a critical role in determining the drug resistance in MDR-expressing cells-only compounds that have a tertiary amine on both side arms are not cross-resistant . To further elucidate the biochemical and pharmacological impact of the side arm structures, one of the 9-aza-anthrapyrazole compounds, BBR 3422 {2-(2-aminoethyl)-5-(2-methylaminoethyl)indazolo{4,3-g,h}isoquinoline-6(2H)-one}, was selected to be photolabeled with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) . In comparison to the parental compound, the photolabeled BBR 3422 was not as cytotoxic or DNA active, but it competed better than the parental compound against azidopine on P-glycoprotein labeling . In addition, confocal microscopic studies showed that BBR 3422 was clustered mainly in the cell nucleus, but its photolabeled analogue was located in the cytoplasm of the human breast cancer cell line MCF-7 . Only a trace amount of both compounds was detected in the doxorubicin-derived resistant cell line MCF-7/ADR . The treatment of MCF-7/ADR cells with verapamil increased the intracellular amounts of both compounds.

J Med Chem, 2001 Nov 22, 44(24), 4137 - 56
Novel erythromycin derivatives with aryl groups tethered to the C-6 position are potent protein synthesis inhibitors and active against multidrug-resistant respiratory pathogens; Ma Z et al.; A novel series of erythromycin derivatives has been discovered with potent activity against key respiratory pathogens, including those resistant to erythromycin . These compounds are characterized by having an aryl group tethered to the C-6 position of the erythronolide skeleton . Extensive structural modification of the C-6 moiety led to the discovery of several promising compounds with potent activity against both mef- and erm-mediated resistant Streptoccoccus pneumoniae . Preliminary mechanistic studies indicated that the new macrolides are potent protein synthesis inhibitors, which interact with methylated ribosomes isolated from resistant organisms . In experimental animal models, these compounds exhibited excellent in vivo efficacy and balanced pharmacokinetic profiles.

J Med Chem, 2001 Nov 22, 44(24), 4092 - 113
The biological effects of structural variation at the meta position of the aromatic rings and at the end of the alkenyl chain in the alkenyldiarylmethane series of non-nucleoside reverse transcriptase inhibitors; Xu G et al.; In an effort to elucidate a set of structure-activity relationships in the alkenyldiarylmethane (ADAM) series of non-nucleoside reverse transcriptase inhibitors, a number of modifications were made at two locations: (1) the meta positions of the two aromatic rings and (2) the end of the alkenyl chain . Forty-two new ADAMs were synthesized and evaluated for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cell culture and for inhibition of HIV-1 reverse transcriptase . The size of the aromatic substituents was found to affect anti-HIV activity, with optimal activity appearing with Cl, CH(3), and Br substituents and with diminished activity occurring with smaller (H and F) or larger (I and CF(3)) substituents . The substituents at the end of the alkenyl chain were also found to influence the antiviral activity, with maximal activity associated with methyl or ethyl ester groups and with diminished activity resulting from substitution with higher esters, amides, sulfides, sulfoxides, sulfones, thioesters, acetals, ketones, carbamates, ureas, and thioureas . Twelve of the new ADAMs displayed submicromolar EC(50) values for inhibition of the cytopathic effect of HIV-1(RF) in CEM-SS cells . Selected ADAMs, 19 and 21, were compared to previously published ADAMs 15 and 17 for antiviral efficacy and activity against the HIV-1 reverse transcriptase enzyme . All four ADAMs were found to inhibit HIV-1 reverse transcriptase enzyme activity, to inhibit the replication of a variety of HIV-1 clinical isolates representing syncytium-inducing, nonsyncytium-inducing, and subtype representative isolates, and to inhibit HIV-1 replication in monocytes . Subsequent assessment against a panel of site-directed reverse transcriptase mutants in NL4-3 demonstrated no effect of the K103N mutation on antiviral efficacy and a slight enhancement (6- to 11-fold) in sensitivity to AZT-resistant viruses . Additionally, ADAMs 19 (44-fold) and 21 (29-fold) were more effective against the A98G mutation (found in association with nevirapine resistance in vitro), and ADAM 21 was 5-fold and 2-fold more potent against the Y181C inactivation mutation than the previously reported ADAMs 15 and 17, respectively . All four ADAMs were tested for efficacy against a multidrug-resistant virus derived from a highly experienced patient expressing resistance to the reverse transcriptase enzyme inhibitors AZT, ddI, 3TC, d4T, foscarnet, and nevirapine, as well as the protease inhibitors indinavir, saquinavir, and nelfinavir . ADAM 21 was 2-fold more potent than ADAM 15 and 6-fold more potent than ADAMs 17 and 19 at preventing virus replication . Thus, we have identified a novel series of reverse transcriptase inhibitors with a favorable profile of antiviral activity against the primary mutation involved in clinical failure of non-nucleoside reverse transcriptase inhibitors, K103N, and that retain activity against a multidrug-resistant virus.

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 62 - 8
Overcoming MDR by ultrasound-induced hyperthermia and P-glycoprotein modulation; Liu Y et al.; We assessed the effects of combining ultrasound-induced hyperthermia (USHT) with the P-glycoprotein modulator PSC 833 on cellular retention and cytotoxicity of rhodamine 123 (R123) and doxorubicin in the parent and multidrug resistance (MDR) variants of two human cancer lines . USHT significantly increased cellular uptake of R123 and doxorubicin . Without PSC 833, release of R123 and doxorubicin from both USHT-treated and untreated cells was rapid . As expected, PSC 833 (0.5 microM) only slowed their release into the MDR lines . Interestingly, despite the differences in their starting amounts, PSC 833 was effective in prolonging R123 and doxorubicin release from both USHT-treated and untreated MDR cells . PSC 833 did not augment the cytotoxicity of doxorubicin in parent lines but did cause a significant increase in cytotoxicity of doxorubicin in the MDR lines . However, the combined effect of USHT and PSC 833 on cytotoxicity of doxorubicin far exceeded that produced by USHT or PSC 833 alone .

J Cardiovasc Pharmacol, 2001 Dec, 38(6), 900 - 8
Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular; Bodin P et al.; In response to increased shear stress, vascular endothelial cells release adenosine triphosphate (ATP) by an unknown mechanism . We have investigated this mechanism using different approaches . First, we discovered that quinacrine, used to locate intracellular stores of ATP bound to peptides, displayed a granular fluorescence, typical of vesicular storage . Second, we found that two inhibitors of vesicular transport (monensin and N-ethylmaleimide) produced a highly significant reduction in the release of ATP from vascular endothelial cells in response to increased shear stress . Preliminary experiments using inhibitors of the cystic fibrosis transmembrane regulator, the sulfonylurea receptor, and the multidrug resistance protein showed no involvement of these ATP-binding cassette transporter proteins (previously characterized in endothelial cells) in the mechanism of release of ATP . We suggest, therefore, that the release of ATP from vascular endothelial cells, like that of nerve cells, is probably by vesicular exocytosis.

Anticancer Drugs, 2001 Nov, 12(10), 807 - 19
Synthetic 1,4-anthracenediones, which block nucleoside transport and induce DNA fragmentation, retain their cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines; Wu M et al.; Anthracene-1,4-dione and 6,7-dichloro-1,4-anthracenedione (code names AQ1 and AQ4, respectively) are cytostatic (IC50: 53 and 110 nM, respectively) and cytotoxic (IC50: 100 and 175 nM, respectively) in wild-type drug-sensitive HL-60-S tumor cells at day 4 in vitro . Therefore, the antitumor effects of these drugs were assessed and compared to those of daunorubicin (DAU) in HL-60-RV and HL-60-R8 tumor cells, which are, respectively, P-glycoprotein-positive and -negative multidrug-resistant (MDR) sublines . In contrast to DAU, which loses its cytostatic {resistance factors (RFs): 30.3-31.8} and cytotoxic (RFs: 48.8-58.1) activities in MDR sublines, AQ1 inhibits cell proliferation (RFs: 0.9-1.3) and cell viability (RFs: 1.4-1.6) as effectively in HL-60-RV and HL-60-R8 as in HL-60-S cells . Similarly, DAU decreases the rate of DNA synthesis less effectively in MDR sublines (RFs: 8.0-13.3) but AQ1 inhibits the incorporation of {3H}thymidine into DNA to the same degree in HL-60-S as in HL-60-RV and HL-60-R8 cells (RFs: 0.9-1.1) . In contrast to DAU, which is ineffective, the advantage of AQ1 is its ability to block the cellular transport of purine and pyrimidine nucleosides in HL-60-S cells, an effect which persists in the MDR sublines (RFs: 1.1) . AQ4, which mimics to a lesser degree all the antitumor effects of AQ1, except the inhibition of adenosine transport, also retains its effectiveness in MDR sublines (RFs: 1.1-3.1) . The peaks of DNA cleavage caused by DAU and AQ1 in HL-60-S cells shift to lower concentrations with increasing times of drug exposure but DAU loses most of its ability to induce DNA fragmentation in MDR sublines, whereas the levels of AQ1-induced DNA cleavage at 16 and 24 h are nearly equivalent in HL-60-S, HL-60-RV and HL-60-R8 cells . Because they not only mimic the antitumor effects of DAU in the nM range but also block nucleoside transport and remain effective in tumor cells that have developed different mechanisms of MDR, AQ1 and AQ4 analogs might be valuable to develop new means of polychemotherapy.

Anticancer Drugs, 2001 Nov, 12(10), 801 - 6
Increased cytotoxicity and stability of Lipiodol-pirarubicin emulsion compared to classical doxorubicin-Lipiodol: potential advantage for chemoembolization of unresectable hepatocellular carcinoma; Favoulet P et al.; There is no well-defined curative treatment for advanced and unresectable hepatocellular carcinoma . The widely used transarterial chemoembolization (TACE) with a doxorubicin-Lipiodol emulsion has not been shown to improve survival in randomized studies . Further, obstruction of the hepatic artery used in the procedure is badly tolerated in patients with cirrhosis . Drugs with a more rapid penetration into the cancer cells are likely to eliminate the need for obstruction of the hepatic artery . We therefore compared the cytotoxicity of another anthracycline pirarubicin with that of the commonly used doxorubicin . In this report, we show that pirarubicin has a greater in vitro cytotoxic effect than doxorubicin on the HepG2 and Hu-H7 human hepatoma cell lines . Pirarubicin emulsion with Lipiodol is more stable at 37 degrees C than doxorubicin-Lipiodol . Moreover, pirarubicin accumulates at a greater extent in the oil phase, permitting Lipiodol to act as a slow-releasing vector for the anthracycline . Further, amiodarone, a multidrug resistance inhibitor, was shown to decrease the intrinsic resistance of HepG2 and Hu-H7 cells to both anthracyclines, and the presence of polysorbate 80 in the amiodarone preparation increased the stability of the anthracycline-Lipiodol emulsions . We therefore conclude that pirarubicin is a better candidate for TACE than doxorubicin . The rapid and increased cytotoxicity of pirarubicin on hepatoma cancer cells and the stability of the pirarubicin-Lipiodol amiodarone emulsion could avoid the complete obstruction of the hepatic artery by Gelfoam sponges, and provide a better tolerated method of TACE in patients with latent liver insufficiency.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 14078 - 83 Epub 2001 Nov 13.
Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of P-glycoprotein; Kondratov RV et al.; By screening a chemical library for the compounds protecting cells from adriamycin (Adr), a series of small molecules was isolated that interfered with the accumulation of Adr in mouse fibroblasts by enhancing efflux of the drug . Isolated compounds also stimulated efflux of Rhodamine 123 (Rho-123), another substrate of multidrug transporters . Stimulation of drug efflux was detectable in the cells expressing P-glycoprotein (P-gp), but not in their P-gp-negative variants, and was completely reversible by the P-gp inhibitors . A dramatic stimulation of P-gp activity against Adr and Rho-123 by the identified compounds was accompanied by suppression of P-gp-mediated efflux of other substrates, such as Taxol (paclitaxel) or Hoechst 33342, indicating that they act as modulators of substrate specificity of P-gp . Consistently, P-gp modulators dramatically altered the pattern of cross-resistance of P-gp-expressing cells to different P-gp substrates: an increase in resistance to Adr, daunorubicin, and etoposide was accompanied by cell sensitization to Vinca alkaloids, gramicidin D, and Taxol with no effect on cell sensitivity to colchicine, actinomycin D, puromycin, and colcemid, as well as to several non-P-gp substrates . The relative effect of P-gp modulators against different substrates varied among the isolated compounds that can be used as fine tools for analyzing mechanisms of drug selectivity of P-gp . These results raise the possibility of a rational control over cell sensitivity to drugs and toxins through modulation of P-gp activity by small molecules.

J Biol Chem, 2002 Jan 25, 277(4), 2908 - 15 Epub 2001 Nov 12.
Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor; Kast HR et al.; The multidrug resistance-associated protein 2 (MRP2, ABCC2), mediates the efflux of several conjugated compounds across the apical membrane of the hepatocyte into the bile canaliculi . We identified MRP2 in a screen designed to isolate genes that are regulated by the farnesoid X-activated receptor (FXR, NR1H4) . MRP2 mRNA levels were induced following treatment of human or rat hepatocytes with either naturally occurring (chenodeoxycholic acid) or synthetic (GW4064) FXR ligands . In addition, we have shown that MRP2 expression is regulated by the pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) . Thus, treatment of rodent hepatocytes with PXR or CAR agonists results in a robust induction of MRP2 mRNA levels . The dexamethasone- and pregnenolone 16alpha-carbonitrile-dependent induction of MRP2 expression was not evident in hepatocytes derived from PXR null mice . In contrast, induction of MRP2 by phenobarbital, an activator of CAR, was comparable in wild-type and PXR null mice . An unusual 26-bp sequence was identified 440 bp upstream of the MRP2 transcription initiation site that contains an everted repeat of the AGTTCA hexad separated by 8 nucleotides (ER-8) . PXR, CAR, and FXR bound with high affinity to this element as heterodimers with the retinoid X receptor alpha (RXRalpha, NR2B1) . Luciferase reporter gene constructs containing 1 kb of the rat MRP2 promoter were prepared and transiently transfected into HepG2 cells . Luciferase activity was induced in a PXR-, CAR-, or FXR-dependent manner . Furthermore, the isolated ER-8 element was capable of conferring PXR, CAR, and FXR responsiveness on a heterologous thymidine kinase promoter . Mutation of the ER-8 element abolished the nuclear receptor response . These studies demonstrate that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5'-flanking region of this gene.

Oncogene, 2001 Oct 25, 20(48), 7006 - 20
beta(2)-microglobulin induces apoptosis in HL-60 human leukemia cell line and its multidrug resistant variants overexpressing MRP1 but lacking Bax or overexpressing P-glycoprotein; Wu CH et al.; In this study, we examined whether exogenous beta(2)-microglobulin (beta(2)m) can induce apoptosis in the drug sensitive HL-60 leukemia cell line and its drug resistant variants and investigated the molecular mechanism of beta(2)m-induced apoptosis . Our data revealed that beta(2)m is very significantly down-regulated in two multidrug resistant variants of the HL-60 cells: (a) the MRP1-bearing, Bax-deficient HL-60/ADR cell line, and (b) the P-glycoprotein (P-gp) overexpressing HL-60/VCR cell line . However, exogenous beta(2)m induced similar levels of apoptosis in HL-60 cells and these drug resistant variants . beta(2)m-induced apoptosis in HL-60 and HL-60/VCR cells was associated with decreased mitochondrial membrane potential (Deltapsim) but did not affect Deltapsim in HL-60/ADR cells . Surprisingly, cyclosporin A (CsA), a known inhibitor of the mitochondrial permeability transition (MPT) pore, inhibited beta(2)m-induced apoptosis in HL-60/ADR cells but not in HL-60 and HL-60/VCR cells, suggesting that the pro-apoptotic effect of beta(2)m in these cells is not through MPT pore formation . Furthermore, beta(2)m induced the release of cytochrome c and the apoptosis-inducing factor (AIF) from mitochondria in HL-60 and HL-60/VCR cells, but not in HL-60/ADR cells . Additionally, Z-VAD-fmk, a general inhibitor of caspases which inhibited cytochrome c release in HL-60 and HL-60/VCR cells, had no effect on AIF release in any of these cell lines, but inhibited beta(2)m-induced apoptosis in all three cell lines . However, Western blot analysis revealed that caspases-1, -3, -6, -8, and -9 are not activated during beta(2)m-induced apoptosis in these cells . Therefore, beta(2)m-induces apoptosis through an unknown caspase-dependent mitochondrial pathway in HL-60 and HL-60/VCR cells and by a Bax-independent, non-mitochondrial, caspase-dependent pathway in HL-60/ADR cells.

Int J Radiat Oncol Biol Phys, 2001 Nov 15, 51(4), 1050 - 7
Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation; Nielsen D et al.; PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells . METHODS AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy) . Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA . The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively . For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method . RESULTS: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr . EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR . Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr . In EHR2/irr, the steady-state accumulation of 3H-VCR and 3H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged . The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells . The ATPase activity was neither stimulated by vinblastine nor VER . CONCLUSION: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells . This phenotype was characterized by increased expression of Mrp1 mRNA, Mrp1, and PGP but decreased expression of mdr1a + b mRNA . The influence of irradiation on PGP and Mrp1 expression seemed to be different.

Rev Panam Salud Publica, 2001 Sep, 10(3), 174 - 80
Genotypic resistance mutations to antiretroviral drugs in HIV-1 B and non-B subtypes from Cuba; Ruibal-Brunet IJ et al.; OBJECTIVES: To determine the prevalence of drug resistance and to analyze the subtyping in HIV-1 samples from Cuba . METHODS: From an estimated total number of 1,950 HIV-1-infected persons in Cuba, a sample of 103 patients were studied, 76 of whom had received drug treatment for HIV and 27 who had not . The RNA plasma viral load was measured, and automated sequencing was used to assess resistance mutations to reverse transcriptase inhibitors (RTIs) and to protease inhibitors (PIs) . Subtyping in the V3 region was performed using heteroduplex mobility assay (HMA) . In order to corroborate the HMA results, sequencing of env (C2-V3-C3) was done with one-third of the samples in each of the subtype groups detected by HMA . RESULTS: Out of the 103 samples, 81 of them (78.6%) were classified as subtype B, 19 (18.5%) as subtype A, and 3 (2.9%) as subtype C . The prevalence of resistance mutations was 26.2% to RTIs, none to PIs alone, and 3.9% to both categories of drugs . The prevalence of resistance to nucleoside RTIs (NRTIs) was 27.6% in treated patients and 7.4% in the untreated patients, and for nonnucleoside RTIs (NNRTIs) it was 5.3% and 0%, respectively . Among treated patients a low frequency (2.6%) of dual resistance to zidovudine (ZDV) plus lamivudine (3TC) and abacavir (ABC) was detected, and multidrug resistance to NRTIs was not found . In relation to PIs together with RTIs, the prevalence of resistance was 5.3% for treated patients and 0% for untreated patients . CONCLUSIONS: Even though Cuba is generally considered an area where subtype B is dominant, we detected a high proportion of non-B subtype viruses . The low prevalence of resistance mutations to RTIs and PIs reflects the delay in introducing these drugs to Cuba . Multidrug resistance to RTIs was not found, so, as of now, the use of these drugs continues to be an option for Cuban patients.

Plant Cell, 2001 Nov, 13(11), 2441 - 54
Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development; Noh B et al.; Arabidopsis possesses several genes related to the multidrug resistance (MDR) genes of animals, one of which, AtMDR1, was shown to be induced by the hormone auxin . Plants having mutations in AtMDR1 or its closest relative, AtPGP1, were isolated by a reverse genetic strategy . Auxin transport activity was greatly impaired in atmdr1 and atmdr1 atpgp1 double mutant plants . Epinastic cotyledons and reduced apical dominance were mutant phenotypes consistent with the disrupted basipetal flow of auxin . The auxin transport inhibitor 1-naphthylphthalamic acid was shown to bind tightly and specifically to AtMDR1 and AtPGP1 proteins . The results indicate that these two MDR-like genes of Arabidopsis encode 1-naphthylphthalamic acid binding proteins that are required for normal auxin distribution and auxin-mediated development.

Hematology (Am Soc Hematol Educ Program) . 2000;:69-89.
New Developments in the Therapy of Acute Myelocytic Leukemia; Gorin NC et al.; Current conventional treatment for patients with acute myelogenous leukemia results in a high percentage of clinical responses in most patients . However, a high percentage of patients still remain refractory to primary therapy or relapse later . This review examines the search for new agents and new modes of therapy . In Section I, Dr . Estey discusses new agents directed at various targets, such as CD33, angiogenesis, inappropriately methylated (suppressor) genes, cell cycle checkpoints, proteosomes, multidrug resistance (MDR) gene, mitochondrial apoptotic pathway . He also reviews preliminary results of phase I trials with the nucleoside analog troxacitabine and liposomal anthracyclin and suggests new strategies for trials of new agents . In Section II, Dr . Jones revisits differentiation therapy and presents results of preclinical and clinical studies that demonstrate that a variety of clinically applicable cell cycle inhibitors (interferon, phenylbutyrate, vitamin D, retinoids, bryostatin-1) preferentially augments growth factor-mediated induction of myeloid leukemia terminal differentiation, as well as blocks growth factors' effects on leukemia proliferation . The combination of cell cycle inhibition plus myeloid growth factors may offer a potential treatment for resistant myeloid leukemias . In Section III, Drs . Levitsky and Borrello address the question of tumor vaccination in AML and shows that, although tumor rejection antigens in AML have not been formally identified to date, a growing number of attractive candidates are ripe for testing with defined antigen-specific vaccine strategies . Interestingly, the ability to drive leukemic blasts to differentiate into competent antigen presenting cells such as dendritic cells may be exploited in the creation of cellular vaccines . Ultimately, the successful development of active immunotherapy for AML will require integration with dose-intensive chemotherapy, necessitating a more complete understanding of host immune reconstitution . In Section IV, Dr . Slavin reviews the concept of delivering non-myeloablative stem cell transplantation (NST) and delayed lymphocyte infusion (DLI) to increase tolerance in particular in high risk and older patients, and take advantage of the graft-versus-leukemia (GVL) effect . All these approaches hold promise in reducing morbidity and mortality and differ from the older concepts aiming at delivering the highest possible doses of chemotherapy and/or total body irradiation to reach maximum leukemia cell kill, whatever the toxicity to the patient.

Org Lett, 2001 Nov 15, 3(23), 3723 - 5
Sugar amino acid containing somatostatin analogues that induce apoptosis in both drug-sensitive and multidrug-resistant tumor cells; Gruner SA et al.; {carbohydrate structure--see text} Resistance to chemotherapy has become a major problem in cancer therapy . The new sugar amino acid (SAA) containing somatostatin analogues presented possess antiproliferative and apoptotic activity against both multidrug-resistant and drug-sensitive hepatoma carcinoma cells . Synthesis, design, and biological evaluation of the cyclic analogues and of the furanoid SAA used will be discussed . Four analogues have IC(50) values in the low microM range, making them promising leads for chemotherapeutic drugs against multidrug-resistant carcinoma.

Leuk Lymphoma, 2001 Jul, 42(3), 371 - 8
Acute myeloid leukemia in the setting of low dose weekly methotrexate therapy for rheumatoid arthritis; Kolte B et al.; Methotrexate is in widespread use as second-line therapy for rheumatoid arthritis . Treatment with methotrexate in this and other settings has not been associated with the development of therapy-related leukemias . Four patients with rheumatoid arthritis are reported who developed acute myeloid leukemia (AML) while receiving low dose weekly methotrexate therapy in the absence of previous or concomitant treatment with known leukemogenic agents . AML in these four patients was of different morphologic subtypes and was associated with heterogeneous cytogenetic abnormalities, cell surface marker expression and multidrug resistance protein expression . None of the recognized features of therapy-related leukemia were present in these four nor in five previously-reported patients . It is likely that the occurrence of AML in patients with rheumatoid arthritis in the setting of methotrexate therapy represents the coincidence of these two diseases, and does not reflect a causal relationship.

Leuk Lymphoma, 2001 Jun, 42(1-2), 177 - 85
Interactions between P-glycoprotein and drugs used in the supportive care of acute myeloid leukemia patients; Mollgard L et al.; Multidrug resistance due to overexpression of P-glycoprotein (Pgp) leads to reduced intracellular drug accumulation and makes the cells resistant to chemotherapy . In this study we focused on how drugs used in the supportive care of acute myeloid leukemia (AML) patients interfere with Pgp . The effect on intracellular accumulation of the fluorescent dye Rhodamine 123 (Rh 123) was studied in the human promyelocytic leukemia cell line HL-60 and two anthracycline resistant, Pgp expressing, sublines . Each drug was used at two different concentrations: plasma peak concentration and half the plasma peak concentration . Drugs which increased the Rh 123 uptake by > 10% were included in the second part of the study where the cytotoxic effect was tested in combination with daunorubicin . In the Rhodamine assay none of the tested drugs had any significant effect on the Rh 123 efflux in the resistant cell lines . Amphotericin B, cefuroxime, erythromycin and dixyrazin had minor effects on Rh 123 uptake but showed a significant additive effect to the toxicity of daunorubicin suggesting other mechanisms of action than reversal of Pgp . In conclusion this in vitro model where Rh 123 uptake was studied in an anthracycline resistant leukemia cell line could not demonstrate any significant interactions with Pgp for the tested drugs.

Ann Oncol, 2001 Sep, 12(9), 1239 - 45
The expression of P-glycoprotein and multidrug resistance proteins 1 and 2 (MRP1 and MRP2) in human malignant mesothelioma; Soini Y et al.; BACKGROUND: Malignant mesothelioma is a malignancy with a primary resistance to chemo- and radiotherapies for reasons which are still unclear . Multidrug resistance proteins might explain the observed resistance, but no studies have assessed their expression in mesothelioma . PATIENTS AND METHODS: Immunohistochemical expression of P-glycoprotein (P-gp), and the multidrug resistance proteins 1 and 2 (MRP1 and MRP2) were investigated in 36 cases of malignant mesothelioma and in samples from normal mesothelium . RESULTS: P-gp immunopositivity was found in 61%, MRP1 immunopositivity in 58% and MRP2 positivity in 33% of the cases . Normal mesothelium did not express these multidrug-resistant proteins . There was a significant association between P-gp and MRP2 (P = 0.022) expression . No or weak P-gp, MRP1 or MRP2 immunostaining was significantly more frequent in sarcomatoid mesothelimas than in epithelial or biphasic mesotheliomas (P = 0.031, P = 0.034 and P = 0.024, respectively) . There was no significant association between patient survival and expression of the multidrug-resistant proteins . CONCLUSIONS: The results show that P-gp, MRP1 and MRP2 are induced and expressed in malignant mesothelial cells . Regardless of their expression no association with survival of the patients was seen, suggesting that the primary resistance of malignant mesotheliomas is not solely dependent on their expression or function.

Can J Physiol Pharmacol, 2001 Oct, 79(10), 876 - 84
Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells; Lee G et al.; The objective of this study was to examine effects of interleukin-6 (IL-6) on the expression and activity of the drug resistance transporters (MDR1 and MRP) in human hepatoma cell lines . Expression and activity of MDR1 and MRP transporters were examined in IL-6-treated and control HuH 7 and HepG2 cells using semi-quantitative RT-PCR analysis and by rhodamine 123 and 5-carboxyfluorescin efflux assays . Results from RT-PCR demonstrated expression of MRP3, MRP6, and MDR1 in HuH 7 cells and expression of MRP1, MRP2, MRP3, MRP6, and MDR1 in HepG2 cells . Compared with controls, treatment of HuH 7 cells with IL-6 (10 ng/mL, 24 h) resulted in a 1.8-fold increase in MRP-mediated efflux of 5-CF with a corresponding 1.5-fold induction of MRP3 mRNA levels (p < 0.05) . Similarly, in HepG2 cells, a 2-fold increase in MRP functional activity and a 1.8-fold induction of MRP1 mRNA levels were seen in the IL-6 treated cells (p < 0.05) . Treatment of cells with IL-6 was also found to cause significant reductions in the expression and activity of MDR1 in HuH 7 cells, but not in HepG2 cells . Our data suggest that IL-6 induces MRP expression and activity in human hepatoma cell lines . Suppressive effects of IL-6 on MDR1 expression and activity were also observed in HuH 7 cells . This underscores the importance of examining the regulation of multiple drug resistance proteins as these proteins may have opposing regulatory mechanisms in malignant cells.

Leuk Lymphoma, 2001 Aug, 42(4), 775 - 87
Correlation between decreased apoptosis and multidrug resistance (MDR) in murine leukemic T cell lines; Lopes EC et al.; Cancer cells may frequently develop cross-resistance to structurally dissimilar chemotherapeutic agents . However, the molecular mechanisms for sensitivity and resistance of tumor cells towards chemotherapy are still partially understood . Antineoplasic drugs have been shown to induce apoptosis in chemosensitive leukemias and solid tumors . In this work, cross-resistance among vincristine (VCR), doxorubicin (DOX) and other antineoplasic agents commonly used in the treatment of leukemia such as etoposide (VP-16), methotrexate (MTX), cyclophosphamide (CTX), dexamethasone (DEX), cytarabine (Ara-C) and L-asparaginase on vincristine resistant (LBR-V160), doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemic T cell lines, was determined . The effect of antineoplasic agents was assayed by tritiated thymidine incorporation . Our results showed that VCR exhibited cross-resistance with DOX, VP-16, DEX and MTX, while DOX demonstrated cross-resistance with VCR, VP-16 and MTX . Ara-C failed to present cross-resistance with any cell line . Apoptosis induced by the above drugs on the same cell lines was analyzed by acridine orange and ethidium bromide staining, DNA hypoploidy (flow cytometry) and oligonucleosomal fragmentation of nuclear DNA showing that therapeutic concentrations of these chemotherapeutic agents induced apoptosis in the LBR- cell line . Our results demonstrated that, except for DEX, none of the drugs presenting cross-resistance were able to induce cell death on LBR-V 160 or LBR-D 160 cell lines.

Leuk Lymphoma, 2001 Aug, 42(4), 739 - 46
All-trans retinoic acid (ATRA) differentiates acute promyelocytic leukemia cells independently of P-glycoprotein (P-gp) related multidrug resistance; Takeshita A et al.; Here the relationship between all-trans retinoic acid (ATRA)-resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) is discussed in acute promyelocytic leukemia (APL) . First, the remission rates of ATRA therapy are similar in relapsed/refractory APL to the preceding chemotherapy given and in newly diagnosed APL . Second, MDR1 cDNA-transduced NB4 (NB4/MDR) cells accumulate less Rhodamine-123 (Rh123) than NB4 cells, but there is no difference in the intracellular ATRA concentration between them . PSC833 or MS209 . MDR modifiers, increases the intracellular accumulation of Rh123 in NB4/MDR and APL cells expressing P-gp, but not of ATRA . Third, the expression of CD11b, the NBT reduction activity, the proportion of apoptotic cells and the morphology are not different between NB4/MDR and NB4 cells, and between APL cells expressing P-gp and not . APL cells express little P-gp, and mainly express CD33 but no CD34 . Despite previous reports that ATRA-resistant APL cells express more P-gp than ATRA-sensitive ones, P-gp and ATRA-resistance seems to exist independently.

Leuk Lymphoma, 2001 Aug, 42(4), 721 - 9
Amifostine does not inhibit the toxic effects of anthracycline derivates or mitoxantrone on MDR tumor cell lines; Michieli M et al.; In this work three human cell lines with multidrug resistance (MDR) caused by a P-glycoprotein (PGP) overexpression, CEM VLB, HL60 DNR, LOVO DX and two cell lines with MDR associated with a multidrug related protein (MRP) or a lung resistance-related protein (LRP) overexpression named GLC4 ADR and SW1573/2R120 were tested for Amifostine protection against Daunorubicin, Doxorubicin, Idarubicin and Mitoxantrone toxicity . This class of anticancer agents was chosen because they are commonly used in the first line treatments of acute leukemias where a PGP, an LRP or an MRP overexpression often occurs even at onset . A 7-day incubation with escalating doses of anticancer agents with or without a 15 minute preincubation in Amifostine or its active metabolite WR-1065 were used . In conclusion, in none of the MDR positive and negative cell lines did Amifostine modify the toxicity of the anticancer drugs . The observation that even the WR-1065 metabolite gave no protection against Anthracyclines toxicity strengthened the data and provided confirmation for the further in vivo testing of the safety and efficacy of Amifostine in leukemias.

J Membr Biol, 2001 Oct 1, 183(3), 165 - 73
Evidence for multidrug resistance-1 P-glycoprotein-dependent regulation of cellular ATP permeability; Roman RM et al.; The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined . Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells . Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release approximately 3-fold . The MDR1 inhibitors cyclosporine A (10 microm) and verapramil (10 microm) inhibited ATP release by 69% and 62%, respectively (p < 0.001) . Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl- current density from -33.1 +/- 12.5 pA/pF to -2.0 +/- 0.3 pA/pF (-80 mV, p < or = 0.02) . In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates . Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release . Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.

In Vivo, 2001 Sep-Oct, 15(5), 437 - 42
Biological activity of a fruit vegetable, "Anastasia green", a species of sweet pepper; Motohashi N et al.; Russian green sweet pepper (Anastasia Green) was successively extracted with hexane, acetone, methanol and 70% methanol and the extracts were further separated into a total of twenty fractions by silica gel or ODS column chromatographies . The biological activities of these extracts and fractions were compared . The extracts and fractions showed higher cytotoxic activity against two human oral tumor cell lines than against normal human gingival fibroblasts, suggesting their tumor-specific action . Several fractions {H3, H4, A4} reversed the multidrug resistant gene (MDR1) against L5178 mouse T-cell lymphoma more effectively than (+/-) verapamil (positive control) . All extracts and fractions showed no anti-human immunodeficiency virus (HIV) nor anti-Helicobacter pylori activity . These data suggest the medicinal importance of an Anastasia Green extract.

J Acquir Immune Defic Syndr, 2001 Nov 1, 28(3), 250 - 3
Genotypic drug resistance and cause of death in HIV-infected persons who died in 1999; Beinker NK et al.; We analyzed the relationship between viral drug resistance and causes of death in 29 HIV-1-infected patients who had been followed in an HIV-outpatient clinic and died in 1999 . Six patients (21%) died with plasma HIV-RNA levels <1000 copies/ml . Seven (24%) died with wild-type (WT) virus in plasma, 6 (21%) had reverse transcriptase (RT) mutations only, 10 (34%) had multidrug-resistant (MDR) virus . The causes of death were not differently distributed among these groups; however, 8 of 16 patients (50%) with resistant viruses died of end-organ failure versus 2 of 7 patients (29%) with WT virus . Seventeen of 32 patients (53%) were thought by their physicians to be noncompliant with prescribed therapy . Major resistance mutations to antiretroviral drugs were present in viruses from at least 55% of our HIV-1-infected patients who died in 1999 . Nonetheless, deaths also occurred among patients with well-controlled HIV infection and among patients with WT virus in plasma . Infections related to incomplete immune restoration, inability to maintain suppressive antiretroviral drug levels, and end-organ failures all contribute to mortalities in the era of highly active antiretroviral therapy.

Clin Infect Dis, 2001 Dec 1, 33(11), 1922 - 30 Epub 2001 Oct 25.
The impact of human immunodeficiency virus type 1 on infectiousness of tuberculosis: a meta-analysis; Cruciani M et al.; To assess if the relative infectiousness of patients with tuberculosis is enhanced by coinfection with human immunodeficiency virus type 1 (HIV-1), data from 6 studies of 1240 health care workers who had contact with tuberculosis patients were analyzed . Overall rates of tuberculin skin test conversion were similar regardless of HIV-1 positivity of tuberculosis patients (odds ratio {OR}, 1.04; 95% confidence interval {CI}, 0.23-1.84) . However, when only 3 studies during nosocomial outbreaks of multidrug-resistant Mycobacterium tuberculosis were analyzed, rates of skin test conversion were higher among contacts of HIV-1-positive index cases (OR, 2.85; 95% CI, 1.85-3.85; P=.0002) . A second meta-analysis included data from 11 studies of 10,714 household contacts of tuberculosis patients . Prevalence of both skin test positivity (OR, 0.45; 95% CI, 0.20-1.03) and active disease (OR, 1.17; 95% CI, 0.78-1.56) were similar regardless of HIV-1 positivity of index cases . These data suggest that tuberculosis patients with HIV-1 infection are not intrinsically more infectious to their contacts than are HIV-1-negative tuberculosis patients.

Cancer Res, 2001 Nov 1, 61(21), 7763 - 9
P-glycoprotein does not reduce substrate concentration from the extracellular leaflet of the plasma membrane in living cells; Chen Y et al.; P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is an important mediator of multidrug resistance in cancer . Pgp exhibits a very broad specificity for substrates . These substrates share a common feature of being amphipathic and can orient into either leaflet of the membrane bilayer . Current evidence suggests that Pgp recognizes and extracts substrates from the membrane bilayer, but from which leaflet is unresolved . To directly test whether Pgp can decrease substrate concentration in the extracellular leaflet of the plasma membrane in living cells, we used the fluorescent lipid analogue 1-{4-(trimethylamino)phenyl}-6-phenylhexa-1,3,5-triene (TMA-DPH) . TMA-DPH in the extracellular solution rapidly partitions into the extracellular leaflet of the plasma membrane and exhibits slow transbilayer flipping into the cytoplasmic leaflet . Because TMA-DPH fluorescence is confined to the extracellular leaflet in early time points after addition but labels intracellular membranes after longer incubation, we can assess the effect of Pgp on TMA-DPH concentration from both extracellular leaflet and intracellular membranes . Transient transfection with a Pgp and the green fluorescence protein (GFP) fusion protein generated cells with heterogeneous expression levels of Pgp-GFP . Compared with nonexpressing cells, cells expressing Pgp-GFP showed decreased accumulation of TMA-DPH in intracellular membranes but similar levels of accumulation in the extracellular leaflet of the plasma membrane . Additionally, in drug-selected MCF7/Adr cells, which constitutively express high levels of Pgp, inhibition of Pgp by cyclosporin A resulted in significantly increased accumulation of TMA-DPH in intracellular membranes but no difference in its accumulation in the extracellular leaflet of the plasma membrane . These data indicate that whereas Pgp can extract TMA-DPH from the cytoplasmic leaflet of the membrane, any activity Pgp may possess in the extracellular leaflet is insufficient to decrease TMA-DPH concentration there and, therefore, does not contribute to lowering the cellular levels . Pgp is the prototype of an increasing number of clinically important ATP-binding cassette transporters of amphipathic drugs and lipids . These results may help decipher a common mechanism of these transporters.

J Control Release, 2001 Nov 9, 77(1-2), 77 - 86
Possibility of the reversal of multidrug resistance and the avoidance of side effects by liposomes modified with MRK-16, a monoclonal antibody to P-glycoprotein; Matsuo H et al.; For cancer chemotherapy, avoiding the side effects of chemotherapeutic agents is difficult . Multidrug resistance is one of the major obstacles to successful cancer chemotherapy . P-Glycoprotein (P-gp) serves as an efflux pump and plays a key role in the multidrug resistance . We examined the effect of MRK-16, a monoclonal antibody against P-gp, modified liposomes (MRK-Lip) on the human myelogenous leukemia K-562 cells and its adriamycin resistance cell line K-562/ADM cells to avoid the side effects and to reverse the multidrug resistance . The uptake of vincristine (VCR) by K-562/ADM cells was lower than that by K-562 cells . This low uptake was increased in the presence of verapamil and MRK-16, however, it was not increased in the presence of control antibody, IgG2A . The binding of MRK-Lip to K-562/ADM cells was higher than that of IgG2A-modified liposome (IgG-Lip) and liposome without modification (Cont-Lip) . Moreover, the cytotoxicity of VCR-encapsulated MRK-Lip to K-562/ADM cells was higher than that of VCR-encapsulated IgG-Lip and Cont-Lip . These results suggest that the interaction between liposomes and multidrug resistance cells was increased by the modification of liposomes with MRK-16 . Consequently, the usefulness of MRK-Lip in cancer chemotherapy as a potent carrier was suggested.

Arch Pharm (Weinheim), 2001 Sep, 334(8-9), 269 - 74
Ellipticine analogues and related compounds as inhibitors of reverse transcriptase and as inhibitors of the efflux pump; Sharples D et al.; Ten polycyclic derivatives related to ellipticine have been synthesised and tested for their intercalating, reverse transcriptase (RT) inhibitory and multidrug resistance efflux pump inhibitory properties . The intercalating activity and the RT inhibitory activity of the derivatives suggest that ellipticine analogues bind at an allosteric binding site on RT or that this inhibition could be controlled at the DNA level . The MDR efflux pump inhibitory activities of these derivatives, however, appears to be unrelated to the DNA binding ability.

Cancer Gene Ther, 2001 Oct, 8(10), 803 - 14
Hammerhead ribozyme against gamma-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins; Iida T et al.; Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for GSH . We constructed a hammerhead ribozyme against a gamma-GCS heavy subunit (gamma-GCSh) mRNA transcript and transfected it to human colonic cancer cells (HCT8DDP) resistant to cisplatin (CDDP) . The effect of the ribozyme transfection on the drug resistance of cancer cells was studied . (a) Transfection of the ribozyme decreased the GSH level and the efflux of CDDP-GSH adduct, resulting in higher sensitivity of the cells to CDDP . (b) The transfection suppressed the expression of ATP-binding cassette (ABC) family of transporters such as MRP1, MRP2, and MDR1, and stimulated the expression of mutant p53 . (c) An electrophoretic mobility shift assay showed that mutant p53 suppresses the SP1-DNA binding activity, suggesting that this mutant p53 is functional and it, in turn, suppresses the expression of ABC transporters . Collectively, transfection of anti-gamma-GCSh ribozyme reduced the synthesis of GSH and the expression of ABC transporters, which causes an increase in the sensitivity of cancer cells to anticancer drugs . Suppression of the SP1-DNA binding activity by p53 may be a factor of down-regulation of ABC transporters.

Respir Res, 2001, 2(3), 164 - 8 Epub 2001 Apr 05.
The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis; Somoskovi A et al.; Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have emerged worldwide . In many countries and regions, these resistant strains constitute a serious threat to the efficacy of tuberculosis control programs . An important element in gaining control of this epidemic is developing an understanding of the molecular basis of resistance to the most important antituberculosis drugs: isoniazid, rifampin, and pyrazinamide . On the basis of this information, more exacting laboratory testing, and ultimately more appropriate and timely treatment regimens, can be developed.

Biomed Pharmacother, 2001 Oct, 55(8), 466 - 74
Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines; Sekiguchi M et al.; Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies . We established cultured cell lines from four untreated tumors . The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities . The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors . The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein . The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant . One cell line produced G-CSF . The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents . The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs . This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism . The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

Leuk Res, 2001 Dec, 25(12), 1127 - 35
Detection of P-glycoprotein in cell lines and leukemic blasts: failure of select monoclonal antibodies to detect clinically significant Pgp levels in primary cells; Taylor BJ et al.; Multidrug resistance (MDR) is a salient feature of chemotherapy failure in pediatric patients . One of the most common and well-studied mechanisms implicated in causing MDR is P-glycoprotein (Pgp), an ATP-dependent, transmembrane drug efflux pump . Accurate and reproducible detection of this MDR protein is necessary as it may have important clinical implications . In this study comparing the directly conjugated anti-Pgp monoclonal antibodies UIC2-PE and 15D3-PE to the unconjugated anti-Pgp mAb MRK16, we analyzed cell lines, normal peripheral blood cells, and bone marrow cells from pediatric patients diagnosed with acute myeloid leukemia and acute lymphoblastic leukemia; all samples were also analyzed for Pgp function using rhodamine 123 in order to correlate results from antibody staining with functional activity . For all patient samples evaluated, only MRK16 correlated well with the rhodamine 123 assay . Both the directly conjugated antibodies UIC2-PE and 15D3-PE failed to detect Pgp in almost all cases . Pre-treatment of cells with neuraminidase did not provide a consistent enhancement of antigen detection . Based on these results, we suggest that while UIC2-PE and 15D3-PE may be able to detect the very high levels of Pgp expressing laboratory-cultured cell lines, they are not suitable for clinical application in their currently available conjugated form . When assaying patient samples for Pgp expression and function using flow cytometry, the rhodamine 123 functional assay should be performed in concert with staining with MRK16.

Br J Pharmacol, 2001 Nov, 134(5), 931 - 8
Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells; Terlouw SA et al.; 1 . The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver . Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved . The objective of the present study was to characterize glutathione conjugate efflux from rat kidney proximal tubule cells (PTC), and to determine the contribution of Mrp2 . 2 . We used isolated PTC in suspension, as well as grown to monolayer density . For comparison, transport characteristics were also determined in the human intestinal epithelial cell line Caco-2, an established model to study MRP2-mediated transport . The cells were loaded with monochlorobimane (MCB) at 10 degrees C . MCB enters the cells by simple diffusion and is conjugated with glutathione to form the fluorescent glutathione-bimane (GS-B) . 3 . In primary cultures of rat PTC, no indications for a transporter-mediated mechanism were found . The efflux of GS-B from Caco-2 cells and freshly isolated PTC was time- and temperature-dependent . Furthermore, GS-B transport in both models was inhibited by chlorodinitrobenzene (CDNB), with an inhibitory constant of 46.8+/-0.9 microM in freshly isolated PTC . In Caco-2 cells, the inhibitory potency of CDNB was approximately 20 fold higher . Finally, efflux of GS-B from freshly isolated PTC from Mrp2-deficient (TR(-)) rats was studied . As compared to normal rat PTC, transport characteristics were not different . 4 . We conclude that in freshly isolated rat PTC glutathione conjugate excretion is mediated by other organic anion transporters rather than by Mrp2.

Med Clin North Am, 2001 Nov, 85(6), 1565 - 81
Controversies in the diagnosis of ventilator-acquired pneumonia; Waterer GW et al.; The appropriate investigation of patients with suspected VAP is controversial . Because it is unlikely that any new diagnostic technique will become available in the near future with better performance characteristics than those currently available, physicians need to tailor their diagnostic approach depending on individual patients and clinical scenarios . The most crucial factor in deciding which diagnostic approach to take is the influence that any test result would have on management . If preliminary screening tests, including Gram stain, are used to determine whether to start antibiotic therapy, invasive diagnostic techniques have an advantage over ETA . Quantitative cultures of respiratory specimens have a higher specificity than qualitative cultures and should be used if there is any possibility that a negative culture result would result in the discontinuation of antibiotic therapy . Physicians are caught between the need to treat VAP promptly with appropriate antibiotics and the undeniable problems of multidrug-resistant bacteria and their association with inappropriate antibiotic use . When clinically possible, a diagnostic strategy should be chosen that maximizes the possibility of limiting broad-spectrum antibiotic use . To give physicians greater comfort in the ability to withhold or discontinue antibiotics safely, further research is needed into the appropriate diagnostic strategies in different clinical settings that make this possible . The studies by Fagon et al and Singh et al are important steps in this direction.

Gastroenterology, 2001 Nov, 121(5), 1203 - 8
Expression and localization of the multidrug resistance proteins MRP2 and MRP3 in human gallbladder epithelia; Rost D et al.; BACKGROUND & AIMS: The multidrug resistance protein (MRP) isoforms MRP2 (ABCC2) and MRP3 (ABCC3) play a decisive role in the hepatic secretion of endogenous and xenobiotic conjugates and are differentially expressed in hepatocytes and cholangiocytes . The epithelium of the gallbladder considerably modifies the composition of primary hepatic bile by absorption and secretion; however, the underlying transport mechanisms were largely unknown . Localization of MRP2 and MRP3 may provide an explanation of how the products of phase II conjugation are effluxed from gallbladder epithelia . METHODS: Expression and localization of MRP2 and MRP3 were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence microscopy of human gallbladder tissue . RESULTS: Expression of MRP2 and MRP3 was identified in all gallbladders by RT-PCR followed by sequencing of the amplified fragments . Double immunofluorescence microscopy using 2 specific antibodies for the respective MRP isoform showed the simultaneous expression of MRP2 in the apical membrane and MRP3 in the basolateral membrane of gallbladder epithelia . MRP1 protein expression was not detectable . CONCLUSIONS: Our findings show the expression of MRP2 and MRP3 in distinct plasma membrane domains of gallbladder epithelia and provide evidence for the capacity of the gallbladder to secrete xenobiotic and endogenous anionic conjugates into blood via MRP3 and into bile via MRP2.

Clin Biochem, 2001 Sep, 34(6), 463 - 8
Alkaline phosphatase from rat liver and kidney is differentially modulated; Martins MJ et al.; OBJECTIVE: To investigate the effect of inhibitors of alkaline phosphatase (ALP) and modulators of P-glycoprotein (Pgp), multidrug resistance protein (MRP) and hepatic taurocholate uptake on the activity of tissue-nonspecific ALP (TNALP) in liver and kidney . DESIGN AND RESULTS: ALP activity was determined in rat liver and kidney homogenates . Levamisole had a stronger inhibitory effect on renal TNALP than on the hepatic isoform . 1,3-dimethylxanthine (theophylline) almost abolished renal TNALP activity whereas its effect on hepatic TNALP was less intense . 3-isobutyl-1-methylxanthine (IBMX) and lidocaine produced opposite effects, activating hepatic TNALP and inhibiting the kidney isoform . Quinidine significantly inhibited renal TNALP without affecting hepatic TNALP . Kaempferol activated both liver and kidney isoforms, the effect being more pronounced on hepatic TNALP . CONCLUSIONS: a) renal TNALP seems to be more sensitive to inhibition than hepatic TNALP, b) TNALP activity studies should take into account the source of ALP isoform and c) ALP pharmacological manipulation in vivo may produce different and even opposite effects in different tissues/organs.

Jpn J Cancer Res, 2001 Oct, 92(10), 1116 - 26
Intracellular levels of two cyclosporin derivatives valspodar (PSC 833) and cyclosporin a closely associated with multidrug resistance-modulating activity in sublines of human colorectal adenocarcinoma HCT-15; Uchiyama-Kokubu N et al.; P-Glycoprotein, which mediates multidrug resistance (MDR) in cancer chemotherapy, is a principal target of cyclosporin A and {3'-keto-Bmt(1)}-{Val(2)}-cyclosporin (valspodar; PSC 833) . To clarify mechanisms contributing to the different MDR-modulating activities of valspodar and cyclosporin A, we investigated the relation of the intracellular levels of the two cyclosporin derivatives to their modulating effect on MDR in different P-glycoprotein-expressing human colorectal carcinoma HCT-15 cells (parental HCT-15 and adriamycin-resistant sublines) . In this study, valspodar was found to be much more potent than cyclosporin A in both sensitizing resistant cells to MDR-related anticancer drugs (e.g., adriamycin, vincristine and paclitaxel (taxol)) and increasing 2-{6-amino-3-imino-3H-xanthen-9-yl}benzoic acid methyl ester (rhodamine 123) retention and {G-(3)H}vincristine sulfate ({(3)H}vincristine) accumulation in these cells . Furthermore, a good correlation was detected between P-glycoprotein levels and the MDR-reversing effect of valspodar . In contrast, the effects of cyclosporin A could not be linked to P-glycoprotein levels in the MDR cells . In addition, the intracellular accumulation of valspodar was found to be 3 - 6 fold higher than that of cyclosporin A in four sublines and verapamil, an inhibitor of P-glycoprotein-mediated transport, enhanced the accumulation of cyclosporin A, but not valspodar . These results suggested that valspodar accumulation is not actively regulated by the P-glycoprotein-mediated efflux system.

Curr Opin Oncol, 2001 Nov, 13(6), 463 - 9
Drug resistance in hematologic malignancies; Marie JP; Drug resistance eventually occurs in most hematologic malignancies treated with chemotherapy . The mechanisms responsible for drug resistance include expression of transporters of xenobiotics of the adenosine triphosphate-binding cassette protein superfamily (P-glycoprotein, multidrug resistance associated proteins, breast cancer resistance protein), modifications of enzymes like deoxycytidine kinase, and defects in chemotherapy-induced apoptosis . The efforts to overcome this drug resistance have been focused, thus far, on modulation of P-glycoprotein . Several compounds were manufactured for this purpose, and phase III trials of PSC833, one of the most potent P-glycoprotein inhibitors, are completed . The emergence of modulators with several adenosine triphosphate-binding cassette protein targets, like GG120918 (inhibiting P-glycoprotein and breast cancer resistance protein) and VX710 (inhibiting P-glycoprotein and multidrug resistance associated protein 1), are of clinical interest in malignancies often expressing several efflux pumps simultaneously . Another approach is the use of "furtive" drugs like liposomal or nanoparticular anthracyclines.

Biochem J, 2001 Nov 1, 359(Pt 3), 605 - 10
Bile-salt hydrophobicity is a key factor regulating rat liver plasma-membrane communication: relation to bilayer structure, fluidity and transporter expression and function; Asamoto Y et al.; Bile-salt hydrophobicity regulates biliary phospholipid secretion and subselection . The aim of this study was to determine whether bile salts can influence liver plasma membrane phospholipids and fluidity in relation to the ATP-dependent transporter . Rats were depleted of bile salts by overnight biliary diversion and then sodium taurocholate was infused intravenously at a constant rate (200 nmol/min per 100 g of body weight), followed by infusion of bile salts with various hydrophobicities (taurochenodeoxycholate, tauroursodeoxycholate, tauro-beta-muricholate, tauro-alpha-muricholate at 200 nmol/min per 100 g of body weight) . The hydrophobicity of the infused bile salts correlated with that of biliary phospholipids, but was inversely related to that of the canalicular membrane bilayer . Canalicular membrane fluidity (estimated by 1,6-diphenyl-1,3,5-hexatriene fluorescence depolarization) and expression of multidrug-resistance proteins (Mrp2, Mrp3) and apical Na(+)-dependent bile-salt transporter (ASBT) were increased by hydrophilic bile salts, although there was no marked change in the expression of P-glycoprotein subfamilies (Mdr2) . Bile-salt export pump (Bsep) expression was increased along with increasing bile-salt hydrophobicity . Bile salts modulate canalicular membrane phospholipids and membrane fluidity, as well as the ATP-dependent transporter expression and function, and these actions are associated with their hydrophobicity . The cytoprotective effect of hydrophilic bile salts seems to be associated with induction of Mrp2, Mrp3 and ASBT.

Int J Cancer, 2001 Oct 15, 94(2), 157 - 65
Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets; Morjani H et al.; Multidrug-resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer) . In this study, we aim at localizing, at subcellular level, where these lipids accumulate . Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile-red . Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells . Microspectrofluorometric measurements show an increase of the gold-yellow emission intensity in MCF7R cells, corresponding to neutral lipids . Similar observations were made in human MDR vincristine-HL60 and doxorubicin-KB selected cells . Total cellular glucosylceramide (GlcCer) measurements using {(3)H}-palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells . Moreover, MCF7R cells treated with fluorescent GlcCer-bodipy exhibit an accumulation of this lipid in cytoplasmic droplets . Treatment of MCF7R cells with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile-red fluorescence emission emanating from these structures and reverses MDR . Moreover, Golgi compartments stained with fluorescent PPMP-bodipy, show an increase in the Golgi compartments density . Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3'-azido-3'deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP . Treatment of MCF7 and MCF7R with the beta-glucosidase inhibitor conduritol beta-epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells . These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P-glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells .

J Org Chem, 1996 Sep 20, 61(19), 6606 - 6616
Synthesis and Conformational Analysis of the Multidrug Resistance-Reversing Agent Hapalosin and Its Non-N-methyl Analog; Dinh TQ et al.; Hapalosin was initially synthesized by macrolactonization, and a second synthesis was achieved by cycloamidation . In both syntheses, three of the five stereocenters in hapalosin were established by two Brown allylboration reactions . The synthesis of the non-N-Me analog of hapalosin involved chelation-controlled reduction of a gamma-amino-beta-keto ester and cycloamidation . In CDCl(3) at 25 degrees C, synthetic hapalosin exists as a 2.3:1 mixture of conformers, while its non-N-Me analog exists only as a single conformer . (1)H,(1)H-NOESY and computation reveal that the configuration of the amide bond is responsible for the conformations of the two compounds . The major conformer of hapalosin is found to be an s-cis amide, the minor conformer an s-trans amide, and the non-N-Me analog an s-trans amide . Applying distance constraints to protons that exhibit NOESY correlations, computation shows that the major conformer of hapalosin and the non-N-Me analog have very different conformations . By contrast, the minor conformer of hapalosin and the non-N-Me analog have very similar conformations.

Amino Acids, 2001, 21(2), 91 - 117
Sensitivity of taurine uptake to oxygen-derived reactive substances in MDR and non-MDR cells; Wersinger C et al.; In human KB and LoVo cell lines, high affinity taurine uptake was strongly reduced in both a time and dose-dependent manner by cumene hydroperoxide (CH) and to a lesser extent by hydrogen peroxide (H2O2) . Uptake-inhibition was greater in multidrug resistant (MDR) cells than in their non-MDR counterparts . Basal taurine efflux was unaffected by the oxidants . Lipid peroxidation levels closely correlated with the uptake inhibition levels, and were greater in MDR cells than in their non-MDR counterparts . The two oxidants reduced the Vmax and, to a lesser extent, the affinity of the transporter for taurine . They also reduced low affinity taurine uptake and, to a lesser extent, taurine diffusion . The composition of the medium used for cell treatment, especially its pyruvate content, greatly affected the H2O2 effect . H2O2- or CH-induced reduction of the high affinity taurine uptake was unaffected by protein kinase C (PKC) inhibitors and by the calmodulin antagonist W-13, ruling out the involvement of PKC and perhaps of calmodulin kinases in their effect.

J Med Humanit, 1996 Summer, 17(2), 91 - 102
Compliance, coercion, and compassion: moral dimensions of the return of tuberculosis; Booker MJ; Since 1986, we have seen a rise in the occurrence of tuberculosis in the United States . Long considered defeated in this country, the disease is returning with distressing vigor . Outbreaks of MDR-TB, tuberculosis resistant to more than one medication, have been reported around the country . This article analyzes the Centers for Disease Control and Prevention's National Action Plan to Combat Multidrug-Resistant Tuberculosis, with particular focus on the moral dimensions of mandatory directly observed treatment (DOT) and involuntary quarantine . It is proposed that a moral response to the control of tuberculosis must be one which is sustainable and which can effectively curtail the spread of the disease at a minimal cost to individual rights.

Biol Pharm Bull, 2001 Oct, 24(10), 1185 - 7
Circumvention of acquired resistance to doxorubicin in K562 human leukemia cells by oxatomide; Ishikawa M et al.; We studied the effect of oxatomide, an antiallergic drug, on the resistance of K562 cells to doxorubicin . Oxatomide synergistically potentiated the cytotoxicity of doxorubicin in doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 1-10 microM, but had hardly any synergistic effect on the parental cell line (K562) at the same concentration . Oxatomide inhibit P-glycoprotein pump-efflux activity and the binding of {3H}-azidopine to the cell-surface protein P-glycoprotein, in a dose-related manner . These results indicate that oxatomide reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Mol Pharmacol, 2001 Nov, 60(5), 934 - 43
Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2; Cui Y et al.; Vectorial transport of endogenous substances, drugs, and toxins is an important function of polarized cells . We have constructed a double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombinant uptake transporter for organic anions in the basolateral membrane and an ATP-dependent export pump for anionic conjugates in the apical membrane . Basolateral uptake was mediated by the human organic anion transporter 8 (OATP8; symbol SLC21A8) and subsequent apical export by the multidrug resistance protein 2 (MRP2; symbol ABCC2) . Under physiological conditions, both transport proteins are strongly expressed in hepatocytes and contribute to the hepatobiliary elimination of organic anions . Expression and localization of OATP8 and MRP2 in MDCK cells growing on Transwell membrane inserts was demonstrated by immunoblotting and confocal laser scanning microscopy . (3)H-Labeled sulfobromophthalein (BSP) was a substrate for both transport proteins and was transferred from the basolateral to the apical compartment at a rate at least six times faster by double-transfected MDCK-MRP2/OATP8 cells than by single-transfected MDCK-OATP8 or MDCK-MRP2 cells . Vectorial transport at a much higher rate by double-transfected than by single-transfected cells was also observed for the (3)H-labeled substrates leukotriene C(4), 17 beta-glucuronosyl estradiol, and dehydroepiandrosterone sulfate, for the fluorescent anionic substrate fluo-3, and for the antibiotic rifampicin . Inhibition studies indicated that intracellular formation of S-(2,4-dinitrophenyl)-glutathione from 2,4-chlorodinitrobenzene selectively inhibits the transcellular transport of {(3)H}BSP at the site of MRP2-mediated export . The double-transfected cells provide a useful system for the identification of transport substrates and transport inhibitors including drug candidates.

Mol Pharmacol, 2001 Nov, 60(5), 885 - 93
Characterization of a human colorectal carcinoma cell line with acquired resistance to flavopiridol; Smith V et al.; Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases (cdks) and represents the first in this anticancer class to enter clinical trials . In anticipation of the likelihood that, as with other cancer drugs, acquired resistance may limit the drug's efficacy, an acquired resistance model has been established by in vitro drug exposure of the human colon carcinoma cell line HCT116 . This stably resistant line, possessing 8-fold resistance to flavopiridol, showed a lack of cross-resistance to the anticancer agents etoposide, doxorubicin, paclitaxel, topotecan, and cisplatin, and notably to other chemical classes of cdk inhibitors: the aminopurines roscovitine and purvalanol A, 9-nitropaullone, and hymenialdisine . Resistance did not seem to be related to differences in the levels of multidrug resistance drug efflux proteins, P-glycoprotein, and MRP1 . Moreover, there were no changes in overall drug accumulation between the resistant and sensitive cell lines . Flavopiridol induced cell cycle arrest, apoptosis, and inhibition of retinoblastoma gene product phosphorylation on serine 780 in both parental and resistant lines, but the latter required 8-fold higher concentrations to achieve these effects . Cyclin E protein levels and cyclin E-associated kinase activity were increased in the resistant line, suggesting that overexpression of cyclin E may be the mechanism of resistance to flavopiridol . However, transfection of cyclin E to increase expression of this protein did not result in an increase in resistance to flavopiridol . Thus, up-regulation of cyclin E alone does not seem to cause resistance to this cdk inhibitor.

J Med Humanit, 1996 Summer, 17(2), 91 - 102
Compliance, coercion, and compassion: moral dimensions of the return of tuberculosis; Booker MJ; Since 1986, we have seen a rise in the occurrence of tuberculosis in the United States . Long considered defeated in this country, the disease is returning with distressing vigor . Outbreaks of MDR-TB, tuberculosis resistant to more than one medication, have been reported around the country . This article analyzes the Centers for Disease Control and Prevention's National Action Plan to Combat Multidrug-Resistant Tuberculosis, with particular focus on the moral dimensions of mandatory directly observed treatment (DOT) and involuntary quarantine . It is proposed that a moral response to the control of tuberculosis must be one which is sustainable and which can effectively curtail the spread of the disease at a minimal cost to individual rights.

Bone Marrow Transplant, 2001 Sep, 28(6), 551 - 6
Topotecan combined with myeloablative doses of thiotepa and carboplatin for neuroblastoma, brain tumors, and other poor-risk solid tumors in children and young adults; Kushner BH et al.; Topotecan appears to be relatively unaffected by the most common multidrug resistance mechanisms, may potentiate cytotoxicity of alkylators, has good penetration into the central nervous system, is active against a variety of neoplasms, and has myelosuppression as its paramount toxicity . We present our experience with a myeloablative regimen that includes topotecan . Twenty-one patients with poor-prognosis tumors and intact function of key organs received topotecan 2 mg/m2 by 30-min intravenous (i.v.) infusion on days -8, -7, -6, -5, -4; thiotepa 300 mg/m2 by 3 h i.v . infusion on days -8, -7, -6; and carboplatin by 4 h i.v . infusion on days -5, -4, -3 with a daily dose derived from the pediatric Calvert formula, using a targeted area under the curve of seven mg/ml* min ( approximately 500 mg/m2/day) . Stem cell rescue was on day 0 . The patients were 1 to 29 (median 4) years old; 18 were in complete remission (CR) and three in partial remission (PR) . Early toxicities were severe mucositis and erythema with superficial peeling in all patients and a seizure, hypertension, and renal insufficiency followed by veno-occlusive disease in one patient each . Post-transplant treatment included radiotherapy alone (four patients) or plus biological agents (11 patients with neuroblastoma) . With a follow-up of 6+ to 32+ (median 11+) months, event-free survivors include 10/11 neuroblastoma patients (first CR), 4/5 brain tumor patients (second PR or CR), 1/3 patients with metastatic Ewing's sarcoma (first or second CR), and a patient transplanted for multiply recurrent immature ovarian teratoma; a patient with desmoplastic small round-cell tumor (second PR) had progressive disease at 8 months . Favorable results for disease control, manageable toxicity, and the antitumor profiles of topotecan, thiotepa, and carboplatin, support use of this three-drug regimen in the treatment of neuroblastoma and brain tumors; applicability to other tumors is still uncertain.

Cancer Res, 2001 Oct 15, 61(20), 7552 - 5
Cross-resistance to the synthetic retinoid CD437 in a paclitaxel-resistant human ovarian carcinoma cell line is independent of the overexpression of retinoic acid receptor-gamma; Kumar A et al.; Treatment of ovarian carcinomas with the antimitotic antitumor drug paclitaxel is highly efficacious . However, development of drug resistance presents a major obstacle . The common cellular phenotypes associated with paclitaxel resistance are an increased expression of the drug transport protein P-glycoprotein (P-gp), an alteration in the levels of beta-tubulin isotypes, and/or changes in the drug binding affinity of the microtubules . We established two paclitaxel-resistant human ovarian carcinoma cell lines . The 2008/17/4 cells exhibited a "classic" multidrug-resistant phenotype (overexpression of P-gp associated with cross-resistance to natural product drugs), whereas the 2008/13/4 cells were an atypical multidrug-resistant subline (no overexpression of P-gp) . In addition to being paclitaxel resistant (250-fold), the 2008/13/4 cells were also cross-resistant to etoposide (39-fold) and vincristine (460-fold) . To identify the alterations in the gene expression profile associated with the development of atypical paclitaxel resistance, we used the Clontech Atlas Human Cancer cDNA Microarray (spotted with 588 genes) . The expression of retinoic acid receptor (RAR)-gamma was significantly higher in the paclitaxel-resistant (2008/13/4 and 2008/17/4) cells than in the parental (2008) cells . Northern blotting analysis demonstrated that the expression of RAR-gamma was 7-fold higher in the 2008/13/4 and 2008/17/4 cells than in the 2008 cells, whereas the expression of RAR-alpha and RAR-beta was not observed in any cell line . Whereas the 2008, 2008/13/4, and 2008/17/4 cells were found to resist the antiproliferative effects of all-trans-retinoic acid, the paclitaxel-resistant cells were 6- to 7-fold cross-resistant to the antiproliferative effects of CD437 (a synthetic RAR-gamma-selective agonist; 6-{-(1-admantyl)-4-hydroxyphenyl}-2-naphthalenecarboxylic acid) compared with the sensitivity of the parental cells . To further understand the association of paclitaxel and CD437 resistance with the observed RAR-gamma overexpression, we transfected the 2008 cells with a full-length RAR-gamma cDNA construct . Two transfectants with increased expression of the RAR-gamma mRNA and protein were isolated and subjected to growth inhibition assays in the presence of various concentrations of paclitaxel, etoposide, vincristine, and CD437 . The sensitivity of the 2008 transfected clones (displaying increased expression of RAR-gamma) to the cytotoxic effects of paclitaxel, etoposide, vincristine, and CD437 was similar to that observed in the parental 2008 cells . These results suggest that the overexpression of RAR-gamma (observed in the 2008/13/4 and 2008/17/4 cells) by itself is not capable of inducing paclitaxel and CD437 resistance (or resistance to etoposide and vincristine).

Cancer Res, 2001 Oct 15, 61(20), 7525 - 9
The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product P-glycoprotein; Wang E et al.; P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs, particularly cancer chemotherapeutics . Multidrug resistance (MDR) is caused largely by the efflux of therapeutics out of the tumor cell by Pgp, resulting in reduced efficacy of chemotherapy . SCH66336, a farnesyl transferase inhibitor in development for cancer therapy, was examined in the present study for its ability to inhibit Pgp . In a test system consisting of a NIH-G185 cell line presenting an overexpressed amount of the human transporter Pgp, known Pgp inhibitors, such as cyclosporin A, paclitaxel, verapamil, tamoxifen, and vinblastine, were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin . SCH66336 significantly inhibited daunorubicin transport with an IC50 of about 3 microM and similarly affected the transport of rhodamine 123 with a potency similar to cyclosporin A . Additionally, by an ATP-hydrolysis assay, SCH66336 was shown to decrease Pgp-mediated ATP hydrolysis by >70% with a Km of 3 microM . This observation indicates that SCH66336 directly interacts with the substrate binding site of Pgp, a quality unique to SCH66336 and its analogues, although not inherent to farnesyl transferase inhibitors in general . Moreover, low concentrations of SCH66336 exhibit synergy with the Pgp substrate/inhibitors paclitaxel, tamoxifen, and vinblastine respectively by significantly potentiating their inhibition of Pgp . Treatment with SCH66336 would be predicted to be synergistic with coadministered cancer therapeutics that are substrates of Pgp . A further benefit of coadministration of SCH66336 could be reduced chemotherapy dosage, hence, lower exposure to normal cells and, therefore, less undesired toxicity.

Int J Tuberc Lung Dis, 2001 Oct, 5(10), 894 - 902
Characteristics of drug resistance and HIV among tuberculosis patients in Mozambique; Mac-Arthur A et al.; SETTING: The rate of human immunodeficiency virus (HIV) seroprevalence among tuberculosis patients varies between 2% and 53% in Mozambique, depending on the region . Drug resistance surveillance has been performed in only a few cities in Mozambique . OBJECTIVES: To establish the extent of drug resistance in areas of Mozambique with different levels of HIV prevalence, to estimate the prevalence of HIV among tuberculosis (TB) patients, and to examine the association between drug resistance and HIV infection . DESIGN: All tuberculosis patients diagnosed at randomly selected health facilities over 9 months (September 1998 to June 1999) were enrolled in the study . Sputum was collected, smeared and cultured, and drug susceptibility tests were performed . Blood was tested for HIV in the respective provinces, and patients received pre-test and post-test counselling . RESULTS: Of 709 culture-positive cases, 25.5% were HIV-positive . HIV-positive patients were significantly more likely to have a prior history of treatment (OR 2.2; 95% CI 1.9-3.6) and resistance to both isoniazid and streptomycin (OR 2.3; 95% CI 1.3, 4.5) . In patients with no history of prior tuberculosis treatment, the multidrug resistance rate was 3.4% and resistance to isoniazid and streptomycin (HS) was 5.2% . Any drug resistance was significantly more common among those with a history of prior treatment (OR 3.1; 95% CI 2.1-4.7), particularly resistance to HS (OR 4.5; 95% CI 2.6-7.9) . CONCLUSIONS: This study demonstrates substantial levels of drug resistance in Mozambique . Differences in drug resistance between high and low HIV prevalence areas may be related to prior treatment.

Int J Tuberc Lung Dis, 2001 Oct, 5(10), 887 - 93
Determinants of drug-resistant tuberculosis: analysis of 11 countries; Espinal MA et al.; SETTING: Eleven countries/territories . OBJECTIVES: Global information on the determinants of drug-resistant tuberculosis (TB) based on representative data is not available . We therefore studied the relationship between demographic characteristics, prior TB treatment, and human immunodeficiency virus (HIV) infection with anti-tuberculosis drug resistance . METHODS: Population-based representative data on new and previously treated patients with TB collected within an international drug resistance surveillance network . RESULTS: Of 9,615 patients, 8,222 (85.5%) were new cases of TB and 1,393 (14.5%) were previously treated cases . Compared with new cases, previously treated cases were significantly more likely to have resistance to one (OR = 2.5,95% CI 2.1-3.0; P < 0.001), two (OR = 4.6, 95%CI 3.7-5.6; P < 0.001), three (OR = 11.5, 95%CI 8.6-15.3; P < 0.001), and four (OR = 18.5, 95% CI 12.0-28.5; P < 0.001) drugs . An approximately linear increase in the likelihood of having multidrug-resistant tuberculosis (MDR-TB) was observed as the total time (measured in months) of prior anti-tuberculosis treatment increased (P < 0.001, chi2 for trend) . In multivariate analysis, prior TB treatment for 6-11 months (OR = 7.6, 95% CI 2.6, 22.4; P < 0.001) and > or = 12 months (OR 13.7, 95% CI 4.5-41.6; P < 0.001), but not HIV positivity, was associated with MDR-TB . CONCLUSION: This study shows that prior but ineffective treatment is a strong predictor of drug resistance, and that HIV is not an independent risk factor for MDR-TB . The association between length of treatment and drug resistance may reflect longer treatment as a result of treatment failure in patients with drug resistance; it may also reflect irregular prior treatment for TB, leading to drug resistance.

Kansenshogaku Zasshi, 2001 Sep, 75(9), 785 - 91
{Increasing fluoroquinolone low-sensitivity in enterotoxigenic Escherichia coli isolated from diarrhea of overseas travelers in Tokyo}; Matsushita S et al.; Drug resistance trends were investigated for 1,318 enterotoxigenic Escherichia coli (ETEC) isolated from overseas traveler's diarrheal cases in Tokyo during 1988-1999 . A total of 1.6% (21 strains) were nalidixic-acid resistant and fluoroquinolones (NFLX, OFLX, CPFX, LVFX, TFLX, SPFX; FQ) low-sensitive (or low-level-resistant) . None of the strains were high-level-resistant to FQ . The FQ low-sensitive strains were isolated in 1996 for the first time, and increased from 3.4% in 1996 to 15.8% in 1999 . Countries visited by travelers with the FQ low-sensitive ETEC were India (16 cases), Nepal (3 cases), Cambodia (1 case), and Egypt (1 case) . Drug resistance-patterns of the FQ low-sensitive strains, including other drugs (CP, TC, SM, KM, ABPC, ST, NA, and FOM) tested, varied among the 6 types . Among those, multidrug resistant strains accounted for 57.1% (12 strains) . The enterotoxin producing types of strains were LT (4 strains), ST (10 strains), and both (7 strains) . The serotypes of the strains were classified into 16 types . The quinolone resistance determining regions (QRDRs) of the gyrA genes of the FQ low-sensitive strains were sequenced . The mutations of a Ser to a Leu at position 83 (Ser-83-->Leu) was found in 19 strains, and Asp-87-->Tyr was found in 2 strains.

Bull Cancer, 2001 Sep, 88(9), 871 - 6
{Role of high-dose chemotherapy with hemopoietic stem-cell support in the treatment of adult patients with high-grade glioma}; Linassier C et al.; Despite surgery, post-operative irradiation and adjuvant conventional chemotherapy, prognosis of high-grade gliomas remains poor . Carmustine (BCNU) has been shown to have limited activity at conventional dosage but is still the standard chemotherapy . Activity of chemotherapy is limited by the blood-brain barrier impermeability and high levels of expression of multidrug resistance proteins on tumor and/or endothelial cells . Despite high response rates, development of intra-arterial chemotherapy remains limited because of frequent acute brain toxicity related to drug administration . High-dose intravenous chemotherapy rescued by autologous hemopoietic stem cell transplantation is an alternative that might increase drug delivery through the blood-brain barrier and tumor control . Several phase I-II trials using high-dose BCNU were published . The maximum tolerated dose seems to be 800 mg/m2 and interstitial pneumonitis and hepatitis are dose-limiting toxicities . Few phase I-II trials of high-dose therapy were published using drug combinations . High response rates in patients with progressive tumor were observed and in adjuvant setting, encouraging results in terms of median survival time and long survivors were published . No phase III trial was reported to date . Future investigations should include randomized trials comparing high-dose and conventional-dose chemotherapy and development of new high-dose regimens that incorporate new drugs such as temozolomide.

J Pharmacol Exp Ther, 2001 Nov, 299(2), 567 - 74
Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule; Miller DS; The interactions of two antiviral, acyclic nucleoside phosphonates, adefovir and cidofovir, with xenobiotic transporters was studied in intact killifish (Fundulus heteroclitus) renal proximal tubules by using fluorescent substrates, confocal microscopy, and quantitative image analysis . Both drugs reduced in a concentration-dependent manner the transport of fluorescein on the classical organic anion system and transport of fluorescein-methotrexate on multidrug resistance-associated protein 2 (Mrp2) . Neither drug inhibited transport of a fluorescent cyclosporin A derivative on P-glycoprotein . Inhibition of Mrp2-mediated transport was abolished by 50 microM p-aminohippurate, indicating that adefovir and cidofovir entered the cells at the basolateral membrane on the classical organic anion transport system (OAT1) . Comparison of the inhibitory potencies of the nucleoside phosphonates with other substrates and inhibitors showed them to be moderate inhibitors of OAT1- and Mrp2-mediated transport.

J Pharmacol Exp Ther, 2001 Nov, 299(2), 434 - 41
cDNA microarray analysis of multidrug resistance: doxorubicin selection produces multiple defects in apoptosis signaling pathways; Watts GS et al.; Doxorubicin plays an important role in the treatment of leukemias, lymphomas, and a variety of carcinomas . Tumor cell resistance to doxorubicin is often associated with expression of the multidrug resistance gene MDR1, which codes for the drug efflux pump P-glycoprotein, and a multidrug-resistant phenotype . Evidence from multiple sources suggests, however, that additional genes besides MDR1 are involved in development of multidrug resistance . To identify genes involved in the multidrug resistance phenotype, we created a 5760-gene cDNA microarray to search for differentially expressed genes between the human multiple myeloma cell line RPMI 8226 and its doxorubicin-selected sublines 8226/Dox6 and 8226/Dox40, both of which express MDR1 and are multidrug-resistant . The cDNA microarray results identified a set of differentially expressed genes, which included MDR1 as expected . Thirty Northern analyses were used to confirm the results of the cDNA microarrays; comparison with the microarray results showed a 90% agreement between the two techniques . Within the set of differentially expressed genes identified by the cDNA microarrays, 29 were of particular interest as they can participate in apoptotic signaling, particularly as mediated by ceramide and the mitochondrial permeability transition . The functional importance of these changes in gene expression is supported by their explanation of the 8226/Dox cell lines' cross-resistance to substances that are not P-glycoprotein substrates, such as Fas/CD95 ligand and staurosporine . We conclude that doxorubicin selection led to changes in gene expression that reduce the apoptotic response to death-inducing stimuli and thus contribute to the multidrug resistance phenotype.

AIDS Res Hum Retroviruses, 2001 Sep 20, 17(14), 1329 - 32
Expression of P-glycoprotein and multidrug resistance-associated protein in healthy volunteers and HIV-infected patients; Meaden ER et al.; Increased expression of the multidrug efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) has been suggested as a potential mechanism for decreased drug availability at certain intracellular sites that provide sanctuary for HIV . Here we investigate the expression of these transporters in peripheral blood mononuclear cells (PBMCs) of HIV-infected patients and healthy volunteers . Venous blood (30 ml) was taken from healthy volunteers (n = 21) and HIV-infected patients (n = 21; 4 antiretroviral drug naive, 17 antiretroviral drug experienced) . PBMCs were isolated and fixed . To assess P-gp expression, PBMCs were incubated with an isotype control antibody or an antibody directed to an external epitope of P-gp (UIC2) . To assess MRP expression, cells were permeabilized before incubation with either a control antibody or an antibody directed to an internal epitope of MRP (MRPm5) . After washing, a secondary phycoerythrin-bound antibody was incubated . After additional wash steps, samples were fixed and analyzed by flow cytometry . The median fluorescence intensity of 5000 events was recorded . Results are expressed as fold increase between isotype control and UIC2/MRPm5 samples . Expression of P-gp in HIV-infected patients (1.42 +/- 0.36) was significantly lower (p = 0.0021; 95% CI, -0.633 to -0.164) than in healthy volunteers (1.82 +/- 0.55) . However, MRP expression was similar in HIV-infected patients (1.37 +/- 0.34) and healthy volunteers (1.37 +/- 0.21; p = 0.91; 95% CI, -0.148941 to 0.165191) . We conclude that in HIV infection, P-gp expression in total PBMCs is reduced whereas MRP expression appears to be unaltered.

J Gene Med, 2001 Sep-Oct, 3(5), 427 - 36
Optimization of retroviral vector generation for clinical application; Schilz AJ et al.; BACKGROUND: For many inherited and acquired diseases of the blood system, gene transfer into hematopoietic cells is a promising strategy to alleviate disease-related symptoms or even correct genetic alterations . In clinical gene therapy applications, low transduction efficiencies have been a major limitation mainly because of insufficient effective titers of the retroviral supernatants used . Thus, optimization of clinical-grade vector production under current 'Good Manufacturing Practice' (GMP) conditions is a prerequisite for successful gene therapy trials . METHODS: We established stable retroviral producer clones with single integrations of a retroviral vector encoding for the multidrug-resistance gene 1 (MDR1) . Optimization of vector production in multi-tray cell factories (MTCFs) was studied with particular regard to harvest medium, cell density and harvest time point . RESULTS: We demonstrated that high-titer vector stocks could be produced in serum-free medium . By reducing the volume of harvest medium, titers could be increased up to four-fold . Plating optimal cell densities of 1 x 10(4) cells/cm2, repetitive harvests of vector supernatant were feasible over four consecutive days . Combining the most advantageous culture and harvest parameters tested, we were able to produce large quantities of serum-free vector supernatant in 40-tray MTCFs . Highly efficient gene transfer into primary human CD34+ progenitor cells demonstrated the quality of these vector stocks . CONCLUSION: The large-scale vector-production protocol in MTCFs described here is easy to handle, is applicable to a wide range of adherent producer cell lines and, most importantly, complies with current GMP guidelines.

Zhonghua Xue Ye Xue Za Zhi, 1999 Feb, 20(2), 69 - 72
{WT1 gene expression in leukemia patients and its correlation with prognosis and multidrug resistance}; Guo X et al.; OBJECTIVE: To evaluate the value of expression of WT1 gene in predicting the prognosis of leukemia patients, and explore the relationship between WT1 gene expression and multidrug resistance and cell apoptosis . METHODS: Expressions of WT1, MRP and mdr1 were measured in 68 leukemia patients by RT-PCR method . Expression of bcl-2 was measured in 32 AML patients by immunofluorescence flow cytometry . RESULTS: Expression of WT1 was revealed in 36 of 68 leukemia patients and none of 23 normal controls . Complete remission rate (59.46%) was lower in WT1 positive patients than that (87.10%, P = 0.011) in WT1 negative patients . The rate of MRP expression was also higher in patients with WT1 expression (58.33%), than in those without WT1 expression (32.26%, P = 0.033) . Thirty-two AML patients were divided into high-, intermediate-, and low-risk groups according to the expression of WT1 and bcl-2 . CR rates were significantly different among these 3 groups (33.33% for high-, 47.37% for intermediate-, and 100% for low-risk group, P < 0.05) . CONCLUSION: The expression of WT1 can predict the treatment outcome and the prognosis for leukemia patients . Expression of WT1 is the most important risk factor, and the coexpression of WT1 and bcl-2 predicts poor prognosis of AML patients.

Zhonghua Yi Xue Za Zhi, 1999 Mar, 79(3), 224 - 6
{In vitro reversal effect of cyclosporin A in combination with cytokines on multidrug resistant cell line K562/A02}; Pu J et al.; OBJECTIVE: To explore the reversal effect of cyclosporin A(CsA) in combination with cytokines on multidrug resistant cell line K562/A02 . METHOD: The cytotoxicities of daunorubicin(DNR) were assayed by MTT method . Intracellular rhodamine(Rh123) concentration was measured by flow cytometry . P-glycoprotein(p-gp) expression was analyzed for staining with monoclonal JSB-1 . Mdr1 mRNA expression was detected by RT-PCR . RESULTS: The cytotoxicities of DNR to K562/A02 were enhanced by 1 mumol/L CsA, 500 U/ml IFN-alpha and 200 U/ml, IL-2 respectively, and their IC50 was (3.78 +/- 0.03), (13.77 +/- 0.38) mu/ml, (18.5 +/- 0.60) micrograms/ml . Their reversal effect was 6.70, 1.84 and 1.37 times than that of K562/A02 . But IC50 of combined CsA and IFN-alpha was (1.71 +/- 0.19) micrograms/ml; its reverse effect increased in 14.8 times . The combination could increase intracellular Rh123 accumulation significantly as compared with either of them alone, but p-gp and mdr1 mRNA expression were not decreased obviously . CsA in combination with IL-2 didn't show a synergistic effect . CONCLUSION: Mdr could be partially reversed by cytokines or low doses CsA(1 mumol/L), but the combination of CsA and IFN-alpha showed a greater synergistic reversal interaction.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3014 - 20
Family of class 1 integrons related to In4 from Tn1696; Partridge SR et al.; The class 1 integron In28, found in the multidrug resistance transposon Tn1403, was found to be located in the res site of the backbone transposon and is flanked by a 5-bp direct duplication, indicating that it reached this position by transposition . In28 has a backbone structure related to that of In4, but has lost internal sequences, including the sul1 gene, due to an IS6100-mediated deletion . In28 also lacks the partial copy of IS6100 found in In4 and contains different gene cassettes, blaP1, cmlA1, and aadA1 . In1, the class 1 integron found in the multidrug resistance plasmid R46, is also located in a putative res site and belongs to the In4 group . In1 has a shorter internal deletion than In28 and has also lost one end . Additional integrons with structures related to In4 were also found in databases, and most of them had also lost either one end or internal regions or both . Tn610 belongs to this group.

Hum Immunol, 2001 Oct, 62(10), 1073 - 80
Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells; Laupeze B et al.; P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin . The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC . Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes . Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction . In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC . In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC . Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.

J Biol Chem, 2001 Dec 28, 276(52), 49053 - 60 Epub 2001 Oct 11.
MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential; Luker GD et al.; MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs . Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive . To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs . Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells . Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance . Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer . Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance . The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.

Yao Xue Xue Bao, 1997 Jun, 32(6), 401 - 5
{Compared study on Fura-2/AM assay and MTT assay for screening multidrug resistant modulators}; Fu LW et al.; To explore the difference of screening results of reversing multidrug resistance (MDR) by modulators between Fura-2/AM assay and MTT assay, 25 compounds which have active structure were studied for MDR reversal activity with both methods . The fold of MDR reversal was shown to have remarkable relation with the amount of Fura-2 accumulation (Y = -3.66 + 17.5X, gamma = 0.86, P < 0.01) . On the other hand, Fura-2/AM assay has several advantages as compared with MTT assay . Fura-2/AM assay needs shorter time (4 h) than MTT assay (96 h), and the MTT assay needs more steps than the Fura-2/AM assay . Furthermore, Fura-2/AM assay was more reliable than MTT assay for screening MDR modulators because MTT assay was dependent on the viable cells, while Fura-2/AM assay was dependent on the function of P-gp . The results suggest that Fura-2/AM assay may replace MTT assay in the screening of MDR modulators on a large scale.

Yao Xue Xue Bao, 1997 Sep, 32(9), 647 - 51
{Antitumor activity of the clavam peptide antibiotic G0069A}; Xue YC et al.; Antibiotic G0069A, produced by a Streptomyces strain isolated from a soil sample collected in Yunnan Province, China, has been verified as a clavam peptide . Determined by MTT assay, G0069A showed highly potent cytotoxicity to cancer cells with multidrug resistance . The IC50 values of G0069A to KB and KB/VCR cells were 0.60 and 0.46 mumol.L-1, and to MCF-7 and MCF-7/ADM cells were 1.4 and 1.2 mumol.L-1, respectively . G0069A displayed equally potent cytotoxicity to the parent cell lines and their resistant sublines . When administered by i.v . or i.p . route at tolerable doses, G0069A exhibited markedly inhibitory effect on the growth of sarcoma 180 and hepatoma 22 in mice . At dose level of 3 mg.kg-1, i.v., x3, sarcoma 180 and hepatoma 22 were suppressed by 87%(P < 0.01) and 72%(P < 0.01), respectively . The results indicate that G0069A is a beta-lactam antibiotic showing antitumor activity.

Cancer, 2001 Oct 15, 92(8), 2065 - 71
Mitoxantrone in patients with prostate specific antigen progression after local therapy for prostate carcinoma; DiPaola RS et al.; BACKGROUND: Molecular mechanisms of chemotherapy resistance are present in prostate carcinoma, some of which increase after androgen ablation (AA) therapy . Therefore, the authors hypothesized that chemotherapy in patients with prostate specific antigen (PSA) progression after local therapy, before androgen ablation therapy, will have greater antitumor activity . METHODS: Twenty-three hormone-naive patients with PSA progression after prostatectomy or radiation therapy were registered in this study . Twenty-two were treated with 10 mg/m(2) of mitoxantrone initially, followed by 12 mg/m(2) every 3 weeks for a maximum of 8 cycles . Prostatectomy specimens were assessed, when possible, for topoisomerase II alpha, multidrug resistance protein MRP, and bcl-2 by immunohistochemistry . RESULTS: Twenty-two patients received a total of 131 cycles of therapy . Three patients had transient Grade 3 or 4 neutropenia without fever . During treatment, 10 of 22 patients showed a decrease in PSA, without an associated decrease in testosterone . In this group of 10 patients, the mean PSA decrease was 29% at 3 months and 43% at 6 months . Overall, 4 of 22 patients had a decrease in PSA of greater than or equal to 50% . The PSA decreased in three of seven patients whose cancer overexpressed MRP and in three of seven patients who overexpressed bcl-2 . No patient with overexpression of topoisomerase II alpha (n = 4) had a decrease in PSA during the study . CONCLUSIONS: To the authors' knowledge, this is the first reported study of mitoxantrone in patients with hormone-naive prostate carcinoma and PSA progression after local therapy; mitoxantrone was safe and biochemically active, similar to prior studies in hormone refractory prostate carcinoma, suggesting that critical molecular mechanisms of chemotherapy resistance are present independent of AA . Further studies are warranted to determine whether pharmacogenomic assessment of topoisomerase II, MRP, or bcl-2 may predict for response to mitoxantrone .

Clin Cancer Res, 2001 Oct, 7(10), 3193 - 8
In vitro prevention of the emergence of multidrug resistance in a pediatric rhabdomyosarcoma cell line; Cocker HA et al.; We have established preclinical models for the development of drug resistance to vincristine (a major drug used in the treatment of pediatric rhabdomyosarcoma) using cell lines . The RD cell line has a mutant P53 phenotype and does not have detectable P-glycoprotein (P-gp) or multidrug resistance-related protein (MRP) despite expressing low levels of mdr-1 mRNA, which encodes P-gp and mrp1 mRNA . Resistant variants of RD were derived by exposure to increasing concentrations of vincristine . This was repeated on six occasions, resulting in three cell lines which could tolerate 64 x the IC(50) concentration . Six independent agents were tested for their ability to prevent the development of resistance in this model . Despite at least 10 attempts, resistance did not develop in the presence of the multidrug resistance (MDR) modulators PSC833, VX710, and XR9576 . This strongly suggests that these agents may delay or even prevent the development of resistance to vincristine . This was also confirmed in a second rhabdomyosarcoma cell line, Rh30 . In contrast, the agents indomethacin (MRP1 modulator), CGP41251 (protein kinase C inhibitor), and dexrazoxane (putative MDR prevention agent) did not affect the development of resistance in the RD model . Characterization of the resistant cell lines indicated the presence of increased mdr-1 and P-gp expression, which resulted in resistance to the agents doxorubicin, etoposide, and vincristine but not cisplatin . The resistance could be modulated using PSC833 or VX710, confirming that functional P-gp is present . No apparent differences were seen between the resistant cell lines derived in the absence and presence of the various agents . These experiments strongly suggest that the development of MDR may be preventable using modulators of MDR and merit clinical studies to test this hypothesis.

Clin Cancer Res, 2001 Oct, 7(10), 3120 - 6
Prognostic significance of multidrug resistance protein in adult T-cell leukemia; Ohno N et al.; The response of adult T-cell leukemia (ATL) to chemotherapy is poor, and a major obstacle to successful treatment is intrinsic or acquired drug resistance . To determine the clinical significance of multidrug resistance protein (MRP) 1 in ATL, we studied MRP1 expression and its association with clinical outcome . The expression of MRP1 mRNA in leukemia cells from 48 ATL patients was studied by slot blot analysis . The expression level of MRP1 mRNA in chronic-type ATL was significantly higher than that in lymphoma-type ATL (P = 0.033) . There was no correlation between MRP1 expression and age, gender, WBC count, LDH, hypercalcemia, blood urea nitrogen, or performance status . However, the expression of MRP1 mRNA correlated only with peripheral blood abnormal lymphocyte counts (P = 0.008) . The transporting activity of MRP1 was assessed using membrane vesicles . Membrane vesicles prepared from ATL cells with high expression of MRP1 mRNA showed a higher ATP-dependent leukotriene C(4) uptake than did those with low expression of MRP1 mRNA . This uptake was almost completely inhibited by LTD(4) antagonists ONO-1078 and MK571 . In acute- and lymphoma-type ATL, high expression of MRP1 mRNA at diagnosis correlated with shorter survival, and Cox regression analysis revealed that MRP1 expression was an independent prognostic factor . These findings suggest that functionally active MRP1 is expressed in some ATL cells and that it is involved in drug resistance and has a possible causal relationship with poor prognosis in ATL . Multidrug resistance-reversing agents, such as ONO-1078 and MK571, that directly interact and inhibit the transporting activity of MRP1 may be useful for treating ATL patients.

Clin Cancer Res, 2001 Oct, 7(10), 3065 - 70
Lack of correlation of functional scintigraphy with (99m)technetium-methoxyisobutylisonitrile with histological necrosis following induction chemotherapy or measures of P-glycoprotein expression in high-grade osteosarcoma; Gorlick R et al.; In osteosarcoma, some studies have suggested P-glycoprotein expression is a prognostic factor . The clearance of (99m)technetium hexakis-2-methoxyisobutylisonitrile ((99m)Tc-MIBI) has been used in some tumor systems as an in vivo measure of P-glycoprotein-mediated efflux . In this study we explored the correlation between (99m)Tc-MIBI clearance and histological necrosis following induction chemotherapy and P-glycoprotein expression in osteosarcoma . The primary tumors of 20 patients with high-grade osteosarcoma were imaged at diagnosis with (99m)Tc-MIBI, and the uptake ratios and biological half-lives were calculated . P-Glycoprotein expression in the tumor tissue was determined immunohistochemically and by measuring mRNA expression of the multidrug resistance-1 gene . The histological necrosis following induction chemotherapy was assessed by the Huvos grading system . The biological half-life of (99m)Tc-MIBI ranged from 1.4 to 52.5 h . Seven of the 20 tumor samples had a favorable extent of necrosis following induction chemotherapy . The (99m)Tc-MIBI half-life and uptake ratio showed no correlation with histological necrosis following induction chemotherapy . The (99m)Tc-MIBI half-life and uptake ratio did not correlate with either measure of P-glycoprotein expression . The results of this pilot study indicate that (99m)Tc-MIBI imaging is not an effective predictor of histological necrosis following induction chemotherapy in high-grade osteosarcoma . (99m)Tc-MIBI imaging did not correlate with measures of P-glycoprotein expression in the tumor tissue.

Aquat Toxicol, 2001 Nov 12, 55(3-4), 157 - 70
P-glycoprotein in the catfish intestine: inducibility by xenobiotics and functional properties; Doi AM et al.; The p-glycoprotein (pgp)-mediated multixenobiotic resistance (MXR) mechanism of aquatic animals has been associated with protection against pollution . Recent studies in mammals suggest that intestinal pgp may modulate intestinal bioavailability of dietary xenobiotics . In order to further delineate this mechanism in the catfish, these studies: (1) examined the pgp-related distribution in the intestine and liver of catfish, (2) evaluated the MXR response following exposure to various dietary xenobiotics and a prototypic pgp inducer and (3) evaluated pgp functional activity in membrane vesicles, using prototypic substrates and inhibitors . For this purpose, catfish were exposed in vivo to the pgp inducer vincristine (VIN), and the xenobiotics beta-naphthoflavone (BNF), benzo{a}pyrene (BaP), and 3,4,3',4'-tetrachlorobiphenyl (TCB) . Membrane vesicles, prepared from liver and intestine (proximal and distal sections) of control and exposed catfish, were subjected to SDS PAGE, Western Blot, and detection with the pgp C219 monoclonal antibody . Transport activity was evaluated in vitro using the pgp substrate {3H}vinblastine (VBL), and the pgp inhibitor verapamil (VP) . Immunoblot studies demonstrated a pgp-related protein of approximately 170 kDa in the intestine and liver of catfish . This protein appears to be very susceptible to degradation, and was present in higher levels in the liver, in comparison to the intestine, where regional differences were not observed . Dietary exposure to the pgp substrate VIN, or the xenobiotics BNF, BaP, and TCB, did not appear to affect pgp-related reactivity . Transport studies with VBL indicate that the pgp-related protein of the catfish intestine displays classic pgp-mediated multidrug resistance (MDR) characteristics, such as energy-dependency, and sensitivity to VP . These studies suggest that the pgp-related protein in the catfish intestine and liver is not only immunochemically, but also functionally related to the mammalian MDR . Moreover, the results presented indicate that pgp-related reactivity and transport in intestinal vesicles of catfish may be influenced by factors including method sensitivity, sample collection, sample preparation, and immunoblot conditions.

Oncogene, 2001 Sep 20, 20(42), 6048 - 56
Increased and correlated nuclear factor-kappa B and Ku autoantigen activities are associated with development of multidrug resistance; Um JH et al.; In this study, we investigated possible engagement of NF-kappaB and Ku autoantigen (Ku) activation in development of multidrug resistance (MDR) and circumvention of MDR by modulation of NF-kappaB and Ku . The NF-kappaB activity and NF-kappaB p65 subunit level were constitutively higher in MDR cells than in drug-sensitive parental cells . Interestingly, a faster running NF-kappaB DNA binding complex was identified as Ku, a DNA damage sensor and a key double strand break repair protein, and was positively correlated with the NF-kappaB activity in MDR cells and Ku- or both subunits of NF-kappaB-transfected cells . Also both NF-kappaB and Ku activities were activated or inhibited by treatment with etoposide (VP-16) or MG-132 (a proteasome inhibitor), respectively . Furthermore, PKA inhibitor suppressed markedly the constitutive and drug-induced activities of NF-kappaB and Ku in MDR cells and subsequently potentiated the cytotoxic activity of anticancer drugs . Our results proposed that the NF-kappaB and Ku activation could be one of multi-factorial MDR mechanism, and PKA inhibitor, likely via inhibition of NF-kappaB and Ku activities, could enhance the effectiveness of anticancer drugs against MDR cells with high activities of NF-kappaB and Ku.

Mol Med, 2001 Aug, 7(8), 509 - 16
MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer; Bera TK et al.; BACKGROUND: With the completion of the human draft genome sequence, efforts are now devoted to identifying new genes . We have developed a computer-based strategy that utilizes the EST database to identify new genes that could be targets for the immunotherapy of cancer or could be involved in the multistep process of cancer . MATERIALS AND METHODS: Utilizing our computer-based screening strategy, we identified a cluster of expressed sequence tags (ESTs) that are highly expressed in breast cancer . Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses demonstrated the tissue specificity of the computer-generated cluster and comparison with the human genome sequence assisted in isolating a full-length cDNA clone . RESULTS: We identified a new gene that is highly expressed in breast cancer . This gene is expressed at moderate levels in normal breast and testis and at very low levels in liver, brain, and placenta . The gene has two major transcripts of 4.5 kb and 4.1 kb . The 4.5-kb transcript is very abundant in breast cancer, and has an open reading frame of 1382 amino acids . The predicted protein sequence of the 4.5-kb transcript reveals that it has high homology with MRP5, a member of multidrug resistant-associated protein family (MRP) . There are seven reported members in the MRP family; we designate this gene as MRP8 (ABCC11) . The 4.5-kb MRP8 transcript consists of 31 exons and is located in a genomic region of over 80.4 kb on chromosome 16q12.1 . The smaller 4.1-kb transcript of MRP8 is found in testis and may initiate within intron 6 of the gene . CONCLUSION: The selective expression of MRP8 (ABCC11), a new member of ATP-binding cassette transporter superfamily could be a molecular target for the treatment of breast cancer.

Saudi Med J, 2001 Sep, 22(9), 776 - 9
Drug resistance pulmonary tuberculosis in the Eastern Province of Saudi Arabia; Al-Rubaish AM et al.; OBJECTIVE: To evaluate the prevalence and pattern of antituberculous drug resistance and patients with pulmonary tuberculosis in the Eastern Province and its impact on the tuberculosis control program . METHODS: Patients with pulmonary tuberculosis, proven by culture, admitted to Dammam Chest Hospital from November 1993 through May 1996 were reviewed . Patients who had at least one documented isolate of mycobacterium tuberculosis resistant to at least one standard anti-tuberculosis drug were identified . Medical records were reviewed and information was retrieved regarding age, sex, nationality, history of previous tuberculosis, human immune deficiency status, and results of direct smear and chest radiograph abnormalities . RESULTS: A total of 411 cases of culture positive pulmonary tuberculosis with drug susceptibility testing, were identified during the period mentioned, of these 123 (30%) were Saudi nationals and 228 (70%) were non-Saudis . Drug resistance to at least one drug was observed in 43 (10.5%) patients, resistance to isoniazid alone was observed in 25 (6%) patients, whereas resistance to rifampicin was noted in only one (0.2%) patient, and to streptomycin in 3 (1%) patients, multidrug resistance was observed in 11 (3%) patients . CONCLUSION: The study has shown that the overall drug resistance rate of 10.5% in the Eastern Province of Saudi Arabia is the lowest reported in the Kingdom, compared with Riyadh (13%), Taif (23%) and Gizan (44%) . However, it appears to be similar to that reported in neighboring countries . In light of the study findings, and as per the recommendation of the World Health Organization, we suggest that a 4-drug regimen, as an initial treatment for pulmonary tuberculosis should be maintained, as resistance to isoniazid is still higher than the cut off value of 4%, and susceptibility testing for first line antituberculosis drugs should be carried out whenever possible . We also recommend applying stricter medical criteria for tuberculosis screening in newcomers, and for Saudi nationals, application of directly observed therapy should be a priority.

J Biol Chem, 2001 Dec 14, 276(50), 46822 - 9 Epub 2001 Oct 04.
Enhanced expression of the human multidrug resistance protein 3 by bile salt in human enterocytes . A transcriptional control of a plausible bile acid transporter; Inokuchi A et al.; The enterohepatic circulation is essential for the maintenance of bile acids and cholesterol homeostasis . The ileal bile acid transporter on the apical membrane of enterocytes mediates the intestinal uptake of bile salts, but little is known about the bile salt secretion from the basolateral membrane of enterocytes into blood . In the basolateral membrane of enterocytes, an ATP-binding cassette transporter, multidrug resistance protein 3 (MRP3), is expressed, which has the ability to transport bile salts . We hypothesized that MRP3 might play a role in the enterohepatic circulation of bile salts by transporting them from enterocytes into circulating blood through the up-regulation of MRP3 expression, so we investigated the transcriptional control of MRP3 in response to bile salts . MRP3 mRNA levels were increased about 3-fold in human colon cells by chenodeoxycholic acid (CDCA), in a dose- and time-dependent manner . In the promoter assay, the promoter activity of MRP3 was increased about 3-fold over the basal promoter activity when treated with CDCA, and the putative bile salt-responsive elements exist in the region -229/-138 including two alpha-1 fetoprotein transcription factor (FTF)-like elements . Constructs with a specific mutation in the consensus sequence of FTF elements showed no increase in basal transcriptional activity following CDCA treatment . In electrophoretic mobility shift assay with nuclear extracts, specific binding of FTF to FTF-like elements was observed when treated with CDCA . The expression of FTF mRNA levels were also markedly enhanced in response to CDCA, and overexpression of FTF specifically activated the MRP3 promoter activity about 4-fold over the basal promoter activity . FTF thus might play a key role not only in the bile salt synthetic pathway in hepatocytes but also in the bile salt excretion pathway in enterocytes through the regulation of MRP3 expression . MRP3 may contribute as a plausible bile salt-exporting transporter to the enterohepatic circulation of bile salts.

Oral Oncol, 2001 Dec, 37(8), 652 - 9
Expression of multidrug resistance-related genes in oral squamous cell carcinomas; Cho YS et al.; To characterize the multidrug resistance (MDR) phenotype in human oral squamous cell carcinomas (OSCCs), the expression levels of four MDR-related genes (multidrug resistance, mdr1; multidrug resistance-associated protein, MRP; glutathione S-transferase-pi, GST-pi; and DNA topoisomerase II, topoII) were analyzed in OSCCs . Fifty-two OSCC tissues and 22 normal oral mucosal tissues were involved in this study . The expression of each gene was analyzed with a reverse-transcription polymerase chain reaction (RT-PCR) method using beta(2)m microglobulin (beta(2)m) mRNA as an endogenous control . The mean values of mdr1, MRP, GST-pi, and topoII gene expression relative to the beta(2)m gene in OSCC tissues were 0.37, 0.75, 0.66, and 1.11; those of normal oral mucosa were 0.40, 0.27, 0.62, and 0.91, respectively . The averaged expression levels of the MRP and topoII gene in OSCC tissues were higher than those of normal oral mucosas (P=0.001 and P=0.02, respectively) . The expression levels of four MDR-related genes in OSCCs were not related with the degree of histologic cell differentiation, tumor stage, primary or recurred tumor, or the presence or absence of chemotherapy . Linear regression analysis showed a correlation between the expression levels of MRP and GST-pi in normal oral mucosas (r=0.596, P=0.003) and in OSCCs (r=0.287, P=0.039) . The results suggest that MRP expression is activated during the tumorigenesis of OSCCs and that this may play a role in de novo drug resistance in OSCCs . These results should provide further insight into the complex role postulated for MDR-related genes in chemotherapy, carcinogenesis and tumor progression.

Eur J Biochem, 2001 Oct, 268(19), 5011 - 26
The role of the TolC family in protein transport and multidrug efflux . From stereochemical certainty to mechanistic hypothesis; Sharff A et al.; Gram-negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy-driven pumps to transport molecules directly across this double-layered partition from the cell interior to the extra-cellular environment . One component of these pumps is embedded in the outer-membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli . A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as alpha-hemolysin, to small toxic compounds, such as antibiotics . TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance . These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner-membrane-associated periplasmic component, an integral inner-membrane translocase and the substrate itself . TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates . We review here the structure and function of TolC and the other components of the efflux/transport pump.

Am J Respir Crit Care Med, 2001 Sep 15, 164(6), 953 - 7
Tuberculosis in the foreign-born population of Tarrant county, Texas by immigration status; Weis SE et al.; The epidemiology of tuberculosis is changing in the United States as a result of immigration, yet the extent to which different classes of immigrants contribute to overall morbidity is unknown . Tuberculosis in nonimmigrant visitors is of particular interest as they are currently exempt from screening requirements . We conducted a prospective survey of all culture-positive tuberculosis patients in Tarrant County, Texas from 1/98 to 12/00 . Immigration status of foreign-born patients was classified as permanent residents, undocumented, or nonimmigrant visitors . Of 274 eligible participants, 114 (42%) were foreign-born; of these, 67 (59%) were permanent residents, 28 (25%) were undocumented, and 19 (17%) were nonimmigrant visitors . Among the foreign-born, we observed significant differences by immigration status in multidrug resistance (p = 0.02), human immunodeficiency virus (HIV) infection (p = 0.0007), and hospitalization (p = 0.03 for ever/never, 0.01 for duration) . Compared with other immigrants, more nonimmigrant visitors were multi-drug-resistant (16 % versus 11% of undocumented residents and 1% of permanent residents), were HIV-positive (32% versus 0% of undocumented and 5% of permanent residents), were hospitalized (47% versus 36% of undocumented and 19% of permanent residents), and had lengthy hospitalizations (median {midspread} days = 87 {25 to 153} versus 8.5 {4 to 28} for undocumented and 10 {7 to 24 d} for permanent residents) . We found nonimmigrant visitors to be an important source of tuberculosis morbidity in Tarrant County . Further studies in other regions of the U.S . are needed to determine if screening and treatment recommendations of persons who spend extended periods in the U.S . should be raised to the standards set for permanent residents.

Cancer Chemother Pharmacol, 2001 Aug, 48 Suppl 1, S45 - 52
Acute myeloid leukemia in adults: where do we go from here?
Schiffer CA.
Although 30-40% of newly diagnosed younger patients with acute myeloid leukemia (AML) can be cured with current approaches, the overall outcome has not improved in recent years . In addition, the outcome in adults > 60 years of age remains dismal with < 10% of patients achieving remission remaining alive and disease free . Results of randomized clinical trials in AML evaluating high-dose cytosine arabinoside, changes in anthracyclines, the use of hematopoietic growth factors, stem cell transplantation in first remission, and modulation of the multidrug resistance phenotype are reviewed . New directions for clinical trials include the use of nonmyeloablative allogeneic stem cell transplantation as a form of "immunotherapy", refinements in autologous stem cell transplantation, and possibly manipulations of neoangiogenesis in the bone marrow and incorporation of newer agents, such as gemtuzumab zogamicin into treatment regimens . It is likely, however, that future advances will be a consequence of a better understanding of the biology of leukemic stem cells, and issues related to such studies are discussed.

Leukemia, 2001 Oct, 15(10), 1554 - 63
Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia; van der Pol MA et al.; Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD) . In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up . Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML . A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity . Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations . In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10) . This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

Leukemia, 2001 Oct, 15(10), 1544 - 53
Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia; van der Kolk DM et al.; The multidrug resistance proteins (MRPs) MRP1, MRP2, MRP3, MRP5 and P-glycoprotein (P-gp) act in concert with each other to give a net resultant pump function in acute myeloid leukemia (AML) . The aim of the present study was to analyze the activity of these proteins, which might be upregulated at relapse as compared with de novo AML due to clonal selection . The mRNA expression and activity of P-gp and the MRPs were determined with RT-PCR and flow cytometry, in conjunction with phenotype, as measured with the monoclonal antibodies CD34, CD38 and CD33, in 30 paired samples of de novo and relapsed AML . P-gp and MRP activity varied strongly between the cases (rhodamine 123 efflux-blocking by PSC833: 5.4+/-7.7, and carboxyfluorescein efflux-blocking by MK-571: 4.3+/-6.7, n = 60) . P-gp and MRP activity were increased in 23% and 40% of the relapse samples, and decreased in 30% and 20% of the relapse samples, respectively (as defined by a difference of >2 x standard deviation of the assays) . Up- or downregulation of mRNA expression was observed for MDR1 (40%), MRP1 (20%), MRP2 (15%), MRP3 (30%), and MRP5 (5%) . Phenotyping demonstrated a more mature phenotype in 23% of the relapsed AML cases, and a more immature phenotype in 23% of the relapses, which was independent of the karyotypic changes that were observed in 50% of the studied cases . P-gp and MRP activity correlated with the phenotypic changes, with higher P-gp and MRP activities in less mature cells (r = -0.66, P < 0.001 and r = -0.31, P = 0.02, n = 58) . In conclusion, this study shows that P-gp and MRP activity are not consistently upregulated in relapsed AML . However, P-gp and MRP activities were correlated with the maturation stage as defined by immune phenotype, which was observed to be different in 46% of the relapses.

J Nucl Med, 2001 Oct, 42(10), 1476 - 83
Expression of multidrug resistance protein and messenger RNA correlate with (99m)Tc-MIBI imaging in patients with lung cancer; Zhou J et al.; In vitro studies have shown that (99m)Tc-sestamibi (MIBI) is a transport substrate for the P-glycoprotein (Pgp) pump and the multidrug resistance-associated protein (MRP) pump . However, whether MRP and lung resistance protein (LRP) affect tumor accumulation and efflux of (99m)Tc-MIBI in lung cancer is not known . In this study, we explored whether Pgp and the other pumps, MRP and LRP, affect tumor accumulation and efflux of (99m)Tc-MIBI in lung cancer . METHODS: Thirty-four lung cancer patients who underwent surgery were examined . Before surgery, (99m)Tc-MIBI SPECT was performed 15 min and 180 min after injection, and early uptake, delayed uptake (L/Nd), and washout rate (L/Nwr) of (99m)Tc-MIBI were obtained . Pgp, MRP, and LRP expression were investigated by immunohistochemistry . The messenger RNA (mRNA) level of Pgp, MRP, and LRP was determined by real-time reverse-transcription polymerase chain reaction . The lung cancer (99m)Tc-MIBI images were correlated with protein and mRNA expression . RESULTS: The mean L/Nd of the Pgp (-) group was significantly higher than that of the Pgp (++) group (P = 0.0324) . The Pgp (++) group had a higher L/Nwr than did the Pgp (-) group (P = 0.0269) . The mean L/Nd of the Pgp mRNA low-expression group was significantly higher than that of the Pgp mRNA high-expression group (P = 0.0127) . The Pgp mRNA high-expression group had a higher L/Nwr than did the Pgp mRNA low-expression group (P = 0.0825) . No appreciable correlation was found between the lung cancer (99m)Tc-MIBI images and the expression of MRP or LRP on the level of protein or mRNA . CONCLUSION: These data suggest that an increased level of Pgp expression correlates with a low accumulation on delayed scans and a high L/Nwr of (99m)Tc-MIBI in lung cancer . Neither MRP nor LRP expression on the level of either protein or mRNA correlated significantly with tumor accumulation or efflux of (99m)Tc-MIBI in lung cancer.

Cancer Res, 2001 Oct 1, 61(19), 7248 - 54
Biochemical genetic analysis of indanocine resistance in human leukemia; Hua XH et al.; Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug-resistant cancer cell lines . We demonstrated that indanocine specifically induces apoptosis in malignant B cells from patients with chronic lymphocytic leukemia . To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymphoblastoid cells . The resistant cells displayed a stable indanocine-resistant phenotype for at least 9 months in drug-free culture . The cloned cells are cross-resistant to colchicine and vinblastine, but not to paclitaxel, and do not have increased expression of the multidrug-resistant p170 glycoprotein . In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization . In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions . Both extract mixing and cell fusion experiments suggested that a stable structural change in microtubules, rather than a soluble factor, was responsible for indanocine resistance . Sequence analysis of parental and resistant cells revealed a single point mutation in the M40 isotype of beta-tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.

Cancer Res, 2001 Oct 1, 61(19), 7225 - 32
Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport; Zeng H et al.; We have recently determined that human multidrug resistance protein (MRP) 3, which confers resistance to certain natural product agents and methotrexate (MTX), is competent in the MgATP-energized transport of MTX and the monoanionic bile constituent glycocholate as well as several glutathione and glucuronate conjugates . Of these capabilities, the facility of MRP3 in conferring resistance to and mediating the transport of MTX is of particular interest because it raises the possibility that this pump is a component of the previously described cellular efflux system for this antimetabolite . However, if this is to be the case, a critical property of cellular MTX efflux that must be addressed is its ability to mediate the export of MTX but not that of its intracellular polyglutamylated derivatives . Here we examine the role of MRP3 in these and related processes by determining the selectivity of this transporter for MTX, MTX polyglutamates, and physiological folates . In so doing, we show that MRP3 is not only active in the transport of MTX but is also active in the transport the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin) and that polyglutamylation of MTX abolishes transport . Both FA and leucovorin are subject to high-capacity (V(max(FA)), 1.71 +/- 0.05 nmol/mg/min; V(max(leucovorin)), 3.63 +/- 1.20 nmol/mg/min), low-affinity (K(m(FA)), 1.96 +/- 0.13 mM; K(m(leucovorin)), 1.74 +/- 0.65 mM) transport by MRP3 . Addition of a single glutamyl residue to MTX is sufficient to diminish transport by >95% . We also show that polyglutamylation similarly affects the capacity of MRP1 to transport MTX and that physiological folates are also subject to MgATP-stimulated transport by MRP1 . On the basis of the capacity to transport MTX but not MTX-Glu(2), it is concluded that MRP3 and MRP1 represent components of the previously described cellular efflux system for MTX . The capacity of MRP3 to transport folates indicates that it may reduce intracellular levels of these compounds and thereby indirectly influence antifolate cytotoxicity, and it also implies that this pump may play a role in the response to chemotherapeutic regimens in which leucovorin is a component.

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