Multidrug resistant

Anticancer Res, 2000 Mar-Apr, 20(2B), 1061 - 7
P-glycoprotein expression is a marker for chemotherapy resistance and prognosis in advanced ovarian cancer; Baekelandt MM et al.; BACKGROUND: It was our aim to study the prevalence and possible prognostic and predictive significance of the expression of P-glycoprotein, a transmembrane transport protein related to classical multidrug resistance, in patients with advanced ovarian cancer . STUDY DESIGN: Tumor tissue from 73 previously untreated patients with FIGO stage 3 ovarian cancer was examined with immunohistochemistry for the expression of P-glycoprotein before and after chemotherapy . Response to 4 cycles of combination chemotherapy with cisplatin and epirubicin was assessed with second look laparotomy . The log rank test was used for univariate survival and the Cox proportional hazards regression model for multivariate survival analysis . RESULTS: P-glycoprotein expression was detected in 47% of untreated cases, and correlated with unfavourable prognostic factors such as advanced age, presence of ascites and larger residual disease deposits after primary surgery . P-glycoprotein negative cases responded significantly better to chemotherapy (P < .001) . In the multivariate survival analysis P-glycoprotein expression was an independent predictor of both overall (P = .045) and progression free (P = .006) survival . When P-glycoprotein expression and residual disease status were considered together, the patients could be divided in three clearly distinct prognostic groups (P = .0009) . CONCLUSION: P-glycoprotein expression is a predictor of response and survival in a uniformly treated and followed cohort of advanced ovarian cancer patients.

Anticancer Res, 2000 Mar-Apr, 20(2A), 1029 - 31
Oncocalyxones A and C, 1,4-anthracenediones from Auxemma oncocalyx: comparison with anticancer 1,9-anthracenediones; Leyva A et al.; Oncocalyxones A and C are 1,4-anthracenediones isolated from Auxemma oncocalyx (Boraginaceae) that have been shown to be cytotoxic to tumor cells in vitro . The present study compared the cytotoxicity of these compounds with that of two conventional anticancer agents doxorubicin and mitoxantrone, both 1,9-anthracenediones, in a panel of human tumor cell lines . The effect on cell growth was examined using an MTT microtiter assay in two leukemia lines, five solid tumor lines of different histological origin, and two multidrug-resistant sublines of a lung tumor line . The oncocalyxones showed much lower potency than the 1,9-anthracenediones, but were similarly more cytotoxic to leukemia cells compared to solid tumor lines . However, in the multidrug-resistant cells with 10 to 500 times decreased sensitivity to doxorubicin, the cytotoxicity of oncocalyxones A and C was only modestly reduced by about twofold, 1,4-Anthracenediones may be a promising novel class of chemotherapeutic agents effective against multidrug resistant tumors.

Anticancer Res, 2000 Mar-Apr, 20(2A), 987 - 96
Multiwavelength videomicrofluorometric study of cytotoxic effects of a marine peptide, didemnin B, on normal and MDR resistant CCRF-CEM cell lines; Rocchi E et al.; The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy . Didemnin B, a marine cyclic depsipeptide, displays interesting biological properties: antiviral activity, inhibition of DNA, RNA and protein synthesis, initiation of apoptosis and ability to block the cell cycle . As very little is known about its mode of action, we studied the effect of increasing doses of Didemnin B on sensitive and resistant human leukemic lymphoblast cell lines . The fluorescence of living cells simultaneously stained with Hoechst 33,342, Rhodamine 123 and Nile Red, were analyzed in a multiparametric approach involving multiwavelength microfluorometry . High concentrations of Didemnin B induced, in the sensitive cell line, a very early decrease in the energetic state of the mitochondria that occurs before a significant decrease of nuclear DNA content, observed simultaneously on sensitive and resistant cells, that could be related to an apoptosis process . Furthermore low Didemnin doses (50 nM) affected CEM-WT and CEM VLB differently, while higher doses (200 nM-250 nM and over) affected the two cell lines in the same way . This indicated that, at these doses, the membranar Pgp has no effect on the mode of action of Didemnin, suggesting that Didemnin does not need to be internalized to be active.

Anticancer Res, 2000 Mar-Apr, 20(2A), 861 - 7
Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates; Molnar J et al.; In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions . Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells . Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL . Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells . The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells . Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression . However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL . The crocin had no antiviral effect {on HSV-2 infected vero cells} up to 25 micrograms/mL concentration . Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump . Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells . It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.

Anticancer Res, 2000 Mar-Apr, 20(2A), 799 - 808
Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells; Wang F et al.; Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability . In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells . The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3{H}-thymidine assay . The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR . The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf) . These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation . The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively . However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM . ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR . Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively . These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells . The distribution of ADR in both cell lines was examined by fluorescence microscopy . It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells . However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells . Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene . Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression.

J Intern Med, 2000 May, 247(5), 521 - 34
Multidrug resistance in haematological malignancies; Sonneveld P; The development of refractory disease in acute myeloid or lymphoblastic leukaemias (AML, ALL) and multiple myeloma (MM) is frequently associated with the expression of one or several multidrug resistance (MDR) genes . MDR1, MRP1 and LRP have been identified as important adverse prognostic factors in AML, T-ALL and MM . Recently, it has become possible to reverse clinical multidrug resistance by blocking P-glycoprotein-mediated drug efflux . The potential relevance of these reversal agents of MDR and potential new approaches to treat refractory disease are discussed.

Eur J Clin Invest, 2000 May, 30(5), 447 - 53
The 16p11 breakpoint in myxoid liposarcomas might affect the expression of the LRP gene on 16p11.2 encoding the multidrug resistance associated major vault protein; Plaat BE et al.; BACKGROUND: Chromosome breakage could influence the expression of genes . This has been noticed in specific cases of acute myeloid leukaemia, where the 16p13 breakpoint affects the expression of the multidrug resistance related protein (MRP) . Myxoid liposarcomas (LPS) are characterized by the t(12; 16)(q13; p11), which leads to the formation of a FUS-CHOP fusion transcript . This study investigates the relationship between the cytogenetically detected breakpoint 16p11 in myxoid LPS, the presence of the FUS-CHOP fusion transcript in nonmyxoid LPS and the expression of the lung resistance major vault protein (LRP) gene on 16p11.2 . MATERIALS AND METHODS: Of 16 cases with a diagnosis of a (possible) liposarcoma with an abnormal karyotype, fresh frozen tumour material was available for immunohistological detection of LRP . Cases without a cytogenetically detected t(12; 16)(q13; p11), were analyzed for the presence of a FUS-CHOP fusion transcript by RT-PCR . RESULTS: In all 9 myxoid LPS a t(12; 16)(q13; p11) was found and LRP expression was absent or low . In none of the remaining 7 cases was a FUS-CHOP fusion transcript found, and four tumours were LRP positive (P = 0 . 02) . LRP expression in myxoid LPS (mean: 1.3%) was lower (P = 0.07) than in the nonmyxoid tumours (mean: 35.7%) . CONCLUSIONS: These observations indicate a relation between the t(12; 16)(q13; p11), leading to a FUS-CHOP fusion transcript in myxoid LPS, and the low or absent expression of the LRP-gene located on 16p11.2.

Biochem Pharmacol, 2000 Jul 1, 60(1), 83 - 90
Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates; Astriab-Fisher A et al.; Antisense oligonucleotides are potentially a powerful tool for the therapeutic manipulation of genes associated with cancer . However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells . In this study, we have prepared peptide-oligonucleotide conjugates with the intent of improving intracellular delivery . The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells . Two types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition of cell surface expression of P-glycoprotein . Surprisingly, the peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions; this is in striking contrast to most other approaches for intracellular delivery of nucleic acids . Effective inhibition of P-glycoprotein expression was attained with submicromolar concentrations of antisense conjugates under serum-replete conditions . The combination of relatively modest molecular size and good efficacy in the presence of serum proteins suggests that peptide-antisense oligonucleotide conjugates may have significant promise for in vivo therapeutic applications.

Zhongguo Yao Li Xue Bao, 1999 Jul, 20(7), 647 - 50
R-dl-verapamil downmodulates multidrug resistance of KBv200 cells to vincristine and doxorubicin; Fang G et al.; AIM: To study the attenuation of multidrug resistance (MDR) by R-dl-verapamil (R-Ver) and the acute animal toxicity of R-Ver, and to compare these results of R-Ver with the results of dl-verapamil (Ver) . METHODS: Cytotoxicity was determined by tetrazolium (MTT) assay . Cellular accumulation of doxorubicin (Dox) was measured by fluorescence spectrophotometry . Acute animal toxicity was tested by i.p . drug administration in BALB/c mice . RESULTS: R-Ver attenuated MDR of KBv200 cells to vincristine (VCR) and Dox . This attenuation ability was dose-related, and was also dependent on drug exposure time . R-Ver 1.25 mumol.L-1 increased the sensitivity of KBv200 cells to VCR (P < 0.01) with a 24-h period of drug exposure . R-Ver downmodulated MDR and increased cellular Dox accumulation of KBv200 cells as effectively as Ver, but possessed lower acute toxicity in BALB/c mice . While LD50 of Ver was 60 (49-73) mg.kg-1, LD50 of R-Ver was 166 (137-202) mg.kg-1 . CONCLUSION: R-Ver downmodulated the MDR to VCR and Dox at 1.25 mumol.L-1, and this effect on VCR can be realized with drug exposure duration of 24 h.

J Cell Sci, 2000 Jun, 113 ( Pt 11), 2011 - 21
The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2); Litman T et al.; Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown . We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP . The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines . We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry . These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance . High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan . Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression . Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells . Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein . Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance . Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance . Definition of its mechanism of transport and its role in clinical oncology is required.

Acta Neuropathol (Berl), 2000 May, 99(5), 555 - 62
Evidence for a constitutive, verapamil-sensitive, non-P-glycoprotein multidrug resistance phenotype in malignant glioma that is unaltered by radiochemotherapy in vivo; Rieger L et al.; Human malignant gliomas are commonly resistant to chemotherapy . Here, we examined the role of the multidrug resistance (mdr) mechanism in the chemo-resistance of these tumors, using a twofold approach: (i) by assessing a possible mdr phenotype before and after chronic drug exposure of glioma cells in vitro, and (ii) by assessing the modulation of expression of the mdr-associated P-glycoprotein (Pgp) using radiotherapy and serial cycles of chemotherapy in human glioblastoma patients in vivo . T98G, and to a lesser degree, LN-229 human malignant glioma cells exhibit a constitutive mdr phenotype as determined by the modulation of dye transport and by the augmentation of chemosensitivity by the mdr antagonist, verapamil . Thus, coexposure to verapamil enhances the cytotoxicity of vincristine, doxorubicin and VM26 in T98G cells and that of vincristine in LN-229 cells . Chronic exposure of the cells to low concentrations of vincristine and doxorubicin, but not VM26, topotecan or BCNU, moderately enhances the mdr-like phenotype, as assessed by drug expulsion assays . However, chronic exposure to increasing drug concentrations does not significantly alter the sensitivity to the respective drugs . These data are consistent with a constitutive, but not drug-inducible, mdr-like drug resistance in glioma cells in vitro . Immunocytochemical analysis of human malignant gliomas in vivo reveals that Pgp expression is more abundant in endothelial cells within the gliomas, than in the glioma cells proper . Importantly, Pgp expression is unaltered by radiochemotherapy, assessed by comparative immunocytochemistry of glioma specimens obtained serially before and after radiochemotherapy . We conclude that (i) glioma cells exhibit constitutive mdr-like drug resistance that is not significantly altered by chronic drug exposure in vitro; (ii) endothelial cells may play an important role in Pgp-mediated drug resistance of gliomas in vivo; (iii) radiotherapy and repeated chemotherapy cycles do not modulate Pgp expression in human malignant gliomas in vivo; (iv) there is preliminary evidence for a non-Pgp, verapamil-sensitive drug transport activity in glioma cells.

Jpn J Cancer Res, 2000 Apr, 91(4), 439 - 45
Photodynamic inactivation with acridine orange on a multidrug-resistant mouse osteosarcoma cell line; Kusuzaki K et al.; Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients . In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS / ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line . Cultured cells of MOS and MOS / ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long- or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation . Living cells were counted by means of the trypan blue exclusion test . The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS / ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15-min flash exposure to AO at concentrations above 1.0 microg/ml plus 10-min illumination with blue light . Even after flash exposure to AO at concentrations above 1.0 microg/ml plus 1-min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/ 1000 within 72 h . Based on these results, we concluded that AO with photo-excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells . These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.

Mol Biochem Parasitol, 2000 Apr 30, 108(1), 13 - 23
The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin; Duraisingh MT et al.; Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear . In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine . Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems . A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin . A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine . The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine . An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine . Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation.

Chem Biol, 2000 May, 7(5), 335 - 43
Novel gallium(III) complexes transported by MDR1 P-glycoprotein: potential PET imaging agents for probing P-glycoprotein-mediated transport activity in vivo; Sharma V et al.; BACKGROUND: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients . Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy . RESULTS: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate . Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry . Radiolabeled (67)Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp(-)) and MDR (Pgp(+)) tumor cells . Compared with control, lead compound 6 . demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells . Futhermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6 . in brain and liver tissues of mdr1a/b((-/-)) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood-brain barrier and hepatocellular biliary cannalicular surface in vivo . CONCLUSIONS: These results indicate that gallium(III) complex 6 . is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate (68)Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET.

Am J Public Health, 2000 May, 90(5), 699 - 701
Helping the urban poor stay with antiretroviral HIV drug therapy; Bamberger JD et al.; Recent studies have documented dramatic decreases in opportunistic infections, hospitalizations, and mortality among HIV-infected persons, owing primarily to the advent of highly active antiretroviral medications . Unfortunately, not all segments of the population living with HIV benefit equally from treatment . In San Francisco, only about 30% of the HIV-infected urban poor take combination highly active antiretroviral medications, as compared with 88% of HIV-infected gay men . Practitioners who care for the urban poor are reluctant to prescribe these medications, fearing inadequate or inconsistent adherence to the complicated medical regimen . Persons typically must take 2 to 15 pills at a time, 2 to 3 times a day . Some of the medications require refrigeration, which may not be available to the homeless poor . Most homeless persons do not have food available to them on a consistent schedule . Therefore, they may have difficulty adhering to instructions to take medications only on an empty stomach or with food . Lack of a safe place to store medications may be an issue for some . In addition, many urban poor live with drug, alcohol, or mental health problems, which can interfere with taking medications as prescribed . Inconsistent adherence to medication regimens has serious consequences . Patients do not benefit fully from treatments, and they will become resistant to the medications in their regimen as well as to other medications in the same classes as those in their regimen . Development of resistance has implications for the broader public health, because inadvertent transmission of multidrug-resistant strains of HIV has been demonstrated . Concern that the urban poor will not adhere to highly active antiretroviral medication regimens has led to debate on the role of clinicians and public health officials in determining who can comply with these regimens . Rather than define the characteristics that would predict adherence to these regimens, the San Francisco Department of Public Health created a program to support adherence among those who may have the greatest difficulty complying with complicated highly active antiretroviral medication regimens . The program, dubbed the Action Point Adherence Project, was conceived through a community planning process in preparation for a city-wide summit on HIV/AIDS that took place in January 1998 . Action Point is funded by the city and the county of San Francisco . Now in its 10th month, the program continues to show promising evidence of improving clients' biological and social indicators.

Biochem Biophys Res Commun, 2000 May 10, 271(2), 534 - 6
Vectors with hidden cloning sites; Welker E et al.; A general strategy is described for using the cleavage site of restriction enzymes in vectors for cloning regardless of how many sites the given enzymes have in the vector . The application of this method allows one to open any vector at its cloning site with protruding ends which can be compatible with almost every commercially available Class II restriction enzyme . By employing this method, the laborious construction of new vectors can be simplified considerably . This general strategy is based on the known ability of Class IIS restriction enzymes to cut any sequence located outside of their recognition site; the introduction of a linker containing recognition site(s) for Class IIS restriction enzyme(s), not present originally in the vector, gives rise to the possibility of opening the vector so as to produce overhangs of arbitrary sequence . In particular, when a symmetrical short sequence representing the protruding end of any Class II enzyme is situated at the cutting position of the Class IIS enzyme, cleavage with the Class IIS enzyme exposes the hitherto hidden, "unique" cloning site . This technique is demonstrated by cloning the cDNA of the multidrug resistance protein to an expression vector .

NMR Biomed, 2000 Apr, 13(2), 92 - 101
Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion; Le Moyec L et al.; The proton NMR spectra of K562 cells contain resonances of lipids . When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed . The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy . Therefore, the K562 drug-sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug-free medium, were used in this study . The P-glycoprotein (P-gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy . The phenotype reversion is due to the decrease of the P-gp function and an increased entrance of anthracycline drug when compared with the resistant cells . The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content . A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids . Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance . These results are in favour of a different lipid organization or of localization within the cell .

Int J Cancer, 2000 May 15, 86(4), 506 - 11
Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines; Harada T et al.; Previous studies have shown that gene re-arrangements play a significant role in tumorigenesis . Gene re-arrangements involving the human multidrug resistance-1 (MDR1) gene have been identified as a mechanism for MDR1 over-expression in human malignant cells . In 2 multidrug-resistant human cancer sublines with high levels of MDR1 and P-glycoprotein (MCF7/TX400 and S48-3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified . To characterize and determine the site of the re-arrangements resulting in generation of hybrid mRNAs, we first constructed a lambda phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1 . In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra-Alu deletion flanked by chi-like sequences at the re-arrangement junction . By contrast, non-homologous recombination was observed in S48-3s/Adr10 cells with Alu repeats near the junction sequence . While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re-arrangements . The gene re-arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1 .

Zhonghua Wai Ke Za Zhi, 1997 Nov, 35(11), 697 - 9
{Drug resistance and its mechanism of intrinsic drug-resistant cell line GRC-1}; Kong X et al.; In order to probe the characteristics of drug resistance and its mechanisms of renal cell carcinoma, drug-resistant spectrum of renal cell carcinoma cell line GRC-1 was detected by in vitro MTT colorimetric assay, the mechanism of drug resistance in GRC-1 was also studied by the methods of both immunocytochemistry assay and flow fluorescence cytometry . The results demonstrated that GRC-1 was cross-resistant to adriamycin, vincrinstine, etoposide and carboplatinium, both mdr1 gene product P-glycoprotein and GST-pi which was an isozyme of glutathione S-transferases were expressed in GRC-1 . The accumulation of net intracellular drugs of GRC-1 was less than that of drug sensitive breast cancer cell line MCF7, and the ability of pumping drugs out of cells was higher than that of MCF7 . The results suggested that there is an intrinsic multidrug resistance in GRC-1 cell line, and both P-glycoprotein and glutathione systems play a role in the development of drug resistance for GRC-1 . GRC-1 is an ideal target cell line for the study of drug resistance.

Cochrane Database Syst Rev . 2000;(2):CD000256.
Artemisinin derivatives for treating uncomplicated malaria; McIntosh HM et al.; BACKGROUND: Artemisinin derivatives are a relatively new group of drugs with antimalarial properties . As resistance to other antimalarial drugs continues to increase, artemisinin drugs may be useful alternatives . OBJECTIVES: The objective of this review was to assess the effects of artemisinin drugs for treating uncomplicated falciparum malaria . SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, the Cochrane Controlled Trials Register, Medline, Embase, Science Citation Index, Lilacs, African Index Medicus; conference abstracts and reference lists of relevant articles . We contacted organisations, researchers in the field and drug companies . SELECTION CRITERIA: Randomised and quasi-randomised trials of artemisinin derivatives, alone or in combination with other antimalarials, compared with standard antimalarial treatments, in adults or children with uncomplicated falciparum malaria . Only trials where treatment was given by mouth or suppository were included . Comparisons between different artemisinin derivatives and treatment regimens were also included . DATA COLLECTION AND ANALYSIS: Eligibility and trial quality were assessed and data were extracted independently by the two reviewers . MAIN RESULTS: Forty-one trials involving over 5000 patients were included . Variation in study design and quality made synthesis of the data problematic . Allocation concealment was adequate in only two trials . Most data were from areas of multidrug resistant falciparum malaria in South East Asia . Compared with standard antimalarial treatments, artemisinin drugs showed fast parasite clearance and high cure rates at follow-up, provided the duration of treatment with artemisinin drugs was adequate . Combination with mefloquine improved sustained parasite clearance and was effective in multidrug resistant areas . When doses were adequate, the combination shortened the duration of treatment . We found no evidence that artemisinin drugs are more harmful than standard treatment drugs over a typical trial period of 28 days . REVIEWER'S CONCLUSIONS: The evidence suggests that artemisinin drugs are effective and safe for treating uncomplicated malaria . There is no evidence from randomised trials that one artemisinin derivative is better than the others . In areas where there is mefloquine resistance, combination therapy with an artemisinin derivative appears to improve sustained parasite clearance compared with either drug alone.

Biochem Pharmacol, 1999 May 1, 57(9), 1047 - 58
pH and drug resistance . II . Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs; Raghunand N et al.; Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer . The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp) . However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs . In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance . We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g . anthracyclines and Vinca alkaloids . The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid . Measured values for these parameters have been inserted into the model . Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange . Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs . The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp . Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein.

Biochem Pharmacol, 1999 May 1, 57(9), 1037 - 46
pH and drug resistance . I . Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines; Martinez-Zaguilan R et al.; A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells . Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump . However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities . Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance . In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells . Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX) . The resting steady-state pHi was similar in the three cell lines . In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity . These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines . The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated . In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did . Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors . Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane . Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells . Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not . Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover . The potential impact of this behavior on drug resistance is examined in a companion manuscript.

Biochem J, 2000 May 15, 348 Pt 1, 183 - 8
Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide; Dombrowski F et al.; Immunohistochemical studies suggest that canalicular secretion via multidrug resistance protein 2 (Mrp2), a conjugate export pump encoded by the Mrp2 gene, is regulated by rapid transporter retrieval from/insertion into the canalicular membrane . The present study was undertaken in order to investigate this suggestion by means of immunogold electron microscopy . Therefore the effects of lipopolysaccharide (LPS) and osmolarity on Mrp2 localization were studied following immunogold labelling in the perfused rat liver by quantitative electron microscopy and morphometric analyses, and by confocal laser scanning microscopy . Mrp2 activity was assessed in the isolated perfused rat liver by measuring the excretion of dinitrophenyl-S-glutathione as a substrate of Mrp2 . Both LPS and hyperosmolarity resulted in a statistically significant decrease in immunogold-labelled Mrp2 in the canalicular membrane and canalicular villi, and an increase in labelling in the pericanalicular cytoplasm . Canalicular morphometric parameters were unchanged under these conditions compared with controls . Under hyperosmolar perfusion Mrp2, but not the canalicular protein dipeptidylpeptidase IV, was found inside the cells, as shown by double immunofluorescence and confocal laser scanning microscopy . The findings suggest a selective retrieval of Mrp2 from the canalicular membrane under the influence of hyperosmolarity and LPS, whereas canalicular morphology remains unchanged.

Aquat Toxicol, 2000 Apr 1, 48(4), 357 - 389
Multixenobiotic resistance as a cellular defense mechanism in aquatic organisms; Bard SM; Multixenobiotic resistance in aquatic organisms exposed to natural toxins or anthropogenic contaminants is a phenomenon analogous to multidrug resistance in mammalian tumor cell lines tolerant of anti-cancer drugs . Multidrug resistance is commonly due to the elevated expression of transmembrane P-glycoproteins (P-gp) which actively transport a wide variety of structurally and functionally diverse compounds . The purpose of this review is to place aquatic ecotoxicological data in context of the larger multidrug resistance field of study . Information on P-glycoproteins structure, mechanism of transport, and substrate specificity gained through traditional mammalian and cell culture models is examined in conjunction with recent work on aquatic species exposed to xenobiotics both in the field and in the laboratory . The physiological function of P-glycoproteins is explored through studies of gene knockout models and expression patterns in normal tissues and tumors . The effect of xenobiotic exposures on P-gp activity and protein titer is examined in wild and captive populations of aquatic invertebrates and vertebrates . Substrate overlap and evidence of co-expression of phase I detoxification enzymes (e.g . cytochromes P450) and P-gp are presented . The role of P-gp chemosensitizers as environmental pollutants and the ecotoxicological consequences of P-gp inhibition are highlighted . The overwhelming evidence suggests that P-glycoproteins provide aquatic organisms with resistance to a wide range of natural and anthropogenic toxins.

Virology, 2000 May 10, 270(2), 310 - 6
Multidrug resistance genotypes (insertions in the beta3-beta4 finger subdomain and MDR mutations) of HIV-1 reverse transcriptase from extensively treated patients: incidence and association with other resistance mutations; Tamalet C et al.; Multiple nucleoside resistance involves specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides . These include a cluster of five mutations in the reverse-transcriptase (RT) coding region (A62V, V75I, F77L, F116Y, and Q151M) generally referred to as multidrug resistance (MDR) mutations, and insertions of one or several amino acid residues between codons 67 and 70 of RT, a flexible region joining two antiparrallel beta sheets (beta3-beta4 insertions) . The objectives of this study were (i) to determine the prevalence of multidrug resistance genotypes (MDR mutations and beta3-beta4 insertions) in a cohort of 632 patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral therapy, and (ii) to analyze the association of multidrug resistance genotypes with other resistance mutations in the RT and protease genes . Among viruses sequenced from these patients, 15 (2.4%) of them contained an insertion and 2 (0.3%) contained a deletion in the beta3-beta4 finger subdomain of RT . In 9 cases, the insertion was associated with a D67S, G, or E mutation . In addition, we identified 13 (2.1%) viruses harboring specific MDR mutations (mainly Q151M and/or A62V, V75I, F116Y) . Interestingly, the A62V mutation was found in 6 of the 15 strains with an insertion, whereas the other MDR mutations were not observed in insertion mutant strains . Especially high levels of resistance to zidovudine were observed for viruses with a beta3-beta4 insertion in the background of A62V, L210W, and T215Y . Otherwise, MDR mutations and beta3-beta4 insertions were found in association with the classic mutations conferring resistance to zidovudine, lamivudine, nonnucleoside RT inhibitors, and protease inhibitors, according to treatment history . Finally, we observed a genome with a deletion of codon 70 associated with a Q151M MDR mutation . These data suggest that the emergence of HIV-1 multidrug resistance, which may occur in various genetic contexts, poses a challenging problem in formulating treatment strategies .

Mol Microbiol, 2000 Apr, 36(2), 402 - 13
Hyperactive forms of the Pdr1p transcription factor fail to respond to positive regulation by the hsp70 protein Pdr13p; Hallstrom TC et al.; Multidrug resistance in Saccharomyces cerevisiae is commonly associated with the overproduction of ATP-binding cassette transporter proteins such as Pdr5p or Yor1p . The Cys6-Zn(II)2 cluster-containing transcription factors Pdr1p and Pdr3p are key regulators of expression of these pleiotropic drug resistance (PDR) loci . Previous experiments have demonstrated that the Hsp70 protein encoded by the PDR13 gene is a positive regulator of Pdr1p function . We have examined the mechanism underlying the control of Pdr1p by Pdr13p . Expression of deletion, insertion and amino acid substitution mutant variants of Pdr1p suggest that the centre region of the transcription factor is the target for Pdr13p-mediated positive regulation . Immunological and fusion protein analyses demonstrate that Pdr13p is located in the cytoplasm, while Pdr1p is found in the nucleus . Biochemical fractionation experiments indicate that Pdr13p is associated with a high-molecular-weight complex and suggest the association of some fraction of Pdr13p with ribosomes.

Br J Haematol, 2000 Mar, 108(4), 703 - 9
P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression in acute promyelocytic leukaemia; Michieli M et al.; We analysed the expression of three drug transporter proteins {p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)} involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution . Cellular daunorubicin accumulation was also evaluated . At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines . Only one case showed LRP overexpression . No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission . In the onset vs . first relapse, only one patient showed an increased (threefold) PGP expression at relapse . At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values . These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline . It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse . The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse . In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.

Br J Clin Pharmacol, 2000 May, 49(5), 437 - 44
In vitro sensitivity of Plasmodium falciparum and clinical response to lumefantrine (benflumetol) and artemether; Tanariya P et al.; AIMS: To assess the sensitivity of 103 Plasmodium falciparum isolates to a combination of lumefantrine (benflumetol) and artemether (CGP 56697), with the objective of determining a correlation between in vitro drug sensitivity and therapeutic outcome . METHODS: Patients suffered from uncomplicated falciparum malaria and came from areas of Thailand affected by multidrug resistance . CGP 56697 was given in the form of tablets containing 20 mg artemether and 120 mg lumefantrine . The standard dose regimen, 4 doses of 4 tablets over 48 h, was compared with two lower dose regimens (4 x 2 tablets and 3 x 4 tablets) . RESULTS: The parasites showed high resistance to chloroquine, fairly advanced resistance to mefloquine and compromised sensitivity to quinine . Sensitivity to artemisinin and lumefantrine prior to treatment was similar in all treatment groups . The 4 x 4 tablet regimen was more effective than the other regimens in coping with infections with relatively low sensitivity to artemisinin and/or lumefantrine . The EC90 for artemisinin is an important determinant of treatment success . Parasite density at the start of treatment was identified as another critical predictor of treatment outcome . CONCLUSIONS: The results indicate that parasite exposure to the drugs may have been inadequate and/or too short in the cases of treatment failure, particularly marked in the lower dose regimens . This could probably be remedied by expanding the dose regimen in areas affected by multidrug resistance and in the case of relatively high parasitaemia.

J Clin Invest, 2000 May, 105(9), 1261 - 7
Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells; Sullivan GF et al.; The expression of several drug-resistance genes, including MRP and p53, increases with advancing stage of human prostate cancer . Altered transcription could account for the genotypic alterations associated with prostate cancer progression, and it was recently reported that the promoter of MRP1 is activated in the presence of mutant p53 . To determine whether there is a relationship between p53 status and the expression of MRP1, a human, temperature-sensitive p53 mutant (tsp Val(138)) was transfected into LNCaP human prostate cancer cells . In the transfected cell line (LVCaP), the wild-type p53 produced growth arrest at the G1/S interface of the cell cycle, inhibited colony formation, and induced p21(waf1/cip1) . Temperature shifting to 38 degrees C (p53 mutant) produced a time-dependent increase in expression of MRP1 . This change in MRP1 expression was also seen in isogenic cell lines in which p53 was inactivated by human papilloma virus (HPV)16E6 protein or by a dominant-negative mutant . Functional assays revealed a decrease in drug accumulation and drug sensitivity associated with mutant p53 and increased MRP1 expression . These results provide the first mechanistic link between expression of MRP1 and mutation of p53 in human prostate cancer and support recent clinical associations . Furthermore, these data suggest a mechanism tying accumulation of p53 mutations to the multidrug resistance phenotype seen in this disease.

J Hematother Stem Cell Res, 1999 Oct, 8(5), 503 - 14
Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs; Omori F et al.; Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance . The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients . We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR . This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml . Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide . Southern blot analyses indicated that most clones had only one proviral integration . Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones . Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase) . Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6 . PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted . To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide . All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR . Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

Yeast, 2000 Apr, 16(6), 561 - 71
The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae; Petrovic S et al.; Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles . Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake . Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains . These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast . Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin .

J Clin Microbiol, 2000 May, 38(5), 1901 - 8
Technical assessment of the affymetrix yeast expression GeneChip YE6100 platform in a heterologous model of genes that confer resistance to antimalarial drugs in yeast; Nau ME et al.; The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization . At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms . The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine . Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis.

J Org Chem, 2000 Apr 21, 65(8), 2479 - 83
Total synthesis of ningalin B utilizing a heterocyclic azadiene Diels-Alder reaction and discovery of a new class of potent multidrug resistant (MDR) reversal agents; Boger DL et al.; A concise, efficient approach to the total synthesis of ningalin B (1) based on a heterocyclic azadiene Diels-Alder strategy (1,2,4,5-tetrazine-->1,2,-diazine-->pyrrole) ideally suited for construction of the densely functionalized pyrrole core found in the natural product is detailed . Examination of the natural product and a number of synthetic intermediates revealed that while lacking inherent cytotoxic activity, many reverse the multidrug-resistant (MDR) phenotype, resensitizing a human colon cancer cell line (HCT116/VM46) to vinblastine and doxorubicin at lower doses than the prototypical agent verapamil.

Eur J Cancer, 2000 May, 36(7), 881 - 8
Incidence of P-glycoprotein overexpression and multidrug resistance (MDR) reversal in adult soft tissue sarcoma; Coley HM et al.; Multidrug resistance (MDR) is a widespread problem in the treatment of neoplastic diseases and may limit the effectiveness of treatment of adult soft tissue sarcomas (STS) . We examined the levels of expression of the MDR marker P-glycoprotein (Pgp) in fresh, surgical material and matched paraffin-embedded tissue using MRK-16 and JSB-1 monoclonal antibodies . Using fresh tumour material in short-term culture an assessment of doxorubicin sensitivity (MTT assay) and MDR modulation using PSC-833 in daunorubicin (DNR) accumulation experiments (FACS analysis) was carried out . 44 patients were studied at various disease stages with a mean follow-up duration of 487 days (range: 45-1095 days) . Immunocytochemistry and immunohistochemistry showed 62% and 58%, respectively, of STS samples were positive for Pgp . Patients showing negative Pgp expression had a median survival of 544 days versus 431 days for Pgp-positive patients (P=0.311), with disease-free survival medians of 508 and 355 days, respectively (P=0.203) . In vitro doxorubicin sensitivity was not informative in this respect and there was no apparent relationship between this and Pgp expression . Eleven out of 29 samples evaluated for MDR modulation showed enhanced tumour cell DNR accumulation . However, the effects of PSC-833 on drug accumulation in clinical material were modest compared with those seen for MDR cell lines, with a maximum of only 20% enhancement . Moreover, there was no relationship between the extent of PSC-833 effects on accumulation and the levels of Pgp seen in the STS samples . Nevertheless, we show evidence that a proportion of cases of STS express moderate to high levels of Pgp . There may be a role for MDR modulating agents in association with doxorubicin in the treatment of these tumours, either in the adjuvant setting or at first relapse.

Mod Pathol, 2000 Apr, 13(4), 407 - 13
Effects of multidrug resistance gene expression in acute erythroleukemia; Mazzella FM et al.; Acute erythroleukemia is a relatively rare disorder of a multilineal nature . Patients with this type of leukemia traditionally have been treated with a standard myeloid protocol, with a wide variation in prognosis between M6a, which has a similar prognosis to acute myelogenous leukemias, and M6b, with an extremely poor outcome despite aggressive therapy . Forty-eight archival cases of acute erythroleukemia, subtypes M6a (the traditional FAB-M6), M6b (pure erythroleukemia), and M6c (>30% myeloblasts and >30% pronormoblasts by FAB exclusion criteria), were evaluated for multidrug resistance gene (MDR-1) status . Findings were correlated with clinical course and karyotypes . Immunohistochemical stain for the protein product of MDR-1, P-glycoprotein, was variably positive in 11 of 23 patients with M6a, as well as in all of the patients with M6b (strongly positive) and M6c (weakly positive) . P-glycoprotein expression positively correlated with unfavorable cytogenetic aberrations, poor response to chemotherapeutic agents, and short survival . Most significant was that P-glycoprotein expression demonstrated a negative additive effect on response to treatment and prognosis with unfavorable cytogenetic anomalies . P-glycoprotein expression and multiple cytogenetic anomalies most probably contribute to the resistance to chemotherapy and poor survival characteristic of the patients with M6b (mean survival, 3.15 +/- 4.2 mo) and M6c (mean survival, 10.5 +/- 12.7 mo) . Because patients with M6b and M6c have increased numbers of pronormoblasts in their bone marrow and past chemotherapeutic attempts have failed, chemotherapy directed at these cells is appropriate . Additional therapy directed toward the MDR-1 gene and its protein product seems indicated from our findings.

J Clin Oncol, 2000 May, 18(9), 1867 - 75
Mitoxantrone, etoposide, and cyclosporine therapy in pediatric patients with recurrent or refractory acute myeloid leukemia; Dahl GV et al.; PURPOSE: To determine the remission rate and toxicity of mitoxantrone, etoposide, and cyclosporine (MEC) therapy, multidrug resistance-1 (MDR1) status, and steady-state cyclosporine (CSA) levels in children with relapsed and/or refractory acute myeloid leukemia . PATIENTS AND METHODS: MEC therapy consisted of mitoxantrone 6 mg/m(2)/d for 5 days, etoposide 60 mg/m(2)/d for 5 days, and CSA 10 mg/kg for 2 hours followed by 30 mg/kg/d as a continuous infusion for 98 hours . Because of pharmacokinetic interactions, drug doses were decreased to 60% of those found to be effective without coadministration of CSA . MDR1 expression was evaluated by reverse transcriptase polymerase chain reaction, flow cytometry, and the ability of CSA at 2.5 micromol/L to increase intracellular accumulation of (3)H-daunomycin in blasts from bone marrow specimens . RESULTS: The remission rate was 35% (n = 23 of 66) . Overall, 35% of patients (n = 23) achieved complete remission (CR), 12% of patients (n = 8) achieved partial remission, and 9% of patients (n = 6) died of infection . Exposure to CSA levels of greater than 2,400 ng/mL was achieved in 95% of patients (n = 56 of 59) . Toxicities included infection, cardiotoxicity, myelosuppression, stomatitis, and reversible increases in serum creatinine and bilirubin . In most who had relapsed while receiving therapy or whose induction therapy had failed, response was not significantly different for MDR1-positive and MDR1-negative patients . CONCLUSION: Serum levels of CSA capable of reversing multidrug resistance are achievable in children with acceptable toxicity . The CR rate of 35% achieved in this study is comparable to previously reported results using standard doses of mitoxantrone and etoposide . The use of CSA may have improved the response rate for the MDR1-positive patients so that it was not different from that for the MDR1-negative patients.

Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 209 - 16
Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using variable treatment schedules; Dassow H et al.; OBJECTIVE: The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs . Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment . METHODS: The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined . Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT) . In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C . RESULTS: Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs . 10% variability after free phosphorothioates) . Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs . 22% MRK16 staining) . Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed . In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control) . CONCLUSION: The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs . A more promising approach seems to be the prophylactic treatment with AS-ODNs.

Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 196 - 203
Resistance modulation in CHO cells by R-verapamil and bile salts is associated with physical and chemical changes in the cell membrane; Dolderer JH et al.; OBJECTIVES: Changes in multidrug resistance by resistance modifiers such as R-verapamil cause changes in fluidity of the cell membrane . The extent to which these changes involve structural alterations in membrane lipids has been investigated in CHO cells . METHODS: Sensitive (AUXB1) and resistant (CH(R)C5) chinese hamster ovary cells (CHO) were grown in culture . Incubations were carried out with R-verapamil (0-10 microM) or the membrane perturbing agents tauro-cheno-deoxycholate (0-1.6 mM, TCDC) and tauro-urso-deoxycholate (0-3.5mM, TUDC) . Cell membrane fluidity was determined by electron-paramagnetic resonance spectroscopy and membrane lipids by HPLC and TLC . RESULTS: The resistant CH(R)C5 subline had a higher cell membrane order (lower fluidity, S = 0.7234) in the interface region of the cell membrane than sensitive AUXB1 cells (S = 0.6984) determined using EPR . The MDR-modulator R-verapamil and TCDC, but not TUDC, lowered cell membrane order in a concentration-dependent manner and increased membrane fluidity of the resistant CH(R)C5 subline . TCDC and R-verapamil were without effect on the cell membrane fluidity of AUXB1 cells . These changes were accompanied by alterations in the fatty acid composition of the plasma membrane . Untreated sensitive AUXB1 cells had higher levels of unsaturated fatty acids than resistant CH(R)C5 cells . In CH(R)C5 cells, R-verapamil increased the content of poly-unsaturated fatty acids and TCDC, but not TUDC, increased the content of mono-unsaturated fatty acids . CONCLUSIONS: The results demonstrate that resistance modifiers such as verapamil may influence cytostatic drug action by producing structural changes to lipid domains in the plasma membrane.

Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 187 - 95
Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP); Lehne G et al.; Multidrug resistance (MDR) to cancer chemotherapy is frequently associated with decreased drug accumulation in cancer cells due to drug expulsion by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP) . The novel resistance modifying agents PSC 833, 280-446, and LY 335979 are primarily targeted at inhibition of Pgp, and their MRP inhibitory potential is largely unknown . OBJECTIVE: In the present study we addressed the effect of these agents on MRP-derived drug resistance . MATERIALS: Drug-resistant human leukemia cells with Pgp+/MRP- (KG1a/200, K562/150) and Pgp-/MRP+ (HL60/130) phenotypes were maintained in suspension cultures for experimental studies of drug accumulation and drug sensitization by Pgp inhibitors . METHODS: Intracellular accumulation of the fluorescent anthracycline daunorubicin was measured by flow cytometry and fluorescence detection . Daunorubicin dose-response curves were generated by non-linear regression of electronically measured cell counts of 72- - 96-h cultures . The half-maximal growth inhibitory dose (GI50) was used as measure of growth inhibition . RESULTS: All MDR phenotypes studied exercised significant resistance to daunorubicin . PSC 833, 280-446 and LY335979 were equal in sensitizing Pgp+/MRP- cells to daunorubicin-induced growth inhibition (p < 0.0001) . The Pgp-/MRP+ cells responded to PSC 833 and 280-446 by increased accumulation of daunorubicin (p = 0.0022 and p = 0.0005, respectively) and sensitization to the drug (p = 0.0009 and p = 0.0007, respectively) . Conversely, LY335979 did not affect accumulation of daunorubicin in Pgp-/MRP+ cells nor sensitize these cells to daunorubicin . CONCLUSION: Pgp inhibitory agents have differential effects on MRP-derived drug resistance which could be exploited in treatment of multidrug resistance in cancer patients.

Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 180 - 6
Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry; Muller MR et al.; OBJECTIVE: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML) . METHODS: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP . The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells . Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux . The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/WT and they express different amounts of PGP . Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) . Cells were then incubated for 60 min with rho123 (10 microM) and analyzed for rhodamine and AMCA-derived fluorescence . The decrease in rho123 fluorescence was determined after a further period of 30 min . RESULTS: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line . PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method . PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML) . CONCLUSION: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells.

Acta Clin Belg, 2000 Jan-Feb, 55(1), 34 - 6
Multidrug-resistant tuberculosis spondylitis; Cherifi S et al.; We report a case of multidrug-resistant spinal tuberculosis complicated by epiduritis and paraspinal abscess in a 68-year-old black woman . Multidrug-resistant tuberculous spondylitis is still rare in Belgium . Two others cases were reported from 1992 to 1997 . The optimal therapy is not standardized and the mandatory duration of treatment is not known . Clinical presentation, radiological findings, and treatment are presented . The need for prompt diagnosis and optimal therapy is emphasized.

Curr Opin Pulm Med, 2000 May, 6(3), 193 - 7
Advances in the management of tuberculosis: clinical trials and beyond; Grange JM et al.; Modern short-course treatment for tuberculosis is highly effective and cost-effective, yet the disease remains a leading cause of suffering and death . The problem has been exacerbated in recent years by the human immunodeficiency virus (HIV) pandemic and the increasing prevalence of multidrug-resistant tuberculosis . Improvements in diagnosis, vaccination, chemoprophylaxis, and therapy are thus urgently needed . Molecular techniques are facilitating the development of rapid and sensitive diagnostic tests and the rational approach to the production of new vaccines . New forms of treatment are being investigated and there is also considerable emphasis on optimizing the deployment of the available treatment regimens . This has resulted in the World Health Organization's five-point directly observed therapy, short course (DOTS) strategy and proposed modifications (DOTS-plus) for the management of multidrug-resistant (MDR) tuberculosis . Despite these advances, it is becoming abundantly clear that the failure to control tuberculosis is a direct consequence of the gross inequities in the distribution of wealth and health care provision worldwide, which do not allow for putting advances in the management of tuberculosis into practice . The control of tuberculosis will therefore require attention to justice and human rights as well as greatly increased technical and financial support from the developed nations.

Curr Opin Pulm Med, 2000 May, 6(3), 174 - 9
Dynamics and control of the global tuberculosis epidemic; Bleed D et al.; Studies of disease burden have reaffirmed that tuberculosis is among the top 10 causes of death in the world . The tuberculosis epidemic in most countries could eventually be brought under control by implementing the World Health Organization's (WHO) directly observed therapy, short course (DOTS) strategy, although tuberculosis linked to human immunodeficiency virus (HIV) in Africa and multidrug-resistant tuberculosis (MDR-TB) in the former Soviet Union urgently demand adaptations and extensions of DOTS . Most high-incidence countries have achieved treatment success rates approaching the WHO 85% target in pilot projects . In the long term, we may have better diagnostics, drugs, and vaccines to control the disease; for the next 5 years, the central problem in global tuberculosis control is to expand DOTS coverage in high-incidence countries . Improved case finding and diagnosis, coupled with best-practice short-course chemotherapy, could quickly and dramatically cut the number of years of healthy life lost due to tuberculosis, especially by preventing death.

Leuk Res, 2000 Jun, 24(6), 535 - 41
In vitro leukemia cell models of Ara-C resistance; Funato T et al.; Cells of the human leukemia line K562 were continuously exposed to cytosine arabinoside (Ara-C) at increasing concentrations for 3 months . The resulting cell line, termed K562/AC, showed 48-fold resistance to Ara-C, compared with the parental K562 cells . The sensitivities of K562/AC to adriamycin (ADR), vincristine (VCR) and etoposide (VP16) were similar to those of parental K562 . Gene analysis revealed that this cell line lacked expression of the deoxycytidine kinase (dCK) gene, which was evident in Ara-C-sensitive cells . As in K562 cells, multidrug resistance (MDR-1) and multidrug resistance protein (MRP) genes were not expressed in K562/AC . We also established an in vitro model of Ara-C resistance using phosphorothioate antisense oligonucleotides to dCK (dCK-AS) . Treatment of K562 with dCK-AS caused decreased dCK expression and 6- to 10-fold increases in resistance to Ara-C, compared with that in cells treated with sense oligonucleotides to dCK (dCK-S) or in non-transfected cells . The cells described here may contribute to the study of a novel mechanism associated with Ara-C resistance, in which reduced dCK activity may play an important role.

J Biol Chem, 2000 Jul 7, 275(27), 20280 - 7
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1; Hou Y et al.; Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs) . Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required . In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A . E., al-Shawi, M . K., and Urbatsch, I . L . (1995) FEBS Lett 377, 285-289) . To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein . Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2 . Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping . Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1 . Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP . Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

Cancer Lett, 2000 May 29, 153(1-2), 95 - 100
Expression of mediated P-glycoprotein multidrug resistance related to Tc-99m MIBI scintimammography results; Sun SS et al.; We prospectively studied a total of 24 patients with breast cancer to evaluate the relationship between the degree of accumulation of technetium-99m sestamibi (Tc-99m MIBI) and p-glycoprotein (Pgp) expression in tumor tissues . All 24 patients underwent Tc-99m MIBI scintimammography before surgery or biopsy . Immunohistochemical studies were performed on multiple non-consecutive sections of the same tumor using a Pgp specific monoclonal antibody, JSB-1 . Planar images were started 10 min after injection of Tc-99m MIBI . Tumor to background (T/B) ratios calculated from the planar images were correlated with Pgp expression as determined by immunohistochemical studies . The T/B ratios were significantly lower for tumors in eight patients with positive Pgp expression (Group 1) than in 16 patients with negative expression (Group 2) (1.40+/-0.11 and 2.76+/-0.60, P<0 . 05) . Our data confirmed that Tc-99m MIBI scintimammography is useful for determination of the presence of multidrug resistance due to Pgp expression in patients with breast cancer.

Blood, 2000 May 1, 95(9), 2897 - 904
P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway; Pallis M et al.; P-glycoprotein (pgp), which is the product of the MDR1 (multidrug resistance-1) gene, has an established role as a mediator of cytotoxic drug resistance in acute myeloid leukemia (AML) . To study the role of pgp in mediating apoptosis resistance in AML cells deprived of serum and growth factors, apoptosis was quantified by flow cytometry using uptake of the dye 7-amino-actinomycin D (7-AAD) alongside low forward scatter . In pgp+ve primary AML samples, there was a significant increase in apoptosis in the presence of the pgp-specific antibody UIC2 (mean increase: 58%; range: 11%-95%; P < . 05) . Likewise, apoptosis in growth factor-deprived TF1 cells cultured for 30 hours increased 2.5-fold in the presence of 25 microg/mL UIC2 . The pgp reversal agent PSC-833 (1 micromol/L) augmented in vitro apoptosis by a median of 52% in pgp+ve patient samples and to a comparable degree in 6 pgp-ve samples . To determine whether the sphingomyelin-ceramide (SM-ceramide) pathway of apoptosis occurs in AML blasts in response to cytotoxic drugs, cells were incubated with daunorubicin at the patient-specific IC(30) (the concentration of daunorubicin that caused apoptotic cell death in 30% of cells) in the presence of the ceramide synthase inhibitor fumonisin B1, which inhibited apoptosis by 18%-81% (median: 40%) . Exogenous SM failed to augment apoptosis induced by growth factor withdrawal in pgp+ve TF1 cells and was significantly more effective at augmenting apoptosis in pgp-ve patient blasts (median increase in cell death: 33%; range: 19%-88%) than in pgp+ve samples (median: 7%; range: 0%-27%; P =.028) . Cellular accumulation of exogenous SM was associated with apoptosis and also occurred in nonapoptotic patient cells treated with PSC-833 . However, this effect was not seen following treatment with the UIC2 antibody . These results indicate that pgp is able to exert a protective effect on AML cell viability and that this is associated with a reduced effect of exogenous SM on apoptosis . The pgp reversal agent PSC-833 acts, at least in part, by a pgp independent mechanism to alter SM distribution and to augment apoptosis induced in AML cells by serum and growth factor withdrawal . (Blood . 2000;95:2897-2904)

J Assoc Physicians India, 1999 Sep, 47(9), 883 - 5
An open study to evaluate the efficacy of artemether in severe falciparum malaria; Sharma P et al.; An open clinical trial was conducted in 30 patients of severe falciparum malaria with heavy parasitaemia (parasitized erythrocytes above 5%) . Artemether (methyl ether of dihydroartemisinin-active principle isolated from Chinese plant Qinghaosu) was administered as 80 mg intramuscular injection twice on first day and then single dose of 80 mg intramuscular on 2nd to 5th day . The trial could be completed in 28 patients and two patients expired . In our observation falciparum malaria affected the young adults in their most productive period of life i.e . 25-44 yrs . All patients became afebrile by the 4th day with fever clearance time approximately 31.92 +/- 15.30 hr . Twenty-five patients (83.33%) became parasite free by 5th day with mean parasite clearance time approximately 47.04 +/- 19.95 hr . Deranged liver function and renal profile was observed in 63% and 50% patients respectively . Two patients, who died had very high degree of parasitaemia (50% and 16%) with cerebral malaria . One died due to multiorgan failure and other due to massive hematemesis and shock . The type of response achieved by artemether therapy was analysed as per WHO criteria suggested for chloroquine resistance . S response was observed in 25 patients (cure rate 83.33%) . Two patients (6.66%) patients showed R II response, one patient (3.33%) showed R III response and R I response was not observed in any patient . No significant side effects were noted . This pilot study demonstrated that intramuscular artemether is a useful addition to antimalarial drugs in this era of multidrug resistant P . falciparum malaria showing high clinical potency with virtually no side effect.

J Biol Chem, 2000 Apr 28, 275(17), 13098 - 108
Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1; Gao M et al.; Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics . The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates . It is not known whether the two NBDs of MRP1 are functionally equivalent . To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-{(32)P}ATP and to trap 8-azido-{(32)P}ADP in the presence of orthovanadate . We found that NBD1 was preferentially labeled with 8-azido-{(32)P}ATP, while trapping of 8-azido-{(32)P}ADP occurred predominantly at NBD2 . Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide . Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2 . Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70% . Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-{(32)P}ATP.

J Virol, 2000 May, 74(10), 4621 - 33
Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain; Sei S et al.; Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed . Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2) . A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly . Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype . This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.

Joint Bone Spine, 2000 Jan, 67(1), 40 - 8
Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders . Part II: Increased P-glycoprotein activity in lymphocytes from systemic lupus erythematosus patients might affect steroid requirements for disease control; Diaz-Borjon A et al.; BACKGROUND: Over-expression of the membrane glycoprotein called P-glycoprotein has been widely observed in a variety of both normal and neoplastic cells . P-glycoprotein is a pump molecule that transports hydrophobic drugs (including steroids) and toxins outside the cells, thus inhibiting their therapeutic or toxic effects . The gene encoding P-glycoprotein is named multidrug resistance-1 (MDR-1) . OBJECTIVE: To evaluate the functional activity of P-glycoprotein in lymphocytes and monocytes from patients with systemic lupus erythematosus . METHODS: 30 systemic lupus erythematosus patients and 20 healthy controls were studied . Peripheral blood mononuclear cells isolated by gradient centrifugation were incubated in the presence of daunorubicin (a fluorescent drug extruded by P-glycoprotein) at 37 degrees C or 4 degrees C for 30 min . P-glycoprotein activity was then analyzed using flow cytometry . Results were expressed as the percentage of lymphocytes or monocytes with high P-glycoprotein activity (i.e., low fluorescence) . RESULTS: Mean fluorescence values for lymphocytes and monocytes were comparable between patients and healthy controls . However, because our method allowed to measure P-glycoprotein function at the single-cell level, we were able to show that the mean percentage of lymphocytes with high P-glycoprotein activity was increased in the patients (11.51% +/- 14.3%) as compared to the healthy controls (0.71% +/- 0.57%) (P < 0.05) . Moreover, P-glycoprotein activity was lower in the patients in clinical remission than in those with active disease . CONCLUSIONS: Our results suggest that P-glycoprotein function might affect glucocorticoid requirements in systemic lupus erythematosus.

Joint Bone Spine, 2000 Jan, 67(1), 30 - 9
Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders . Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid arthritis patients might influence disease outcome; Llorente L et al.; BACKGROUND: Multidrug resistance (MDR) is characterized by overexpression of P-glycoprotein, a pump molecule that decreases intracellular drug concentrations by increasing drug efflux from cells . OBJECTIVE: To look for correlations between clinical status and P-glycoprotein activity and/or TNF-alpha mRNA levels in patients with rheumatoid arthritis . METHODS: Sixteen patients were studied . Based on response to therapy, eight were refractory and eight nonrefractory to treatment . Findings were compared to those in 24 healthy controls . Flow cytometry was used to evaluate P-glycoprotein activity in peripheral blood mononuclear cells isolated by gradient centrifugation and incubated with the P-glycoprotein substrate daunorubicin . TNF-alpha mRNA levels were determined using quantitative PCR . RESULTS: Patients with rheumatoid arthritis showed an increased number of lymphocytes with high P-glycoprotein activity (p = 0.0001) as compared to the normal controls . P-glycoprotein activity was higher in the refractory than in the non-refractory patient subgroup (p = 0.006) . Also, TNF-alpha mRNA levels were markedly higher in the refractory subgroup than in the nonrefractory subgroup, and were undetectable in the normal controls . CONCLUSIONS: Enhanced P-glycoprotein activity may be closely related to an unfavorable clinical course and a poor response to treatment . Increased TNF-alpha expression and chronic exposure to various drugs, including glucocorticoids, may contribute to increase P-glycoprotein activity . Both high P-glycoprotein activity and excessive amounts of TNF-alpha seem associated with poor outcome in rheumatoid arthritis.

Nucl Med Biol, 2000 Feb, 27(2), 135 - 41
Assessment of the in vitro and in vivo properties of a (99m)Tc-labeled inhibitor of the multidrug resistant gene product P-glycoprotein; Bergmann R et al.; Overexpression of P-glycoprotein (Pgp), which is present in the plasma membrane of various tumor cells and in several normal cell types, contributes to the multidrug resistance (MDR) phenotype of many human cancers . As a prerequisite for therapy, the expression of Pgp must be studied . The available clinical radiopharmaceuticals for studying the expression of Pgp include the lipophilic (99m)Tc cations (sestamibi, tetrofosmin) as well as {(99m)Tc}Q57, {(99m)Tc}Q58, and {(99m)Tc}Q63 . Here we describe the in vitro and in vivo properties of the structurally different complex (3-thiapentane-1, 5-dithiolato){{N-(3-phenylpropyl)-N-2(3-quinazoline-2, 4-dionyl)-ethyl}amino-ethylthiolato inverted question mark oxotechnetium(V) ((99/99m)Tc1) as a potential inhibitor of Pgp . (99)Tc1 enhances the net cell accumulation of Pgp substrates {(3)H}vinblastine, {(3)H}vincristine, {(3)H}colchicine, {(99m)Tc}sestamibi, and {(99m)Tc}tetrofosmin in rat brain endothelial cells (RBE4), an immortalized endothelial cell line that expresses Pgp . In addition, the cell accumulation of (99m)Tc1 could be increased by verapamil and reserpine, which are known Pgp inhibitors . A multitracer approach was used to study the side effects of (99)Tc1 on cell metabolism . The cells were simultaneously incubated with {(99m)Tc}sestamibi, 2-{(18)F}fluoro-2-deoxyglucose ({(18)F}FDG), and various (3)H-labeled tracers . Two-dimensional scatter plots of {(99m)Tc}sestamibi uptake/{(18)F}FDG uptake show typical changes of known Pgp inhibitors including (99)Tc1 . The effects of (99)Tc1 on the in vivo distribution of {(99m)Tc}sestamibi and {(18)F}FDG in rats also are comparable with the effects of verapamil, an established Pgp inhibitor and calcium channel blocker . We conclude that (99/99m)Tc1 is a transport substrate and a potential inhibitor of Pgp . Our approach may be useful in the design of further radiotracers with specificity to Pgp.

Int J Antimicrob Agents, 2000 Apr, 14(3), 193 - 201
Chemistry and biological activity of new 3-benzazepines; Kawase M et al.; This review summarizes our experiments investigating structure-activity relationships of 3-benzazepines . Three 7, 8-dihydroxy-3-benzazepines {7-9} were cytotoxic to human promyelotic leukaemia HL-60 cells . Compound {9} showed the highest cytotoxicity and the activity was twice as high as that of dopamine (DA, {11}) . Three active compounds {7-9} produced radicals, whereas other less potent benzazepines {1-6, 10} did not produce radicals . Furthermore, cytotoxic 3-benzazepines {7-9} also enhanced the decay of ascorbic acid in rat brain homogenate . Two 7,8-dimethoxy-3-benzazepines {5, 10} were able to form a complex with the replicative form of plasmid DNA . The multidrug resistance (MDR) P-glycoprotein (Pgp) efflux pump of mouse lymphoma cells was inhibited by three compounds {5, 8, 10} . Compound {8} has the highest activity in MDR reversal and is two times more potent than verapamil . Three cytotoxic 3-benzazepines {7-9} showed inhibitory effects against reverse transcriptase (RT) of Moloney leukemia.

Int J Antimicrob Agents, 2000 Apr, 14(3), 173 - 6
Phenothiazines: an alternative to conventional therapy for the initial management of suspected multidrug resistant tuberculosis . A call for studies; Amaral L et al.; Increased frequency of multidrug resistant strains of Mycobacterium tuberculosis results from inappropriate treatment and lack of patient compliance . The Center for Disease Control/American Thoracic Society (CDC/ATS) guidelines issued for the management of newly diagnosed cases of tuberculosis (TB) will not be totally effective regardless of adherence to the guidelines and patient cooperation . The long interim period between the diagnosis of TB and confirmation of antibiotic susceptibility contributes to the infection rate . Consequently, the use of an adjuvant that is known to inhibit all encountered multidrug resistant strains of M . tuberculosis may be helpful until antibiotic susceptibility is known . Phenothiazines such as chlorpromazine, methdilazine and thioridazine are effective against strains of M . tuberculosis in vitro and in vivo . It is recommended that studies be designed and conducted for the purpose of managing new cases of TB that emanate from areas known to harbour multidrug resistant strains of M . tuberculosis, with phenothiazines as adjuvants to the regimen recommended by the CDC/ATS guidelines until antibiotic susceptibility is defined . Because the normal maximum period for obtaining conventional antibiotic susceptibility results is less than 7 or 8 weeks, the probability of serious side effects from the use of a phenothiazine is remote.

J Pharmacol Exp Ther, 2000 May, 293(2), 530 - 8
Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1); Loe DW et al.; Multidrug resistance in tumor cells is often associated with reduced drug accumulation resulting from increased expression of the 190-kDa multidrug resistance protein 1 (MRP1) or the 170-kDa P-glycoprotein . However, unlike P-glycoprotein, MRP1 is a primary active transporter of many conjugated organic anions, including the cysteinyl leukotriene LTC(4) . Moreover, agents such as verapamil that reverse P-glycoprotein-mediated resistance are often poorly, or not at all, effective in MRP1-overexpressing cells . In the present study, we investigated the effects of verapamil on MRP1-mediated transport processes . We found that verapamil inhibited LTC(4) transport into inside-out membrane vesicles prepared from MRP1-transfected cells in a competitive manner, but only in the presence of reduced glutathione (GSH) or its nonreducing S-methyl derivative . In the presence of 1 mM GSH, the apparent K(i) for verapamil was 1.2 microM, and in the presence of 100 microM verapamil, the apparent K(i) for GSH was 77 microM . Verapamil itself was not transported by MRP1 in either intact cells or membrane vesicles . However, verapamil strongly stimulated MRP1-mediated GSH uptake by membrane vesicles in a concentration-dependent and osmotically sensitive manner that was inhibitable by MRP1-specific monoclonal antibodies . In the presence of 100 microM verapamil, the apparent K(m) and V(max) for GSH uptake were 83 microM and 55 pmol mg(-1) min(-1), respectively . It is proposed that the variable ability of verapamil to modulate MRP1-mediated resistance in different cell lines may be more closely linked to its effect on the GSH status of the cells than on its ability to inhibit the MRP1 transporter itself.

Drug Metab Dispos, 2000 May, 28(5), 522 - 8
In vitro flow cytometry method to quantitatively assess inhibitors of P-glycoprotein; Wang EJ et al.; P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs . A simple and reliable in vitro method to identify inhibitors of Pgp helps to prevent the potential of drug interactions . Using daunorubicin as a fluorescent marker and vanadate as a positive control compound, a functional flow cytometry method for assessing the ability of a drug to inhibit Pgp-mediated drug efflux from CR1R12 multidrug-resistant cells has been evaluated . Quantitation of the relative fluorescence was used to compare potency of individual inhibitors . Known Pgp inhibitors, such as cyclosporin A, nicardipine, verapamil, quinidine, terfenadine, tamoxifen, and vinblastine were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin . Cyclosporin A and terfenadine were the most potent inhibitors among the compounds tested . Tetraphenylphosphonium and alpha-tocopherol had little inhibitory effect . Progesterone produced significant inhibition at relatively high concentrations . This study demonstrated that this in vitro flow cytometry method is a simple, sensitive, and quantitative tool to assess the capacity of a drug to inhibit Pgp transporters, and is useful for screening or identifying inhibitors of Pgp as well as evaluation of potential for drug interactions.

Southeast Asian J Trop Med Public Health, 1998 Dec, 29(4), 696 - 701
A clinical trial of combination of artesunate and mefloquine in the treatment of acute uncomplicated falciparum malaria: a short and practical regimen; Wilairatana P et al.; The difficulties in treating drug-resistant falciparum malaria in Thailand are compounded by the necessity of giving antimalarials over long periods of time . The resultant fall in patient compliance not only lowers cure rates but also predisposes to the further spread of drug-resistance . Sequential treatment with artesunate given over 5 days followed by mefloquine produced 100% cure rates in previous study, but might not be a suitable regimen for field treatment . We conducted a clinical trial of a combination of artesunate and mefloquine given twice daily for 2 days in 150 patients with acute uncomplicated falciparum malaria . The dose of artesunate (200 mg) and mefloquine (312.5 mg) were given simultaneously in a separate package . All patients were admitted to a hospital in Bangkok for 28 days to exclude re-infection and monitor the possible adverse effects . One hundred and thirty patients completed the study with 28 days follow up . Twenty patients (13%) left the hospital prior to completion of follow-up for reasons unrelated to their treatment . Cure rate was 97% (126/130) . There were no RII or RIII failures and all four patients with treatment failures were of the RI type . The mean parasite clearance time and fever clearance time were 46.4 and 42.5 hours, respectively . All patients were tolerated the combination drugs well and there were no serious toxic adverse reactions . The results indicate that combination of artesunate and mefloquine given twice daily for 2 days is effective and well tolerated in patients with acute, uncomplicated falciparum malaria and suitable as an alternative treatment for multidrug resistant falciparum malaria.

Southeast Asian J Trop Med Public Health, 1998 Dec, 29(4), 669 - 84
Application of geographical information systems to co-analysis of disease and economic resources: dengue and malaria in Thailand; Indaratna K et al.; Two vector-borne communicable diseases, malaria and dengue, are among a number of diseases of particular importance in relation to economic development in Southeast Asia and thus need to be assessed in relation to economic parameters in the region . Geographical Information Systems (GIS) provide one means of comparing disease and resource data versus time and place, to facilitate rapid visualization by planners and administrators . Given that Thailand is a global epicenter of multidrug resistant falciparum malaria and of dengue hemorrhagic fever, both of which are mosquito-borne, application of GIS methods to these two diseases gives opportunity for comparison of resource needs and allocation in relation to disease epidemiologic patterns . This study examined per capita gross provincial product (GPPpc) and health care resources in relation to geographic distribution of malaria and dengue in Thailand . The two diseases vary greatly in overall seasonal patterns and in relation to provincial economic status, and present differing demands on resource utilization: planned integration of control of malaria and dengue could utilize such analyses in relation to resource sharing and consideration of allocative efficiency . The concentration of malaria (and to a lesser extent dengue) along international border areas underscores the desirability of multi-country coordination of disease management and control programs . Because socio-economic and disease data are collected by quite different means and in different time frames, there are some limitations to the dynamic interpolation of these two broad data sets, but useful inferences can be drawn from this approach for application to overall planning, at both national and multi-country levels.

Antimicrob Agents Chemother, 2000 May, 44(5), 1328 - 32
Susceptibility to PNU-140690 (Tipranavir) of human immunodeficiency virus type 1 isolates derived from patients with multidrug resistance to other protease inhibitors; Rusconi S et al.; In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs) . The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690 . We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC(50)) and IC(90), PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products . Phenotypic resistance to PIs was invariably related to genotypic mutations . The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M . Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC(50)s, >0.1 microM) . We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration . The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T . Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC(90) of 0.18 +/- 0.02 microM in multiple experiments . The average mean +/- standard error of mean IC(90) for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 +/- 0.055 microM (range, 0.31 to 0.86 microM) . Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies.

Anticancer Res, 2000 Jan-Feb, 20(1A), 373 - 7
3,5-diacetyl-1,4-dihydropyridines: synthesis and MDR reversal in tumor cells; Shah A et al.; Eleven 4-phenyl-3,5-diacetyl-1,4-dihydropyridines (AcDHPs) {G1-11} substituted at the phenyl ring were synthesized and compared for their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems . Among them, compound {G7} showed the highest cytotoxic activity against human promyelocytic leukemia HL-60 and human squamous cell carcinoma HSC-2 cells . However, no compounds tested produced radicals at pH 7.4-12.5 . The activity of P-glycoprotein (Pgp) responsible for MDR in tumor cells was reduced by compounds {G2, 3, 6, 5, 8, 1, 11}, verapamil {VP} and nifedipine {NP} . However, compounds {G4, 7, 10} were hardly active while G9 did not show a MDR reversing effect at 2.0-20.0 micrograms/mL . These data show a relationship between chemical structures and MDR-reversing effect on tumor cells.

Anticancer Res, 2000 Jan-Feb, 20(1A), 139 - 50
Cytosine arabinoside (ara-C) resistance confers cross-resistance or collateral sensitivity to other classes of anti-leukemic drugs; Martin-Aragon S et al.; The major limitation of treatment with antimetabolite drugs is that they produce resistant clones both in vitro and in patients who either do not respond to treatment or relapse soon after response has been documented . To better understand the phenomenon of cross-resistance, we developed seven CEM/ara-C-resistant leukemic clones from the CEM/0 (wt) cell line . These clones ranged from 4- to 3.5 x 10(8)-fold more resistant to ara-C than the wt CEM/0 cell line . Using this model, we determined IC50 concentrations to several chemotherapeutic agents and gamma radiation, and we also studied pro- (p53) and anti-apoptotic (bcl-2) proteins, as well as P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) . The cell viability assays showed that these clones were cross-resistant to 6-thioguanine (6-TG) or 6-mercaptoguanosine (6-TGuo) from 1.1- to 8.8-fold with ara-C; cross-resistance to vincristine (VCR) was from 200- to 1 x 10(4)-fold with ara-C . Taxotere (TXR) showed cross-resistance with ara-C from 1.39- to 3.03 x 10(3)-fold; dexamethasone (DEX) also showed a significant degree of cross-resistance from 27.4- to 3.87 x 10(7)-fold . Gamma radiation treatments from 0.77 Gy to 12.3 Gy showed a radiation dose-dependent cross-resistance with ara-C from 1.43- to 2.93-fold . Idarubicin was collaterally sensitive with ara-C from 4.6- to 1 x 10(9)-fold in these cell lines . The CEM/ara-C/G resistant cell line was 3-fold more sensitive to 6-TG or VCR than CEM/0 (wt), and 5-fold more sensitive to 6-TGuo . This cell clone expressed p53 and did not overexpress bcl-2 protein . All of the cell lines studied, CEM/0 (wt) and the ara-C resistant clones, showed functional p53 protein . The cell treatment with 0.1, 1 and 10 microM ara-C for 48 hours showed increased p53 protein expression in most of these lines . No increase in bcl-2 protein expression was seen in the wt cell line after ara-C treatment for 48 hours . Three cell lines resistant to ara-C (CEM/ara-C/B, CEM/ara-C/D and CEM/ara-C/I) showed an important increased expression of bcl-2 protein after treatment with 1 microM ara-C, but not after 10 microM . This alteration may lead to resistance to apoptosis and enhanced cell survival . The ratio of bcl-2 to p53 was increased significantly in these three clones, thus favoring an anti-apoptotic drive . All of the cell lines examined were negative for MRP expression and only two, CEM/ara-C/B and CEM/ara-C/J, were positive for MRP functional activity . However, three ara-C resistant cell clones, CEM/ara-C/7A, CEM/ara-C/B and CEM/ara-C/G, were positive for P-gp expression and functional activity . It is apparent that selection for ara-C resistance confers cross-resistance to many other classes of drugs and gamma radiation, probably due to bcl-2 protein overexpression or P-gp and MRP expression, as independent mechanisms.

Lancet . 2000 Apr 8;355(9211):1246.
Warsaw conference on emerging infections in central and eastern Europe; Balinska MA; PIP: On March 28-29, 2000, epidemiologists and microbiologists convened in Warsaw, Poland, to discuss emerging, re-emerging, and drug-resistant infections in central and eastern Europe . Delegates were from the Czech Republic, Greece, Hungary, Latvia, Poland, Romania, Russia, and Yugoslavia, with the exception of the US and the UK and other worst affected countries in central-eastern Europe . It has been documented that the cause of diphtheria epidemics in several countries in the mid-1990s resulted from the breakdown of vaccination campaigns following social dislocation . Currently, diphtheria morbidity has declined through targeted vaccination programs, although fundamental socioeconomic problems continue to threaten public health . Other infectious diseases raised during the conference were the evolution of tuberculosis and HIV/AIDS, which remains largely unpredictable . In addition, the growing incidence of multidrug-resistant tuberculosis is documented in several countries . Among the major problems in tuberculosis control include late diagnosis, nonexistent or erratic drug supplies and unreliable reporting . The syringe ecosystem in both healthcare settings and injecting drug users were singled out as the vectors of sharp increase in HIV/AIDS infection . Throughout the conference, the recurrent theme was on the overriding importance of providing specialized training and the building up of networks of public health specialists .

Minerva Ginecol, 1999 Dec, 51(12), 463 - 70
{Expression and prognostic value of the drug resistance markers P-gp, Mrp1, Mrp2, and Lrp in ovarian carcinoma}; Katsaros D et al.; BACKGROUND: Intrinsic and/or acquired chemoresistance is the major obstacle to overcome in the treatment of patients with ovarian carcinoma . The aim of the present study was to investigate the prognostic value of drug resistance associated proteins P-glycoprotein (P-gp), multidrug resistance related protein (Mrp1), canalicular multispecific organic anion trans-porter (c-MOAT or Mrp2) and lung resistance protein (Lrp) in ovarian carcinoma . METHODS: Expression of P-gp, Mrp1, Mrp2 and Lrp was determined by immunohistochemistry of frozen tissue sections of 115 ovarian carcinoma patients and associated to clinico-pathological factors, response to chemotherapy and (progression free) survival . RESULTS: Expression of P-gp was observed in 20 out of 115 (17%), Mrp1 in 51 out of 115 (44%), Mrp2 in 19 out of 115 (16%) and Lrp in 85 out 115 (74%) tumors . Expression of Mrp1 was related to Mrp2 (p < 0.0001) and P-gp (p < 0.001) expression, while Lrp expression was more frequently observed in patients with stage I/II versus stage III/IV tumors (p < 0.01), grade I/II versus III tumors (p < 0.05) and residual tumor < 2 cm versus > 2 cm after laparotomy (p < 0.05) . Lower stage (p < 0.001), small residual tumor after first laparotomy (p < 0.001) and lower differentiation grade (p < 0.05) were related to longer (progression free) survival . P-gp, Mrp1, Mrp2, and Lrp expression was neither related to response to first line chemotherapy (59 evaluable patients) nor to (progression free) survival (all patients) . On multivariate analysis only stage and residual tumor after first laparotomy were independent prognostic factors for (progression free) survival . CONCLUSIONS: In ovarian carcinoma Mrp1 expression is associated with Mrp2 and P-gp expression, while Lrp expression is associated with favorable clinicopathological characteristics . Assessment of P-gp, Mrp1, Mrp2 or Lrp does not allow prediction of response to chemotherapy or (progression free) survival in ovarian carcinoma.

Cancer Gene Ther, 2000 Mar, 7(3), 466 - 75
A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1; Stuart DD et al.; The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo . Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung . We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle . The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN . The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice . The majority of the asODN was cleared from blood, with a half-life of >10 hours compared with <1 hour for free asODN . When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein . No inhibition was found for nontargeted formulations or for free asODN . A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN.

Toxicol Appl Pharmacol, 2000 Apr 15, 164(2), 134 - 42
Low-level doxorubicin resistance in benzo{a}pyrene-treated KB-3-1 cells is associated with increased LRP expression and altered subcellular drug distribution; Cheng SH et al.; The P-glycoprotein (P-gp)-negative epidermoid pharyngeal carcinoma cells KB-3-1 were grown in 0.25 mM benzo{a}pyrene (BaP) for 3 months and increased resistance to doxorubicin, but not to vinblastine, colchicine, or cisplatin, was found . Doxorubicin resistance was not altered by cyclosporin, the P-gp inhibitor . Intracellular accumulation of BaP or calcein, a substrate for P-gp and multidrug resistance protein (MRP), was not altered by inhibitors of the P-gp and MRP . The expression of cytochrome P450 (CYP) 1A1, lung-resistance-related protein (LRP), P-gp, and MRP was investigated . Overexpression of CYP1A and LRP, on the mRNA and protein levels, was found . BaP-treated KB-3-1 cells remained P-gp negative while the level of MRP was not altered . Subcellular accumulation of BaP was found to be localized in the cytoplasm and minimal in the nuclei in BaP treated cells . In contrast, even penetration of BaP to the nuclei and cytoplasm was found in untreated cells . Subcellular distribution of doxorubicin was altered following BaP treatment with localized accumulation of the cancer drug in cytoplasmic organelles but not in the nuclei . Our data suggested that LRP might play a protective role against toxic compounds . The correlation of increased expression of LRP, but not P-gp nor MRP, with decreased doxorubicin accumulation in the nuclear target suggests a pivotal role of this perinuclear transporter in the MDR phenotype of P-gp-negative cancer cells . These results also propose an alternative mechanism of cancer drug resistance emergence, namely, induction of LRP activity following treatment with BaP, an environmental toxicant and a carcinogen .

Urol Oncol, 2000 Apr 1, 5(3), 118 - 121
Expression of the multidrug resistance gene in human prostate cancer; Bhangal G et al.; Prostate cancer has become the most common cancer in males and the second most common cause of male cancer death in England and Wales . Death rates have doubled over the last 20 years . Prostate cancer is characterized by a high initial response rate to hormonal therapy . Drug-resistance is a significant cause of relapse in cancer . The multidrug resistance genes (MDR) encode resistance to a diverse family of cytotoxic chemotherapy agents . There are four known MDR genes, two of which are present in humans . MDR1 encodes for P-glycoprotein, a 170-kDa transmembrane calcium-dependent efflux pump . We examined P-glycoprotein expression by immunocytochemistry in 96 patients with prostate cancer and 20 patients with benign prostatic hypertrophy . A direct correlate was found between tumor grade, stage, and prostate specific antigen levels, indicating the possible significance of this protein in recurrent prostate cancer.

Eur J Cancer, 2000 Apr, 36(6), 803 - 9
Antitumour activity of suramin analogues in human tumour cell lines and primary cultures of tumour cells from patients; Dhar S et al.; Suramin has shown promising antitumour activity against several tumour types, both in vitro and in vivo, but the clinical utility of this compound is hampered by its unfavourable toxicity profile . In the present study, the semi-automated fluorometric microculture cytotoxicity assay (FMCA) was employed for evaluation of the cytotoxicity of seven suramin analogues in vitro in a panel of human tumour cell lines and in primary cultures of tumour cells from patients . Like suramin, the analogues showed little sensitivity to resistance mechanisms involving P-glycoprotein, topoisomerase II, multidrug resistance associated protein and glutathione-mediated drug resistance . In the cell line panel, NF067 and FCE 26644 showed activity comparable with suramin . All analogues were less potent than suramin in patient cells except for FCE 26644 . Correlation to suramin activity patterns in the cell line panel was highest for NF037 and low to moderate for the remaining analogues . In patient cells, high correlation coefficients were obtained for FCE 26644, NF110, NF031 and NF037 . The results indicate that the cytotoxic activity of suramin on patient tumour cells is shared by the analogues with FCE 26644 being the most active . The pharmacophore for cytotoxicity in patient cells may be different from that observed in the cell lines.

Int J Oncol, 2000 May, 16(5), 959 - 69
Overcoming of multidrug resistance by introducing the apoptosis gene, bcl-Xs, into MRP-overexpressing drug resistant cells; Ohi Y et al.; Multidrug resistance associated protein (MRP) is one of drug transport membranes that confer multidrug resistance in cancer cells . Multidrug resistance has been known to be associated with resistance to apoptosis . In this study, using MRP overexpressing multidrug resistant nasopharyngeal cancer cells, we examined the expression of apoptosis related genes including p53, p21WAF1, bax and bcl-Xs between drug sensitive KB and its resistant KB/7D cells . We also examined whether the introduction of apoptosis related gene could increase the sensitivity to anticancer drugs in association with apoptotic cell death . The relative resistances to anticancer drugs in KB/7D cells evaluated by IC50 values were 3.6, 61.3, 10.4 and 10.5 to adriamycin (ADM), etoposide (VP-16), vincristine (VCR) and vindesine (VDS), respectively . The resistance to anticancer drugs in KB/7D cells was associated with the attenuation of internucleosomal DNA ladder formation in apoptosis . Of important, the mRNA expression of bcl-Xs gene in KB/7D cells was decreased in one-fourth as compared to that of KB cells among the apoptosis genes . The mRNA expression of bcl-Xs gene in a bcl-Xs transfected clone (KB/7Dbcl-Xs) was increased about 2-fold compared to that of KB/7Dneo cells, while the mRNA expression of MRP gene was not significantly different in KB/7bcl-Xs and KB/7Dneo cells . The sensitivities to anticancer drugs including ADM, VCR and VDS except VP-16 were increased in KB/7Dbcl-Xs cells, in turn, the relative resistance in KB/7Dbcl-Xs cells was decreased to 1.4, 4.0, and 3.0 in ADM, VCR and VDS, respectively, as compared to those of KB/7Dneo cells . Of interest, the studies on the accumulation of {3H}VCR showed that the decrease of {3H}VCR accumulation in KB/7Dbcl-Xs was not significantly different from that of KB/7Dneo cells . Collectively, these results indicated that the mechanism(s) of drug resistance in KB/7D cells could be explained at least by two factors: a) reduced drug accumulation mediated by MRP; b) resistance to apoptosis due to the decreased expression of the bcl-Xs gene . These results also indicated that the multidrug resistance mediated by MRP might be partially overcome by introducing apoptosis gene into drug resistant cells without modulation of MRP function in drug accumulation.

Am J Physiol Gastrointest Liver Physiol, 2000 Apr, 278(4), G522 - 31
MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells; Cantz T et al.; The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells . Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted . Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane . Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR . Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein . In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump . We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells . Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.

J Infect Dis, 2000 Apr, 181(4), 1421 - 7 Epub 2000 Apr 13.
Multiple drug-resistant Chlamydia trachomatis associated with clinical treatment failure; Somani J et al.; In vitro susceptibility testing and genotyping were done on urogenital isolates of Chlamydia trachomatis from 3 patients, 2 of whom showed evidence of clinical treatment failure with azithromycin and one of whom was the wife of a patient . All 3 isolates demonstrated multidrug resistance to doxycycline, azithromycin, and ofloxacin at concentrations >4.0 microg/mL . Recurrent disease due to relapsing infection with the same resistant isolate was documented on the basis of identical genotypes of both organisms . This first report of clinically significant multidrug-resistant C . trachomatis causing relapsing or persistent infection may portend an emerging problem to clinicians and public health officials.

Cancer Control, 1996 Oct, 3(5), 428 - 433
Gene Therapy of Bladder Cancer; Seigne JD; BACKGROUND: The incidence of bladder cancer has been steadily increasing and, despite improvements in treatment, many patients will not survive five years . Gene therapy is a promising approach that may improve the management of this disease . METHODS: Gene therapy involves two steps: (1) selection of an appropriate gene and (2) transfer of that gene to the target cells . RESULTS: Five broad categories of cancer gene therapy are undergoing clinical evaluation: (1) immunological augmentation by cytokine or (2) foreign gene transfer into tumor cells, (3) insertion of a suicide gene into tumor cells, (4) tumor suppressor gene reconstitution, and (5) bone marrow protection by insertion of a multidrug resistance gene . CONCLUSIONS: Although many gene transfer approaches have great intuitive appeal, several constraints, including problems with methods of gene transfer, must be overcome before this biologic approach can reach its potential.

Am J Trop Med Hyg, 2000 Jan, 62(1), 82 - 5
Antibiotics for prophylaxis of Plasmodium falciparum infections: in vitro activity of doxycycline against Senegalese isolates; Pradines B et al.; The in vitro activities of doxycycline, chloroquine, quinine, amodiaquine, artemether, pyrimethamine, and cycloguanil were evaluated against Plasmodium falciparum isolates from Senegal (Dielmo and Ndiop), using an isotopic, micro, drug susceptibility test . The 71-50% inhibitory concentration (IC50) values for doxycycline ranged from 0.7 to 108.0 microM and the geometric mean IC50 for the 71 isolates was 11.3 microM (95% confidence interval = 9.5-13.4 microM) . The activity of doxycycline did not differ significantly (P = 0.0858) between the chloroquine-susceptible isolates and the chloroquine-resistant isolates . There was no in vitro correlation between the responses to doxycycline and those to artemether, chloroquine, quinine, amodiaquine, pyrimethamine, and cycloguanil, suggesting no in vitro cross-resistance among these drugs . Potency was increased by prolonged exposure . In 96-hr incubations, the activity of doxycycline was 4-5-fold more increased than in 48-hr incubations . The in vitro activity of doxycycline against intraerythrocytic stages of multidrug-resistant P . falciparum, its action against the preerythrocytic forms, the lack of correlation between the responses in vitro of P . falciparum to doxycycline and the other antimalarial drugs, and its original potential site of action are factors that favor its use as antimalarial drug.

Am J Trop Med Hyg, 2000 Jan, 62(1), 65 - 9
A case-control auditory evaluation of patients treated with artemisinin derivatives for multidrug-resistant Plasmodium falciparum malaria; Van Vugt M et al.; The artemisinin derivatives are now used widely in areas with multidrug-resistant Plasmodium falciparum malaria such as Southeast Asia, but concerns remain over their potential for neurotoxicity . Mice, rats, dogs, and monkeys treated with high doses of intramuscular artemether or arteether develop an unusual pattern of focal damage to brain stem nuclei (particularly those involved in auditory processing) . To investigate whether a similar toxic effect occurs in patients treated with these compounds, clinical neurologic evaluation, audiometry and early latency auditory evoked responses were measured in a single-blind comparison of 79 patients who had been treated with > or =2 courses of oral artemether or artesunate within the previous 3 years, and 79 age- and sex-matched controls living in a malaria-endemic area on the northwestern border of Thailand . There were no consistent differences in any of these test results between the cases and controls . This study failed to detect any evidence of significant neurotoxicity in patients treated previously with oral artemether or artesunate for acute malaria.

Jpn J Cancer Res, 2000 Feb, 91(2), 248 - 54
The novel anticancer drug KRN5500 interacts with, but is hardly transported by, human P-glycoprotein; Takara K et al.; The interaction of the novel anticancer drug KRN5500, a spicamycin derivative, with human P-glycoprotein (P-gp) was analyzed from the viewpoint of cellular pharmacokinetics, i.e . by means of {3H}azidopine photoaffinity labeling, cellular accumulation and transcellular transport experiments . In this study, P-gp-overexpressing LLC-GA5-COL150 cells, porcine kidney epithelial LLC-PK1 cells transformed with human MDR1 cDNA, were used, since this cell line constructs monolayers with tight junctions, and would provide sufficient information for analyzing the cellular pharmacokinetics . 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the growth-inhibitory effect of KRN5500 in LLC-GA5-COL150 cells was comparable to that in LLC-PK1 cells (IC50 = 79.4 and 72.7 nM, respectively), but the inhibition of {3H}azidopine binding by KRN5500 was concentration-dependent in the membrane fraction of LLC-GA5-COL150 cells . The cellular accumulation of {14C}KRN5500 after its basal application in LLC-GA5-COL150 cells was slightly lower than that in LLC-PK1 cells, and was restored by the multidrug resistance (MDR) modulator SDZ PSC 833 . The basal-to-apical transport of {14C}KRN5500 in LLC-GA5-COL150 cells was also slightly higher than that in LLC-PK1 cells, and was inhibited by SDZ PSC 833 . However, the basal-to-apical transport of {14C}KRN5500 in LLC-GA5-COL150 cells was only a little higher than the apical-to-basal transport . Consequently, these results demonstrated that KRN5500 interacted with, but was hardly transported via, P-gp . These observations suggested that KRN5500 may be useful even for the treatment of tumors exhibiting P-gp-mediated MDR.

Cent Eur J Public Health, 2000 Feb, 8(1), 24 - 7
The first occurrence of a multi-drug resistant tuberculosis epidemic in the Czech Republic caused by genetically closely related Mycobacterium tuberculosis strains; Kubin M et al.; DNA fingerprinting based on the detection of the insertion sequence IS6110 in Pvull restriction fragments was applied to M . tuberculosis isolates originating in the first microepidemic of multidrug resistant tuberculosis recorded in the Czech Republic . Their disseminators were 21 individuals living in--or roaming between three distant areas . The age of 17 males ranged from 36 to 64 years (average 45 years) and of 4 females aged from 38 to 52 years . The index person was most probably a former male prisoner, aged 49 years, who disseminated multidrug resistant M . tuberculosis over a period of 28 months . In ten of the patients the following risk factors for tuberculosis were found: imprisonment, homelessness, immigration and previous stay in asylum--or in a psychiatric ward . In six cases, M . kansasii infection preceded tuberculosis . Four out of the 21 patients died . The RFLP analysis separated the patients into two distinct groups: group A comprising 14 members of which M . tuberculosis strains were isolated with six IS6110 copies, whereas the isolates of seven individuals of the group B, the RFLP profile displayed highly similar RFLP patterns compared to the isolates of group A, but with two additional IS6110 copies . In one patient, both A and B patterns were found: the first one in a M . tuberculosis strain isolated in 1993 and the second one in the isolate isolated two years later . Both the appearance of pattern B among the isolates of a part of patients and the switch from A to B pattern in one of patients can be plausibly explained by the unstability of DNA genotypes caused by transposition of IS6110 elements.

Int J Clin Pharmacol Res, 1999, 19(3), 65 - 71
Serum pharmacokinetics of antimycobacterial drugs in patients with multidrug-resistant tuberculosis during therapy; Yew WW et al.; Serum samples of 13 patients with multidrug-resistant tuberculosis were taken 0, 1, 2, 4, 8 h after administration of antimycobacterial drugs for assay of levels in order to gain further insight into their basic pharmacokinetics . The drugs assessed were amikacin, kanamycin, ofloxacin, levofloxacin, para-aminosalicylic acid, prothionamide, cycloserine, pyrazinamide and ethambutol . Techniques used for assay were reversephase high-performance liquid chromatography, gas liquid chromatography and fluorescent polarization immunoassay . The results from 12 patients were evaluated . These provided new pharmacokinetic data on high-dose levofloxacin, cycloserine and prothionamide given once daily, and could be useful in guiding the scheduling of drugs . The data obtained might also lead to insights into the development of therapeutic drug monitoring in multidrug-resistant tuberculosis.

Cancer Control, 1998 Jan, 5(1), 25 - 33
Large Granular Lymphocyte Leukemia; Lamy T et al.; BACKGROUND: Clonal diseases of large granular lymphocyte (LGL) disorders can arise from a CD3+ T-cell lineage or from a CD3- NK-cell lineage . CD3+ LGL leukemia is the most frequent form of LGL leukemia and is a distinct entity by FAB and REAL classifications . METHODS: The clinical course, biological features, and recent data on pathogenesis of CD3+ LGL leukemia are reviewed . The spectrum of differential diagnosis is described . RESULTS: T-LGL leukemia affects elderly people . Approximately 60% of patients are symptomatic; recurrent infections secondary to chronic neutropenia, anemia, and rheumatoid arthritis are the main clinical features . The most common phenotype is CD3+, CD8+, CD57+ . Clonality is detected by clonal rearrangement of the T-cell receptor gene . Clinical and molecular remission can be obtained with oral low-dose methotrexate . Serologic findings show frequent reactivity to the BA21 epitope of HTLV-I env p21e, suggesting that a cellular or retroviral protein with homology to BA21 may be important in pathogenesis . Clonal expansion may be facilitated by IL-12 and IL-15 lymphokines . Constitutive expression of Fas ligand by leukemic LGLs support the hypothesis that leukemic cells arise from antigen-activated cytotoxic T cells . Leukemic LGLs express a multidrug-resistance phenotype that could partly explain the chemoresistance observed in aggressive cases . CONCLUSIONS: CD3+ LGL leukemia is a distinct lymphoproliferative T-cell disorder with specific clinicobiological aspects . The clinical spectrum of LGL proliferations is wide and immunophenotypic, and genotypic studies are needed to establish the diagnosis.

Kidney Int, 2000 Apr, 57(4), 1636 - 42
ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2; Leier I et al.; BACKGROUND: Para-aminohippurate (PAH), a widely used model substrate for organic anion transport in proximal tubule epithelia, was investigated as a substrate for the apical multidrug resistance protein MRP2 (symbol ABCC2) . This ATP-dependent export pump for anionic conjugates and additional amphiphilic anions was cloned recently and localized to the apical membrane of proximal tubules in human and rat kidney . METHODS: Membrane vesicles from HEK-MRP2 cells containing recombinant human MRP2 and from control vector-transfected HEK-Co cells were incubated with various concentrations of {3H}PAH, and the net ATP-dependent transport into inside-out vesicles was determined . Comparative studies were performed with membrane vesicles containing recombinant human MRP1 . RESULTS: Transport rates at 10 micromol/L PAH were 21.9 +/- 1.9 and 1.6 +/- 0.4 pmol x mg protein-1 x min-1 (means +/- SEM, N = 10) with membrane vesicles from HEK-MRP2 and HEK-Co cells, respectively . The Km value for PAH was 880 micromol/L . The high-affinity substrate leukotriene C4 and the inhibitor of MRP-mediated transport, MK571, inhibited MRP2-mediated transport of PAH (100 nmol/L) with IC50 values of 3.3 and 4.0 micromol/L, respectively . The nephrotoxic mycotoxin ochratoxin A inhibited MRP2-mediated PAH transport with an IC50 value of 58 micromol/L . Ochratoxin A was itself a substrate for MRP2 . CONCLUSIONS: PAH is a good substrate for the ATP-dependent export pump MRP2 . The localization and function of MRP2 indicate that this unidirectional transport protein contributes to the secretion of PAH and other amphiphilic anions into the lumen of kidney proximal tubules.

Br J Haematol, 2000 Mar, 108(3), 565 - 73
Constitutive levels of cAMP-dependent protein kinase activity determine sensitivity of human multidrug-resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumour necrosis factor alpha; Yin Y et al.; The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) signal pathway regulates cell proliferation, differentiation and cell death . It may also regulate the multidrug resistance (MDR) phenotype in leukaemic cells . These data showed that MDR1+ K/Dau600 cells exhibited a higher basal level of PKA activity than MDR- parental cells . The significance of this on tumour necrosis factor alpha (TNFalpha)-induced apoptosis and cytostasis was investigated further . In comparison with MDR1- parental cells, K/Dau600 cells had a higher expression of PKA regulatory subunit RIalpha and nuclear catalytic subunit PKAcalpha . They were also more susceptible to inhibition of proliferation and induction of apoptosis by TNFalpha and/or forskolin, but this could be attenuated by H89 . An increase in cAMP was associated with the apoptosis in the K/Dau600 cell line . Forskolin inactivated NF-kappaB in K/Dau600 cells but not in K562 cl . 6 cells, whereas TNF activated NF-kappaB in K562 cl.6 cells but not in K/Dau600 cells . 8-Cl-cAMP exhibited similar inhibitory effects on the proliferation of all of the cell lines used via its metabolite 8-Cl-adenosine, which indicates that these effects were independent of residual PKA or cAMP . Therefore, the differential sensitivity to apoptosis and/or growth inhibition could be mediated via cAMP, partly through PKA via NF-kappaB and partly by PKA-independent pathways.

Anticancer Drugs, 2000 Jan, 11(1), 55 - 61
Effects of KR-30035, a novel multidrug-resistance modulator, on the cardiovascular system of rats in vivo and on the cell cycle of human cancer cells in vitro; Lee BH et al.; The present study was performed to evaluate the adverse effects of KR-30035, a multidrug-resistance modulator, on the cardiovascular system in vivo, along with its effect on paclitaxel-induced cell cycle arrest in cultured cancer cells . In anesthetized rats, KR-30035 was about 10-fold less potent than verapamil in lowering blood pressure (i.v . ED20: 0.320+/-0.052 and 0.034+/-0.005 mg/kg, respectively) and in producing electrocardiogram changes . In conscious spontaneously hypertensive rats, verapamil caused a significant antihypertensive effects at the doses tested (p.o . ED20, 7.8+/-4.0 mg/kg), whereas KR-30035 did not significantly change either the blood pressure or the heart rate at any doses tested (up to 100 mg/kg) . The estimated i.v . LD50 values in mice were 5.9 and 48.9 mg/kg for verapamil and KR-30035, respectively . In the presence of 10 microM KR-30035, paclitaxel (1 microM) when added to cultures of HCT15/CL02 human cancer cells greatly shifted the cell population from the G0/G1 phases towards G2/M phases (from 42.4, 30.3 and 27.3 to 14.6, 21.5 and 63.9% for the G0/G1, S and G2/M phases, respectively), with a similar magnitude to that of 10 microM verapamil (14.0, 15.7 and 70.3%, respectively) . These results suggest that KR-30035 has weaker in vivo effects on the cardiovascular system compared with verapamil, while potentiating the G2/M arresting effect of paclitaxel on the cell cycle.

Oncol Res, 1999, 11(7), 303 - 10
Effects of mitomycin C and carboplatin pretreatment on multidrug resistance-associated P-glycoprotein expression and on subsequent suppression of tumor growth by doxorubicin and paclitaxel in human metastatic breast cancer xenografted nude mice; Ihnat MA et al.; Overexpression of P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP), and several other proteins has been associated with development of multidrug resistance by cancer cells, which represents a significant obstacle to successful treatment by chemotherapy . We had previously demonstrated that a single noncytotoxic dose of mitomycin C (MMC), carboplatin, or one of several other DNA cross-linking agents suppressed mRNA expression of the mdr1 gene coding for Pgp, leading to a subsequent suppression of Pgp protein levels and a concomitant decrease in drug efflux . Pretreatment with MMC led to a 5- to 10-fold decrease in the ED50 for cell killing by a subsequent agent such as the Pgp substrate, doxorubicin, but did not affect killing by the non-Pgp substrate, cisplatin . In this study, we report that MMC and carboplatin each significantly suppressed Pgp protein levels in human MDA-MB-435 cells xenografted as solid tumors into the lateral mammary fat pads of female nude mice, with a similar time course as had previously been observed in cell culture . Pretreatment of mice with MMC or carboplatin 48-72 h prior to receiving either doxorubicin or paclitaxel caused a significantly greater reduction in tumor growth rate compared to either agent alone or the combination given simultaneously . These data suggest that a combination chemotherapy regimen consisting of a DNA cross-linking agent given to modulate the MDR phenotype, followed by a second cytotoxic agent, may be an effective treatment for human patients with de novo or late stage acquired multidrug-resistant malignancies.

J Pharm Pharmacol, 2000 Mar, 52(3), 289 - 96
Membrane permeation by multidrug-resistance-modulators and non-modulators: effects of hydrophobicity and electric charge; Castaing M et al.; This study was designed to test the hypothesis that lipophilic cationic drugs with only roughly similar structures mediate the reversal of multidrug-resistance (MDR) by interacting with membrane phospholipids . The permeation properties of MDR-modulators and non-modulators were studied by quantifying their ability to induce the leakage of Sulphan blue through the membrane of negatively charged unilamellar liposomes . Of the 22 compounds under investigation, only those bearing a net positive electric charge per molecule (z) > or = 0.2 induced dye leakage . All these efficient drugs are well-known MDR-modulators: calcium-channel blockers (propranolol, verapamil, diltiazem and dipyridamole), calmodulin antagonists (clomipramine and thioridazine) and antiparasitic agents (mepacrine, thioacridine derivatives and quinine) . The non-modulators tested, including antineoplastic agents and steroids, did not induce any membrane permeation . The permeation process was a co-operative one (1.1 < Hill coefficient < 4.1) and the permeation doses inducing 50% dye leakage (PD50) were 1.9-11.2 mM . The permeation ability of the MDR-modulators (log(1/PD50)) increased significantly with octanol-buffer distributions per unit net electric charge ((logD)/z) . The results provide evidence that a complex interplay occurs between the electric charge and the lipophilicity of the MDR-modulators when a dye leakage is induced through model membranes, and probably also when the MDR is reversed in leukaemic cells.

In Vivo, 2000 Jan-Feb, 14(1), 287 - 96
Growth inhibitors in the treatment of malignant neoplasms; Kaiser HE et al.; Growth inhibitors are an integral part of the regulatory mechanisms involved in the growth and differentiation of cells and tissues . Aberration in the response to growth inhibitors leads to the escape of the cell from the cell cycle control mechanisms and may lead to the development of malignancies . The inactivation of tumor suppressor genes, activation of oncogenes leads to the acquisition of an invasive and increasingly malignant immunophenotype and secretory profile by transformed cells . The commencement of the complex process of carcinogenesis, and subsequent, rapid tumor growth and progression of mammalian neoplasms depends upon the continuous de novo formation of capillaries (angiogenesis) . The generation of a malignant, invasive cellular immunophenotype (CIP) and distant metastases, as aspects of tumor progression, are also NRA-dependent processes . Specific molecules with cytostatic/cytotoxic growth inhibitory effects represent a very diverse group of factors . Growth inhibitors may regulate the cell cycle at various levels, and growth inhibitors comprise a heterogeneous group of agents including cytokines, growth factors, steroid hormones, etc . The phenomenon of multidrug resistance to a wide spectrum of cytostatic/cytotoxic agents has posed a major difficulty in the effective chemotherapeutical treatment of cancer patients . The development of novel therapeutic regimens should be based on the observations of the growth inhibitory profile of the particular malignancy, in addition to its immunophenotype and genotype, and the devisement of 'individualized' combinations of factors, including gene and immuno-therapeutical options, targeting different aspects of the malignant disease.

Int J Tuberc Lung Dis, 2000 Mar, 4(3), 223 - 31
Anti-tuberculosis drug resistance in Portugal; Antunes ML et al.; SETTING: A survey based upon a representative sample of smear-positive pulmonary tuberculosis patients was undertaken in Portugal, as part of the World Health Organization's Global Project on Anti-Tuberculosis Drug Resistance Surveillance . OBJECTIVE: To determine the level of primary antituberculosis drug resistance at both national and regional levels, and to assess its relative weight within the performance of the National Tuberculosis Programme (NTP) . DESIGN: Mycobacterium tuberculosis isolates from 1,105 patients with smear-positive pulmonary tuberculosis admitted to 46 randomly stratified treatment centres all over mainland Portugal were submitted to susceptibility testing with four drugs . Human immunodeficiency virus (HIV) testing was included in the patients' evaluation scheme . RESULTS: Of the strains isolated, 197 (17.8%) were resistant to at least one drug . Primary resistance to isoniazid was 7.7% and to rifampicin 1.9% . Acquired drug resistance was 39.2% in total, any acquired resistance to isoniazid 31.1% and to rifampicin 20.9% . Primary multidrug resistance (MDR) was 1.8% and acquired MDR was 20.9% . HIV testing was positive in 29.2% of MDR-TB cases . CONCLUSIONS: Drug resistance in Portugal is high . Primary MDR and particularly acquired MDR occur in a high proportion of cases, indicating a need for improvement in NTP performance.

Chem Pharm Bull (Tokyo), 1999 Dec, 47(12), 1679 - 84
Synthesis and antitumor activity of novel pyrimidinyl pyrazole derivatives; Naito H et al.; Novel pyrimidinyl pyrazole derivatives were synthesized and examined for cytotoxic and antitumor activity . Mannich reaction was employed to construct this scaffold . Among the compounds synthesized, a series of propene derivatives exhibited a potent cytotoxic activity against some tumor cell lines including multidrug resistant cell lines due to the overexpression of P-glycoprotein . The vinyl bond moiety in the scaffold was believed to be required for the cytotoxic activity . Among them, compound 14 g, when administered intraperitoneally, showed potent antitumor activity against the malignant ascites caused by intraperitoneal inoculation of P388 cells in mice . This compound also showed high activity against a solid tumor Meth A mouse fibrosarcoma when administered both intraperitoneally and orally.

J Biol Chem, 2000 Jun 9, 275(23), 17626 - 30
Nonequivalent nucleotide trapping in the two nucleotide binding folds of the human multidrug resistance protein MRP1; Nagata K et al.; Multidrug resistance protein 1 (MRP1) and P-glycoprotein, which are ATP-dependent multidrug efflux pumps and involved in multidrug resistance of tumor cells, are members of the ATP binding cassette proteins and contain two nucleotide-binding folds (NBFs) . P-glycoprotein hydrolyzes ATP at both NBFs, and vanadate-induced nucleotide trapping occurs at both NBFs . We examined vanadate-induced nucleotide trapping in MRP1 stably expressed in KB cell membrane by using 8-azido-{alpha-(32)P}ATP . Vanadate-induced nucleotide trapping in MRP1 was found to be stimulated by reduced glutathione, glutathione disulfide, and etoposide and to be synergistically stimulated by the presence of etoposide and either glutathione . These results suggest that glutathione and etoposide interact with MRP1 at different sites and that those bindings cooperatively stimulate the nucleotide trapping . Mild trypsin digestion of MRP1 revealed that vanadate-induced nucleotide trapping mainly occurs at NBF2 . Our results suggest that the two NBFs of MRP1 might be functionally nonequivalent.

J Biol Chem, 2000 Jun 2, 275(22), 16857 - 64
Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis; Kremer L et al.; Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease . Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis . In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis . Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM . In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M . tuberculosis . The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M . tuberculosis . Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.

J Antimicrob Chemother, 2000 Apr, 45(4), 489 - 92
Comparative therapeutic efficacy of clinafloxacin in a pneumococcal meningitis mouse model; Shapiro MA et al.; The therapeutic efficacy of clinafloxacin, a fluoroquinolone in clinical trials, was compared with that of ciprofloxacin and ceftriaxone in a novel pneumococcal meningitis mouse model . Mice were challenged by the intracerebral ventricular route with 50 IL of a lethal bacterial suspension and treated subcutaneously 2 h later . Both penicillin-susceptible and multidrug-resistant pneumococcal strains were used for evaluation . Survival percentages were calculated as the median curative dose (CD50) using log-probit statistical methods . Ceftriaxone was the most active agent against the penicillin-susceptible strain (CD50 = 2 mg/kg), but showed a 30-fold decrease in potency against the resistant strain . Clinafloxacin was equally effective against both strains, and proved to be the most active agent against the penicillin-resistant pneumococcus.

J Clin Microbiol, 2000 Apr, 38(4), 1370 - 4
Emergence of drug resistance mutations in human immunodeficiency virus type 2-infected subjects undergoing antiretroviral therapy; Rodes B et al.; The reverse transcriptase (RT) and protease genes from 12 human immunodeficiency virus type 2 (HIV-2)-infected individuals who had been exposed to antiretroviral drugs for longer than 6 months were examined for the presence of mutations which could be involved in drug resistance . Four individuals carried virus genotypes with amino acid substitutions potentially associated with resistance to nucleoside analogues: two at codon 70 (K-->R) and two at codon 184 (M-->V) . Moreover, the latter two patients harbored a codon Q151M mutation which is associated to multidrug resistance in HIV-1, and one of these subjects carried some of the typically linked mutations at codons 65 and 69 . With regard to the protease inhibitors, substitutions associated with resistance to protease inhibitors at codon 46 were observed in all individuals . Moreover, minor resistance mutations, as well as new ones of unknown meaning, were often seen in the protease gene . In conclusion, amino acid changes in the HIV-2 RT and protease genes which could be associated with drug resistance seem to occur at positions identical to those for HIV-1.

J Exp Clin Cancer Res, 1999 Dec, 18(4), 549 - 52
Reversal effect of TTD on human multidrug resistant KBV200 cell line; Xu JY et al.; The reversal effect of TTD (a Chinese medicine) on human multidrug- resistant KBV200 cell line was studied and compared with verapamil (VPL) . The chemosensitivity of KBV200 was detected by MTT assay in vitro and the level of MDR1 mRNA of KBV200 was investigated by RT-PCR . The cytotoxicity of TTD to KBV200 and the parent sensitive cell line KB is very low and nearly same with a concentration of 10(-6) mol x L(-1) . With the concentration TTD increased VCR cytotoxicity on KBV200, 50% inhibitory concentration (IC50) decreased from 1122.5+/-72.36 mol x L(-1) to 31.76+/-5.4 nmol x L(-1) (p<0.001) . It was more effective than VPL was (p<0.01) . The combination of low concentration of TTD (10(-8) mol x L(-1)) and VCR (100 nmol x L(-1)) has significantly increased VCR cytotoxicity on KBV200, cell Surviving Fraction decreased from 0.91+/-0.056 to 0.74+/-0.07 (p<0.02) . TTD did not inhibit the expression of MDR1 mRNA of KBV200 with the concentration of 10(-6) mol x L(-1) . These data indicated that TTD could reverse VCR resistance of KBV200 and may be useful in enhancing the clinical effectiveness of VCR.

Eur J Clin Microbiol Infect Dis, 2000 Feb, 19(2), 132 - 6
Effect of administering short-course, standardized regimens in individuals infected with drug-resistant Mycobacterium tuberculosis strains; Furin JJ et al.; Presented here are the cases of three siblings with multidrug-resistant tuberculosis who demonstrated increased antituberculous-drug resistance during the periods in which they received standard regimens of directly observed, short-course chemotherapy that were administered before the susceptibility patterns of their Mycobacterium tuberculosis isolates had been checked . More specifically, they acquired resistance to drugs they received as part of ineffective standard treatment and retreatment regimens . Development of antituberculous-drug resistance through inadvertent, inadequate therapy appears to be the most likely explanation for the increased resistance seen in these three patients.

Jpn J Cancer Res, 2000 Jan, 91(1), 84 - 90
Expression of drug resistance-related genes in head and neck squamous cell carcinomas and normal mucosa; Hirata S et al.; We examined the expression levels of mRNA for multidrug resistance 1 (MDR1), multidrug resistance-associated protein (MRP), human canalicular multispecific organic anion transporter (cMOAT), lung resistance-related protein (LRP), topoisomerase IIalpha, beta (Topo IIalpha, beta) and topoisomerase I (Topo I) genes in human head and neck squamous cell carcinoma (HNSCC) specimens and mucosa (HNM) specimens, to elucidate their roles in relation to the biological characteristics and drug resistance in vivo . Fifty-eight samples (45 head and neck carcinomas and 13 head and neck mucosa) obtained during surgical resection or biopsy from 38 patients were analyzed using the quantitative reverse transcription-polymerase chain reaction (RT-PCR) method . MDR1, MRP, LRP, Topo IIalpha, Topo IIbeta, and Topo I gene transcripts were detected in all the samples tested, but cMOAT mRNA was not detected in them . Comparisons of the expression levels in HNSCC with those in HNM showed that the Topo IIalpha gene expression level was higher in HNSCC than in HNM (P=0.0298) . Moreover, the Topo IIalpha mRNA level was significantly higher in metastatic lymph node samples of HNSCC than in HNM samples (P=0.0205) . There were no significant differences in the six genes' expression levels between samples exposed to platinum drugs and those not exposed to platinum drugs . These results suggest that it may be effective in anticancer therapy to use topoisomerase-targetting drugs against HNSCC, especially metastatic neck tumors, and that the expression of these genes in HNSCC is not associated with platinum drug exposure.

Bull World Health Organ, 2000, 78(2), 238 - 51
Directly observed treatment, short-course strategy and multidrug-resistant tuberculosis: are any modifications required?
Bastian I, Rigouts L, Van Deun A, Portaels F.
Multidrug-resistant tuberculosis (MDRTB) should be defined as tuberculosis with resistance to at least isoniazid and rifampicin because these drugs are the cornerstone of short-course chemotherapy, and combined isoniazid and rifampicin resistance requires prolonged treatment with second-line agents . Short-course chemotherapy is a key ingredient in the tuberculosis control strategy known as directly observed treatment, short-course (DOTS) . For populations in which multidrug-resistant tuberculosis is endemic, the outcome of the standard short-course chemotherapy regimen remains uncertain . Unacceptable failure rates have been reported and resistance to additional agents may be induced . As a consequence there have been calls for well-functioning DOTS programmes to provide additional services in areas with high rates of multidrug-resistant tuberculosis . These "DOTS-plus for MDRTB programmes" may need to modify all five elements of the DOTS strategy: the treatment may need to be individualized rather than standardized; laboratory services may need to provide facilities for on-site culture and antibiotic susceptibility testing; reliable supplies of a wide range of expensive second-line agents would have to be supplied; operational studies would be required to determine the indications for and format of the expanded programmes; financial and technical support from international organizations and Western governments would be needed in addition to that obtained from local governments.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 1997 Dec, 32(6), 336 - 8
{Detection of P-glycoprotein in squamous cell carcinoma of larynx}; Shi L et al.; The multidrug resistance (MDR) of malignant tumor is a troublesome problem in the chemotherapy . One established mechanism of multidrug resistance is elevated expression of the P-glycoprotein (PGP) . JSB1 (which is a special monoclonal antibody of human MDR, PGP) was used to examine the expression of PGP by immunohistochemistry in 23 specimens of squamous cell carcinoma of larynx . The expression of JSB1 in 10 normal laryngeal tissue specimens was also studied, no positive result was found . Of the 23 laryngeal cancer specimens, positive reaction to JSB1 was showed in 15, and there was no relationship between the positive expression, and the tumor differentiation, T staging and lymph-node metastasis . However, by using another newly developed monoclonal antibody C219, a different result was obtained from specimens of the same patients.

Zhonghua Zhong Liu Za Zhi, 1997 Jan, 19(1), 72 - 5
{Clinical significance of multidrug resistance gene(mdr1) expression in patients with acute leukemia}; Dong Z et al.; OBJECTIVE: To evaluate relationship between drug resistance of leukemia cells and prognosis . METHODS: The expression of multidrug resistance gene(mdr1) mRNA was measured in 85 patients with acute leukemia and in 20 normal controls by using reverse transcription polymerase chain reaction (RT-PCR) . RESULTS: The mdr1 positive rate in untreated group was 44.7% . The CR rate differed significantly between mdr1+ (23.9%) and mdr1-(88.5%)(P < 0.005) . The mdr1 positive rate of refractory-relapsed group was higher than that in CR group (P < 0.001) . The mdr1 mRNA expression level of refractory-relapsed group was higher than that in CR group . A gradually increasing level of mdr1 mRNA expression in CR patients indicated early relapse . The mdr1 positive rate in normal contral and long-term surviver patients was very low . The mdr1 expression was correlated with French-American-Birtish Cooperative Group (FAB) classification . CONCLUSION: The mdr1 expression correlated with chemotherapeutic effect and prognosis . It is an unfavorable prognostic factor for patients with acute leukemia.

Zhonghua Zhong Liu Za Zhi, 1997 Jan, 19(1), 38 - 41
{Criteria of measuring mdr-1 gene expression level in breast cancer by RT-PCR}; Liu X et al.; OBJECTIVE: To formulate criteria of judjing multidrug resistance gene(mdr-1 gene) expression level and to provide basis for predicting chemotherapy response and prognosis . METHODS: Using reverse transcription-polymerase chain reaction (RT-PCR) assay, the expression of mdr-1 gene in 82 breast cancer samples was detected . The data were treated by statistic analysis system (SAS)-singlevariate analysis . RESULTS: The level of mdr-1 gene expression clearly deviated from normal to right distribution (P < 0.0001), and thus might be divided by quantiles P50(mdr-1/beta 2-MG = 0.2) and P75 (mdr-1/beta 2-MG = 0.6), which were taken as the criteria for comparing 56 patients' chemosensitivity to ADM, VDS, VCR in vitro and 32 relapsed metastatic patients' chemotherapy response in vivo, seperately . When mdr-1/beta 2-MG < 0.2, the ratio of coincidence was lower between expression of mdr-1 and drug resistance in vitro and in vivo; When mdr-1/beta 2-MG > or = 0.2-< 0.6, the ratio of coincidence elevated slightly, but in 30%-50% of the cases drug resistance in vitro and in vivo did not correlate . When mdr-1/beta 2-MG > or = 0.6, the ratio of coincidence elevated significantly . According to the above-mentioned results, criteria of evaluating mdr-1 gene expression level was formulated: the mdr-1/beta 2-MG ratio < 0.2(P50) was considered as negative expression, the ratio > or = 0.2-< 0.6(P75) was weakly positive expression, > or = 0.6 was strongly positive expression . CONCLUSIONS: The criteria of mdr-1 gene expression may reflect objectively drug resistance in vitro and chemotherapy response in vivo . The method may also be applicable to other tumours.

Pharm Biotechnol, 1999, 12, 353 - 86
Multidrug-resistance transporters; Silverman JA; P-glycoprotein was initially isolated due to its role in multidrug resistance to cancer chemotherapeutics . Recent work, however, makes it increasingly apparent that this transporter is also involved in the pharmacokinetics of many drugs . P-gp is strategically expressed in the luminal epithelial cells of organs often associated with drug absorption and disposition, for example, hepatocyte canalicular membrane, renal proximal tubules, and the intestinal mucosa . P-gp is also expressed in the endothelial cells comprising the blood-brain barrier . This localization clearly suggests the potential for this protein to serve as a protective mechanism against entry of toxic xenobiotics and also suggests that P-gp is well situated to participate in the removal of therapeutic agents . Numerous investigations with drugs such as digoxin, etoposide, cyclosporine, vinblastine, Taxol, loperamide, dom-peridone, and ondansteron demonstrate that P-gp has an important role in determining the pharmacokinetics of substrate drugs . Pharmacological modulation of P-gp function to increase drug bioavailability, both on a organismal and a cellular level, is one approach currently being explored to enhance therapeutic effectiveness . This approach is not without potential collateral consequences given the wide tissue distribution of P-gp . While animals deficient in P-gp are viable and without obvious abnormalities, the pharmacokinetics and toxic consequences of several compounds are significantly altered in these animals . Thus blockade of the protective P-gp barrier in humans may have adverse effects on substrate drugs . In particular, this situation may arise when several compounds which may be substrates compete for P-gp-mediated transport . Additional multidrug transporters, notably MRP and family members, have been identified and may also determine the fate of pharmaceuticals . Further understanding the physiological role of each of the multidrug transporters is critical for determining their role in pharmacokinetics and for evaluating the consequences of modification of their activities . Such information is also important in the development of novel drugs which may be substrates for these transporters.

J Cancer Res Clin Oncol, 2000 Mar, 126(3), 168 - 72
Modulation of cisplatin sensitivity by taxol in cisplatin-sensitive and -resistant human ovarian carcinoma cell lines; Yamamoto K et al.; PURPOSE: The aim of this study was to determine whether taxol can circumvent cisplatin resistance, using a KF28 cell line derived from human ovarian carcinoma and a cisplatin-resistant line, KFr13, derived from the parental cell line, KF28, and taxol-resistant cell lines, KF28TX and KFr13TX, derived from the respective parental counterpart . METHODS: KF28 is a single-cell clone of the human ovarian carcinoma cell line KF . The cisplatin-resistant KFr13 subline was established by repeated exposure of the parent KF28 cell line to escalating doses of cisplatin . Similarly, KF28TX and KFr13TX were established by repeated exposure of the KF28 and KFr13 cell lines to escalating doses of taxol . A cytotoxicity assay was performed using a crystal violet staining method . Platinum and taxol accumulation were assayed by atomic absorption and reverse-phase high-performance liquid chromatography . The quantitative assay of MDR1 mRNA used polymerase chain reaction . RESULTS: KFr13 cells were about 4.8-fold more resistant to cisplatin and about 1.8-fold more sensitive to taxol than were KF28 cells . When taxol resistance was induced in KF28 and KFr13 cells, sensitivity to cisplatin rose about 1.3- and 1.6-fold respectively . Elevation of sensitivity was correlated with platinum uptake by both KF28TX and KFr13TX cells . Expression of multidrug resistance (MDR1) mRNA, which was not observed in KF28 and KFr13 cells, was observed after induction of taxol resistance . CONCLUSIONS: These results may suggest rational therapeutic strategies for patients with cisplatin-resistant or refractory ovarian carcinoma.

Clin Cancer Res, 2000 Mar, 6(3), 942 - 8
1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitizes neuroblastoma cells for taxol and vincristine; Sietsma H et al.; In this study, we show that an inhibitor of glycosphin-golipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increases the chemosensitivity of neuroblastoma tumor cells for Taxol and vincristine . At noneffective low doses of Taxol or vincristine, the addition of a noneffective dose of PDMP resulted in 70% cytotoxicity, indicating synergy . Such an effect was not observed for etoposide (VP16) . PDMP caused an early (6 h) increase in ceramide (Cer) levels, but the excess Cer was metabolically removed in the long-term (96 h) . However, upon incubation with PDMP in combination with Taxol, but not with etoposide, Cer levels remained elevated at 96 h . These results suggest that neuroblastoma cells are normally able to metabolically remove excess Cer, but lose this capacity upon exposure to microtubule modulating anticancer agents (Taxol or vincristine) . In addition, PDMP treatment resulted in a decreased efflux of {14C}Taxol and {3H}vincristine from neuroblastoma cells, similar to treatment with PSC833 or MK571, suggesting an effect of PDMP on the transporter proteins P-glycoprotein and/or multidrug resistance protein . PDMP did not further reduce {14C}Taxol or {3H}vincristine efflux in PSC833-treated cells, although it did further diminish cell survival under these conditions . We conclude that a combined administration of nontoxic concentrations of PDMP and either Taxol or vincristine results in highly sensitized neuroblastoma cells . This appears to involve a sustained elevation of Cer levels, possibly in concert with increased drug accumulation.

Clin Cancer Res, 2000 Mar, 6(3), 820 - 4
Quickly predicting chemotherapy response to paclitaxel-based therapy in non-small cell lung cancer by early technetium-99m methoxyisobutylisonitrile chest single-photon-emission computed tomography; Kao CH et al.; The purpose of this study was to retrospectively predict the chemotherapy response to paclitaxel in non-small cell lung cancer (NSCLC) using technetium-99m methoxyisobutylisonitrile (Tc-99m MIBI) chest single-photon-emission computed tomography (SPECT) to detect the expression of multidrug-resistance-mediated Mr 170,000 P-glycoprotein . Before chemotherapy with Paclitaxel (Taxol), 30 patients with stage IIIb or IV NSCLC were enrolled in this study . Early chest SPECT 10 min after i.v . injection of Tc-99m MIBI was performed to qualitatively interpret Tc-99m MIBI chest SPECT visually and quantitatively calculate early tumor:normal lung ratios (T:NL) for quick assessment of multidrug-resistant P-glycoprotein expression in NSCLC . On the basis of qualitatively visual interpretation of early Tc-99m MIBI chest SPECT, all of 15 (100%) cases with good response to chemotherapy with Taxol could be detected but 10 (67%) of 15 cases with poor response could not be detected . Early Tc-99m MIBI chest SPECT could correctly predict chemotherapy response in 25 (83%) of 30 of cases . The early T:NL were 3.30 +/- 0.82 for 15 patients with good response and 2.02 +/- 0.19 for 5 patients with poor response . The differences were significant (P < 0.05) by independent Student t tests . However, no significant differences were found for other prognostic factors (age, sex, tumor size, tumor location, stage, and cell type) between good-response and poor-response patients . Early Tc-99m MIBI chest SPECT has the potential to predict chemotherapy response to Paclitaxel.

Cell Prolif, 2000 Feb, 33(1), 51 - 62
Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid; Yatouji S et al.; It is now known that the analysis of chromatin texture can be used in oncology as a sensitive detection method, either to define diagnostic classifications or to locate a lesion along a defined trend curve . However, the functional significance of these variations in textural features remains sometimes unclear . Several drugs have been shown to be able to modulate chromatin structure . Among them, the phosphatase inhibitor okadaic acid at low concentration can increase accessibility to DNA in chromatin of carcinoma cells . This paper demonstrates that short exposures (0-3 h) to a 10-nM dose of okadaic acid induced an increased sensitivity to DNase I digestion in human CEM leukaemic cell nuclei and that this sensitization was associated to variations of nuclear texture characteristics, as evaluated by image cytometry . CEM cells treated with okadaic acid for 0-3h displayed changes in chromatin supraorganization with a more homogeneous and fine chromatin texture, as compared to control cells . This suggests that the appearance of an open configuration of chromatin structure as evaluated by biochemical methods corresponds to a more decondensed texture of nuclei measured by image cytometry . Longer exposures (6-24h) of CEM cells to 10 nM okadaic acid lead to apoptosis . As reported previously for camptothecin-treated HL60 cells, okadaic acid-treated CEM cells display biphasic nuclear chromatin texture changes, i.e . a decondensation phase followed by the appearance of typical apoptotic cells with a smaller nuclear area and a highly condensed chromatin . Finally, using the multidrug-resistant CEM-VLB cell line, it was confirmed that these multidrug-resistant cells also display cross-resistance to okadaic acid, as this compound was unable to induce either increased DNase I sensitivity, apoptosis, or altered nuclear texture in this particular cell line.

Pharmacol Ther, 2000 Mar, 85(3), 217 - 29
Resistance mechanisms associated with altered intracellular distribution of anticancer agents; Larsen AK et al.; The resistance of tumor cells to anticancer agents remains a major cause of treatment failure in cancer patients . The term multidrug resistance (MDR) is used to define a resistance phenotype where cells are resistant to multiple drugs with no obvious structural resemblance and with different molecular targets . It is now clear that MDR is always multifactorial . The intracellular drug distribution is modified in many MDR cell lines, leading to increased drug sequestration in acidic vesicles, such as the trans-Golgi apparatus, recycling endosomes, and lysosomes, followed by transport to the plasma membrane and extrusion into the external medium . Since most anticancer agents target DNA or nuclear enzymes, sequestration of drug in cytoplasmic organelles will lead to decreased drug-target interaction and thereby, decreased cytotoxicity . Altered intracellular drug distribution is usually associated with the expression of drug efflux pumps, such as the P-glycoprotein and the multidrug resistance protein . Another common modification in MDR cells is alkalization of the intracellular pH . The relationship between these different resistance mechanisms is reviewed and a model proposed that suggests why these different resistance mechanisms are co-expressed in multiple cell lines.

Pharmacol Ther, 2000 Mar, 85(3), 191 - 205
The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine; Rosowsky A et al.; N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC) . Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates . Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect . However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype . As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth . The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated . Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.

Exp Cell Res, 2000 Apr 10, 256(1), 300 - 7
Downregulation of JNK/SAPK activity is associated with the cross-resistance to P-glycoprotein-unrelated drugs in multidrug-resistant FM3A/M cells overexpressing P-glycoprotein; Kang CD et al.; In the present study, cross-drug resistance in multidrug-resistant (MDR) cells, which overexpress P-glycoprotein (Pgp), a mdr1 gene product, against Pgp-unrelated drugs, and its relevance to c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) activity were examined . The multidrug-resistant FM3A/M cells overexpressing Pgp were resistant to apoptotic cell death induced either by Pgp-related drugs including vincristine and vinblastine, which are pumped out by Pgp, or by the Pgp-unrelated drugs including 5'-fluorouracil (5-FU) and bleomycin, which are not targets for Pgp, compared with the parental FM3A cells . Verapamil reversed the resistance of FM3A/M cells to apoptosis induced by the Pgp-related drugs but not that induced by the Pgp-unrelated drugs . Interestingly, FM3A/M cells have shown significantly lower basal and drug-stimulated JNK/SAPK activities than FM3A cells . After transfection with pEBG-SEK or pEBG-SAPK constructs, FM3A/M cells recovered the basal and Pgp-unrelated drug-stimulated activities of JNK/SAPK and the susceptibility to Pgp-unrelated drug-induced apoptotic cell death comparable to those of FM3A cells . Furthermore, FM3A cells became resistant to apoptotic cell death induced by vincristine and 5-FU after transfection with pEBG-SEK(K --> R), a dominant negative inhibitory mutant of SEK . These results suggest that downregulation of JNK/SAPK activity appears to confer on Pgp-associated FM3A/M cells a cross-resistance to Pgp-unrelated drugs .

Gan To Kagaku Ryoho, 2000 Mar, 27(3), 388 - 94
{New combination therapies in hematological malignancies}; Takeshita A et al.; New combination therapies against hematological malignancies have recently been reported . Anti-CD20 chimeric antibody adds a therapeutic benefit to standard-dose CHOP therapy without causing significant additional toxicity in the treatment of indolent B cell lymphoma . Multidrug resistant modifiers such as PSC833 and MS209 in combination with chemotherapy are useful for treating poor risk AML patients, whose leukemia/lymphoma cells express P-glycoprotein . Fludarabine containing FL and FLAG therapy were effective in patients with AML in relapse . The early addition of chemotherapy to ATRA, and maintenance therapy with chemotherapy and intermittent ATRA, can reduce the incidence of relapse in cases of acute promyelocytic leukemia.

Int J Clin Pharmacol Ther, 2000 Mar, 38(3), 130 - 40
Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine; Ferry D et al.; INTRODUCTION: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype . METHODS: In this paper we used the 1,4-dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of {3H}-vinblastine transport by P-gp . RESULTS: In cytotoxicity assays, 1 microM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM . In marked contrast, N-methyl-DN was without effect . In intact MCF7 ADR cells, DN reversed the {3H}vinblastine uptake deficit with an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive . In photoaffinity labelling studies using the arylazide {3H}-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect . However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited {3H}-B9209-005 photoaffinity labelling of P-gp . Furthermore, in membrane fragments {3H}-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent {3H}-vinblastine transport into inside-out vesicles . Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp . However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks {3H}-vinblastine transport . CONCLUSION: These data are consistent with the hypothesis that 1,4-DHPs block {3H}-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.

Int J Clin Pharmacol Ther, 2000 Mar, 38(3), 122 - 9
Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein; Wheeler R et al.; BACKGROUND: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents . The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well . Thus detailed evidence may be difficult to obtain from studies on intact cells . MATERIAL AND METHODS: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process . We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means . CONCLUSIONS: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding . We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.

Int J Clin Pharmacol Ther, 2000 Mar, 38(3), 111 - 21
Substrate recognition by P-glycoprotein and the multidrug resistance-associated protein MRP1: a comparison; Seelig A et al.; OBJECTIVES: It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 +/- 0.3 A (type I units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 +/- 0.6 A (type II units) {Seelig 1998} . Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units . In addition, we searched the putative transmembrane domains (TMD 1-12) of P-gp and (TMD 6-17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates . METHODS: The three-dimensional structures of potential MRP1 substrates were modeled with a force-field approach and were then screened for electron donor units . Helical wheel projections of the 12 putative transmembrane domains of P-gp (1-12) and MRP (6-17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with pi-electron systems . RESULTS: MRP1 recognizes compounds with type I and type II units . At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported . Transport increases with increasing number of electron donor units . Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all . An analysis of the putative transmembrane alpha-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non-hydrogen-bonding) side chains on the other side . In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with beta-electron systems . CONCLUSIONS: We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation . Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a beta-electron slide guide.

Int J Clin Pharmacol Ther, 2000 Mar, 38(3), 94 - 110
The prognostic significance of membrane transport-associated multidrug resistance (MDR) proteins in leukemia; van den Heuvel-Eibrink MM et al.; A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents . There are several ways for cancer cells to develop resistance or defense mechanisms against cytotoxic drugs . This review paper will focus on membrane transport-associated multidrug resistance (MDR) . The proteins involved, P-glycoprotein (P-gp), MRP1 and LRP/MVP, share the ability to act as drug transport proteins . Following upregulation of the mdr-1 gene, the energy-dependent transmembrane P-gp overexpression results in diminished intracellular concentrations of anthracyclins, vinca-alkaloids and epipodophyllotoxins . The other transmembrane protein, MRP1, also has intracellular epitopes which are involved in intracellular redistribution and sequestration of drugs . The last named mechanism has also been ascribed to LRP, a protein which only occurs intracellularly . In leukemia patients, cellular drug resistance profiles determined in vitro at the time of presentation show a strong correlation with outcome . In AML, mdr-1 overexpression at diagnosis is a strong independent predictor for CR and long-term survival . In ALL, mdr-1 expression is of minor importance for prediction of outcome . In AML, MRP1 expression at diagnosis is not correlated with clinical response and survival in most studies . In ALL, MRP1 expression at diagnosis is not associated with response and long-term survival in the few studies on this aspect which have been published . The studies on LRP in AML emphasize the importance of the correlation between LRP-expression and anthracycline accumulation and suggest that LRP-expression has prognostic value at diagnosis . However, there is an equal number of studies where a predictive value in the case of LRP-expression in de novo AML cannot be shown . The highest levels of LRP have been reported in multiple relapses of ALL . Furthermore, new membrane-associated drug transport proteins have been reported including the transporter associated with antigen processing (TAP), the anthracyclin resistance-associated protein (ARA), five new homologues of MRP (MRP2, or MOAT, MRP3, MRP4, MRP5, and MRP6), the sister of P-glycoprotein (sP-gp) and breast cancer resistance protein (BCRP) . Studies on the (clinical) significance of these proteins have not yet been reported.

Leuk Res, 2000 Mar, 24(3), 249 - 54
Effect of PSC 833 on the cytotoxicity and pharmacodynamics of mitoxantrone in multidrug-resistant K562 cells; Fukushima T et al.; We examined the effect of PSC 833, a nonimmunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of an anthraquinone antileukemia drug mitoxantrone (MIT) . This was done in P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells and compared with the effect of cyclosporin A (CsA) . We also compared MIT with the effect of PSC 833 on the cytotoxicity of daunorubicin (DNR) and doxorubicin (DOX) . While PSC 833 and CsA had no effect on the cytotoxicity, accumulation and retention of MIT in the parent K562 cells, PSC 833 and CsA restored accumulation and retention of MIT in K562/D1-9 cells dose-dependently . Consequently, there was increased sensitivity of K562/D1-9 cells to MIT . The reversing activity of PSC 833 on the cytotoxicity of MIT was stronger than that of CsA, and was almost the same as the reversing activity of PSC 833 on the cytotoxicity of DNR and DOX . The resistance index of MIT decreased from 43.9-fold to 2.8-fold by 0.4 microM PSC 833, which is a clinically achievable plasma concentration . These results suggest that the combination of PSC 833 with MIT could be a promising treatment in reversing PGP-mediated MDR in leukemia patients.

Cancer, 2000 Apr 1, 88(7), 1614 - 22
Growth inhibitory effects of paclitaxel on human epithelioid sarcoma in vitro: heterogeneity of response and the multidrug resistance phenotype; Reinecke P et al.; BACKGROUND: Epithelioid sarcoma is a highly malignant soft tissue tumor that is largely resistant to conventional chemotherapy and radiotherapy . Because paclitaxel has been proven to be effective in other human malignancies refractory to conventional chemotherapy, the authors analyzed the in vitro growth inhibitory effects of paclitaxel on the human epithelioid-sarcoma cell line GRU-1 and its clonal subpopulations GRU-1A, GRU-1B, and GRU-1C . METHODS: Paclitaxel-induced morphologic alterations were visualized using light microscopy, immunofluorescence microscopy, and transmission electron microscopy . The antiproliferative effects of paclitaxel on the cell lines were determined by 3-{4,5-dimethylthiazol-2-yl}-2, 5-diphenyltetrazolium' bromide (MTT) assay . The extent of paclitaxel-induced apoptosis was determined by light microscopy . The expression and function of P-glycoprotein and the multidrug resistance-associated protein (MRP) were defined by reverse transcriptase-polymerase chain reaction and fluorescence-activated cell sorter analysis . RESULTS: Paclitaxel-induced morphologic alterations such as micronucleus formation and microtubule bundles showed no significant differences between the parental cell line and its clonal subpopulations . A significant (P < 0.05) dose-dependent growth inhibition was observed in GRU-1 and its clonal subpopulations, with the IC(50) (concentration that inhibits 50%) values ranging from 0.04-0.49 microM in the different subpopulations . Paclitaxel-induced growth inhibition was accompanied by a slight increase in apoptosis . All cell lines showed an expression of and an effective function of P-glycoprotein and MRP . CONCLUSIONS: The differential response of GRU-1 and its clonal subpopulations to paclitaxel could not be predicted by the expression and function of P-glycoprotein and MRP, suggesting that other drug resistance mechanisms might be relevant in the heterogenous response observed in the epithelioid sarcoma cell lines in the current study .

Cancer Lett, 2000 Apr 14, 151(2), 161 - 7
Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action; Trindade GS et al.; Photodynamic action has been advocated as an alternative treatment of tumors but the most common used dyes, hematoporphyrin derivatives, are substrate for P-glycoprotein . This study investigated the MDR-reverting properties of methylene blue (MB) and compared the sensitivity to its photodynamic action (PDA) in five cell lines that either express or do not express the MDR phenotype . MB was able to revert the MDR phenotype and there was no difference in sensitivity to MB-PDA between MDR and non-MDR cells, suggesting that MB has the advantage of being used simultaneously as a MDR reverser and a photodynamic agent.

Cancer Lett, 2000 Feb 28, 149(1-2), 153 - 61
Non-ionic detergent Tween 80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx; Yamazaki T et al.; The non-ionic detergent Tween 80, which is used as a solvent for lipophilic drugs such as VP-16 and Taxotere, was found to reverse VP-16 resistance of the P-glycoprotein-associated multidrug resistance phenotype via increasing VP-16 influx . In adriamycin-resistant human chronic myelogenous leukemia K562 cells (K562/ADM), which overexpress mdr1 mRNA, the accumulation of VP-16 was only about 10% that in wild-type K562 cells . Tween 80 enhanced VP-16 accumulation in K562/ADM cells but did not influence VP-16 accumulation in parental K562 cells . VP-16 efflux was rapid and similar in both sensitive and resistant cell lines and was not blocked by Tween 80 or verapamil . Under glucose-free conditions, VP-16 accumulation in K562/ADM cells was only half of that in K562 cells . Tween 80 increased VP-16 accumulation in K562/ADM cells in glucose-free medium . In growth inhibition assay, Tween 80 reversed K562/ADM sensitivity to VP-16 without cell damage . Taken together, Tween 80 reverses VP-16 sensitivity in multidrug-resistant K562 cells by increasing influx, which is considered to be the primary mechanism of VP-16 resistance in K562/ADM cells.

Cancer Lett, 2000 Feb 28, 149(1-2), 143 - 51
SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein; Goulding CW et al.; SDZ PSC 833 (PSC 833) is a new multidrug resistance modulator . Recent studies have shown that the principal mechanism of action of PSC 833 is to bind P-glycoprotein (P-gp) and prevent cellular efflux of chemotherapeutic drugs . We previously reported that PSC 833 increases cellular ceramide levels . The present study was conducted to determine whether the impact of PSC 833 on ceramide generation is dependent on P-gp . Work was carried out using the drug-sensitive P-gp-deficient human breast adenocarcinoma cell line, MCF-7, and drug resistant MCF-7/MDR1 clone 10.3 cells (MCF-7/MDR1), which show a stable MDR1 P-gp phenotype . Overexpression of P-gp in MCF-7/MDR1 cells did not increase the levels of glucosylceramide, a characteristic which has been associated with multidrug resistant cells . Treatment of MCF-7 and MCF-7/MDR1 cells with PSC 833 caused similar ceramide elevation, in a dose-responsive manner . At 5.0 microM, PSC 833 increased ceramide levels 4- to 5-fold . The increase in ceramide levels correlated with a decrease in survival in both cell lines . The EC50 (concentration of drug that kills 50% of cells) for PSC 833 in MCF-7 and MCF-7/MDR1 cells was 7.2 +/- 0.6 and 11.0 +/- 1.0 microM, respectively . C6-Ceramide exposure diminished survival of MCF-7 cells; whereas, MCF-7/MDR1 cells were resistant to this short chain ceramide analog . Preincubation of cells with cyclosporine A, which has high affinity for P-gp, did not diminish the levels of ceramide generated upon exposure to PSC 833 . These results demonstrate that PSC 833-induced cellular ceramide formation occurs independently of P-gp . As such, these data indicate that reversal of drug resistance by classical P-gp blockers may be modulated by factors unrelated to drug efflux parameters.

Biochem Pharmacol, 2000 May 15, 59(10), 1245 - 52
Reversal of MRP-mediated doxorubicin resistance with quinoline-based drugs; Vezmar M et al.; The overexpression of P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) have been shown to confer broad drug resistance in tumor cells . We have demonstrated previously direct binding between MRP and a quinoline-based photoreactive drug (iodo-azido-amino quinoline, IAAQ) (Vezmar et al., Biochem Biophys Res Commun 241: 104-111, 1997) . In this report, we show the reversal of multidrug resistance in two MRP-overexpressing cell lines, HL60/AR and H69/AR, with four quinoline-based drugs . Non-toxic concentrations (5-20 microM) of chloroquine, quinine, quinidine, and primaquine potentiated the toxicity of doxorubicin in a concentration-dependent manner . These quinoline-based drugs showed a 5- to 10-fold decrease in the IC(50) of doxorubicin in H69/AR and HL60/AR cells . Primaquine was the most active, with modulation ratios of 10- and 5-fold versus 8- and 3-fold with MK-571 for H69/AR and HL60/AR, respectively . Moreover, using IAAQ, we showed that molar excesses of chloroquine, quinine, quinidine, and MK-571 inhibit the photoaffinity labeling of MRP . Primaquine and vinblastine showed lesser inhibition of MRP photoaffinity labeling by IAAQ . Taken together, the results of this study demonstrated the reversal of doxorubicin resistance with several quinoline-based drugs . Moreover, these drugs have been shown to reverse P-gp-mediated MDR and are clinically well tolerated.

FEBS Lett, 2000 Mar 24, 470(2), 156 - 60
Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants; DeRisi J et al.; The cDNA from activated mutants of the homologous transcription factors Pdr1p and Pdr3p was used to screen DNA microarrays of the Saccharomyces cerevisiae complete genome . Twenty-six overexpressed targets of the PDR1-3 and/or PDR3-7 mutants were identified . Twenty-one are new targets, the majority of which are of unknown function . In addition to well known ABC transporters, these targets appear to be involved in transport or in membrane lipids and cell wall biosyntheses . Several of the targets seem to contribute to the cell defence against a variety of stresses . Pdr1p and Pdr3p do not act similarly on all targets . Unexpectedly, the expression of 23 other genes appeared to be repressed in the PDR1-3 and/or PDR3-7 mutants . In contrast to the majority of the activated genes, none of the repressed genes contains pleiotropic drug resistance binding sites in their promoter.

J Pharmacol Exp Ther, 2000 Apr, 293(1), 230 - 6
Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4; Takanaga H et al.; The presence in orange juice of compounds that specifically inhibit the P-glycoprotein (P-gp) drug efflux transporter, but not the cytochrome P450 (CYP) isozyme CYP3A4, was investigated . The uptake of {(3)H}vinblastine, a substrate of P-gp, by Caco-2 cells was measured . An ethyl acetate extract of orange juice did not affect the initial uptake rate of {(3)H}vinblastine but significantly increased the steady-state uptake, as did cyclosporin A (20 microM), an inhibitor of P-gp . No significant effect on the uptake of 3-O-{(3)H}methylglucose or {(14)C}phenylalanine by Caco-2 cells was found, compared with the control . When the extract was separated on a Cosmosil column, the eluate with 70% methanol showed the most potent ability to increase {(3)H}vinblastine uptake . Additional separation of the 70% methanol eluate on a silica gel column with hexane-acetone (3:1) gave 3,3',4',5,6,7,8-heptamethoxyflavone (HMF) and 4',5,6,7,8-pentamethoxyflavone (tangeretin) . HMF, tangeretin, and 3',4',5,6,7,8-hexamethoxyflavone (nobiletin), another methoxyflavone contained in orange juice, all increased the steady-state uptake of {(3)H}vinblastine by Caco-2 cells in a concentration-dependent manner . The order of potency of these compounds at the concentration of 50 microM was tangeretin > HMF > nobiletin . None of these methoxyflavones inhibited 6beta-hydroxylation of testosterone catalyzed by CYP3A4 . The ethyl acetate extract of orange juice and these methoxyflavones also increased steady-state {(3)H}vinblastine uptake by LLC-GA5-COL300 cells (a cell line transfected with human MDR1 cDNA) . We conclude that these methoxyflavones enhanced vinblastine uptake by specifically inhibiting drug efflux via P-gp . They may have potential as agents for reversing multidrug resistance or for recovering the bioavailability of certain drugs.

Blood, 2000 Apr 1, 95(7), 2378 - 85
HMBA induces activation of a caspase-independent cell death pathway to overcome P-glycoprotein-mediated multidrug resistance; Ruefli AA et al.; Multidrug resistance (MDR) is often characterized by the expression of P-glycoprotein (P-gp), a 170-kd ATP-dependent drug efflux protein . As well as effluxing xenotoxins, functional P-gp can confer resistance to caspase-dependent apoptosis induced by a range of different stimuli, including Fas ligand, tumor necrosis factor, UV irradiation, and serum starvation . However, P-gp-positive cells remain sensitive to caspase-independent death induced by cytotoxic T-cell granule proteins, perforin, and granzyme B . It is, therefore, possible that agents that induce cell death in a caspase-independent manner might circumvent P-gp-mediated MDR . We demonstrated here that hexamethylene bisacetamide (HMBA) induced equivalent caspase-independent cell death in both P-gp-positive and -negative cell lines at concentrations of 10 mmol/L and above . The HMBA-induced death pathway was marked by release of cytochrome c from the mitochondria and reduction of Bcl-2 protein levels . In addition, we show that functional P-gp specifically inhibits the activation of particular caspases, such as caspases-8 and -3, whereas others, such as caspase-9, remain unaffected . These studies greatly enhance our understanding of the molecular cell death events that can be regulated by functional P-gp and highlight the potential clinical use of drugs that function via a caspase-independent pathway for the treatment of MDR tumors.

Invasion Metastasis, 1998-99, 18(5-6), 229 - 39
Effects of all-trans-retinoic acid incorporated into low-density lipoprotein on invasive properties of multidrug-resistant MCF-7 spheroids; Affoue M et al.; Cultured cells grown as spheroids provide an in vitro model that is closer to an in vivo tumour than conventional monolayer techniques . Previous work from our laboratory has demonstrated that spheroids formed from multidrug-resistant MCF-7 cells exhibit invasive characteristics which were not present in their sensitive counterparts . The treatment of these spheroids by all-trans-retinoic acid (ATRA), a potent inducer of in vitro and in vivo differentiation, decreases their proteolytic activity and ability to invade Matrigel-coated filters . The efficiency of ATRA is enhanced by its incorporation into low-density lipoprotein (LDL) (LDL-ATRA) . Indeed, invasion through a reconstituted basement membrane was reduced by 73% with 10(-6) M ATRA and 3 x 10(-8) M LDL-ATRA . Furthermore, inhibition of invasion was correlated with a decrease in several factors: (1) secreted matrix metalloproteinase-9 and enzymes degrading type IV collagen and Matrigel films, and (2) tissue plasminogen activator . The results observed were found with a concentration of LDL-ATRA 30 times lower than that of ATRA . This could be due to the protective effect of LDL and to a better targeting of cancer cells through their LDL receptors . LDL-ATRA may therefore represent a new and potent inhibitor of invasion that could be developed for clinical trials .

Eur J Pharmacol, 2000 Mar 17, 391(3), 207 - 16
Inhibition of the P-glycoprotein- and multidrug resistance protein-mediated efflux of anthracyclines and calceinacetoxymethyl ester by PAK-104P; Marbeuf-Gueye C et al.; Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or Multidrug Resistance-Associated protein (MRP(1)) . Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins . Overexpression of these transporters by tumor cells is thought to be a significant factor in both intrinsic and acquired resistance to anticancer drugs . Consequently a great deal of interest is focused on identifying chemical agents that can either antagonise drug transport by these proteins or that can inhibit the proliferation of tumors cells despite the expression of these transporters . P-glycoprotein-mediated multidrug resistance is reversed by a variety of compounds, but surprisingly, few agents reverse the MRP(1)-mediated multidrug resistance . However, it has recently been shown that 2-{4-(diphenylmethyl)-1-piperazinyl}ethyl-5-(trans-4,6-dimethyl-1, 3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P oxide (PAK-104P) was able to inhibit the P-glycoprotein and MRP(1)-mediated efflux of several compounds . Understanding of the interactions between transporters and multidrug resistance reversing agents is important in the design of more effective multidrug resistance modulators . We now examined the effect of PAK-104P on Pgp-and MRP1-mediated efflux of three anthracyclines, daunorubicin, pirarubicin, hydroxydoxorubicin and of calcein acetoxymethyl ester and calcein . Our data show that PAK-104P non-competitively inhibits the P-glycoprotein-mediated efflux of anthracycline derivatives and calcein acetoxymethyl ester with an inhibitory constant K(I)=0 . 25+/-0.05 microM . PAK-104P also non-competitively inhibits the MRP(1)-mediated efflux of daunorubicin, pirarubicin, hydroxyrubicin, calcein acetoxymethyl ester and calcein . However, surprisingly, in this case the K(I) values obtained were very different ranging from 0.06 for hydroxyrubicin to 10 microM for calcein . These data strongly suggested the existence of two different mechanisms for the inhibition by PAK-104P of the MRP(1)-mediated efflux of molecules: a first mechanism, involving a low-affinity site for PAK-104P, and which would concern molecules such as calcein, cysteinyl leukotriene LCT(4) etc . whose efflux do not depend on glutathione . A second mechanism involving a high-affinity site for PAK-104P and which would concern molecules such as anthracyclines, calcein acetoxymethyl ester whose efflux depends on the presence of glutathione.

Arch Biochem Biophys, 2000 Apr 1, 376(1), 34 - 46
Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein; Figler RA et al.; Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae . Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form . Yeast cells expressing P-gp were valinomycin resistant . Basal ATPase activity of P-gp in yeast membranes was 0 . 4-0.7 micromol/mg/min indicating excellent functionality . P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated . By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein . The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone . Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter . Glycerol was demonstrated to enhance posttranslational stability of P-gp . Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized . Turnover numbers as high as 12 s(-1) were observed . The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources . In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies .

J Neurooncol, 1999, 45(1), 37 - 46
Increased phosphorylation of DNA topoisomerase II in etoposide resistant mutants of human glioma cell line; Matsumoto Y et al.; The efficacy of the epipodophyllotoxins VP-16 and VM-26 is limited by the occurrence of drug resistance in the tumor cell population . Cellular insensitivity to drugs that stabilize the cleavable complex is frequently expressed as multidrug resistance (MDR) . In some cell lines, overexpression of MDR-1/P-glycoprotein or the multidrug resistance associated protein (MRP) has been demonstrated and implicated as the mechanism of resistance . Typically, these cells have reduced drug accumulation, secondary to increased drug efflux . In other cell lines, an atypical MDR phenotype has been identified, with the predominant mechanism of resistance shown to be qualitative and/or quantitative changes in the levels and activity of topoisomerase II . For VP-16, increased expression of MDR-1 or MRP and alterations in topoisomerase II have been shown to confer tolerance . To further understand resistance to VP-16, T98G-VP(1000) was initially isolated as a single clone from parental cell, T98G, by exposure to VP-16 . Subsequently, a population of cells from this subline was exposed to three-fold higher drug concentration allowing stable sublines to be established at higher extracellular drug concentration . Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections . Reduced topoisomerase II mRNA and protein levels were observed in the initial isolate . This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 47-fold resistance to topoisomerase II poisons . With advancing resistance, MRP expression increased, with increased VP-16 efflux and reduced accumulation . This adaptation allowed for partial restoration of topoisomerase II activity secondary to increased expression and hyperphosphorylation, with a resultant increase in growth rate . In this cell line, hyperphosphorylation coincided with increased casein kinase II mRNA protein levels, without increased PKC protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation . In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase II protein levels secondary to an acquired 615 bp deletion in one topoisomerase II allele, which resulted in reduced protein levels . In this subline, high levels of resistance were attained as a result of synergism between the reduced topoisomerase II levels and MRP overexpression . These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied . Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from additional mechanisms helping to confer high levels of resistance.

J Hepatol, 2000, 32(1 Suppl), 3 - 18
Hepatobiliary transport; Kullak-Ublick GA et al.; The alterations of hepatobiliary transport that occur in cholestasis can be divided into primary defects, such as mutations of transporter genes or acquired dysfunctions of transport systems that cause defective canalicular or cholangiocellular secretion, and secondary defects, which result from biliary obstruction . The dysfunction of distinct biliary transport systems as a primary cause of cholestasis is exemplified by the genetic defects in progressive familial intrahepatic cholestasis or by the direct inhibition of transporter gene expression by cytokines . In both, the hepatocellular accumulation of toxic cholephilic compounds causes multiple alterations of hepatocellular transporter expression . In addition, lack of specific components of bile caused by a defective transporter, as in the case of mdr2/MDR3 deficiency, unmasks the toxic potential of other components . The production of bile is critically dependent upon the coordinated regulation and function of sinusoidal and canalicular transporters, for instance of Na+-taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP) . Whereas the downregulation of the unidirectional sinusoidal uptake system NTCP protects the hepatocyte from further intracellular accumulation of bile salts, the relative preservation of canalicular BSEP expression serves to uphold bile salt secretion, even in complete biliary obstruction . Conversely, the strong downregulation of canalicular MRP2 (MRP, multidrug resistance protein) in cholestasis forces the hepatocyte to upregulate basolateral efflux systems such as MRP3 and MRP1, indicating an inverse regulation of basolateral and apical transporters The regulation of hepatocellular transporters in cholestasis adheres to the law of parsimony, since many of the cellular mechanisms are pivotally governed by the effect of bile salts . The discovery that bile salts are the natural ligand of the farnesoid X receptor has shown us how the major bile component is able to regulate its own enterohepatic circulation by affecting transcription of the genes critically involved in transport and metabolism.

Int J Cancer, 2000 Apr 1, 86(1), 101 - 7
The influence of expression of P-glycoprotein on the penetration of anticancer drugs through multicellular layers; Tunggal JK et al.; The success of chemotherapy in the treatment of solid tumours may be limited by cellular mechanisms leading to drug resistance and/or by the slow penetration of drugs through tissue, resulting in a steep concentration gradient from tumour blood vessels . One mechanism leading to the development of multidrug resistance is overexpression of the membrane-based export pump P-glycoprotein (P-gp) . The relationship between expression of P-gp by constituent cells and the penetration of P-gp substrates through tissue was studied by comparing the penetration of P-gp substrates through multicellular layers derived from either wild-type or P-gp overexpressing cell lines . P-gp reversal agents were added to confirm the contribution of P-gp in influencing the penetration of its substrates . Our data indicate: 1) penetration of the P-gp substrates, 99mTc-sestaMIBI and 14C-doxorubicin, is greater through multicellular layers formed from P-gp overexpressing cell lines as compared with wild-type cells; 2) the addition of agents that inhibit the function of P-gp results in decreased penetration of these substrates through multicellular layers with P-gp expression . There was no effect of P-gp reversal agents on penetration of 14C-sucrose or of 3H-5-fluorouracil (non-substrate controls) . Our data suggest that the administration of agents that inhibit the function of P-gp might have opposing effects on therapeutic index in solid tumours: increased sensitivity of perivascular tumour cells but decreased penetration of P-gp substrates to more distal cells . These effects may explain, in part, the limited therapeutic benefit for solid tumours that has accrued from use of agents that reverse the effects of P-gp.

Int J Cancer, 2000 Apr 1, 86(1), 95 - 100
Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer; Oguri T et al.; To investigate the role of the multidrug resistance-associated protein (MRP1) homologue MRP5 in relation to platinum drug resistance, we examined the steady-state levels of the mRNAs for MRP5 in both lung cancer cell lines and peripheral mononuclear cells (PMN) after exposure to platinum drug and in normal lung and lung cancer tissue specimens . Firstly, we examined MRP5 gene expression levels in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients who had died from lung cancer . Next, we monitored MRP5 gene expression levels within 24 hr in both lung cancer cell lines incubated with cisplatin and in PMN from 10 previously untreated lung cancer patients after carboplatin administration alone . The MRP5 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction or RNase protection assay . The MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs during their lifetime were significantly higher than those in tissues from non-exposed patients . On the other hand, the MRP5 expression levels were not rapidly induced by platinum drugs either in lung cancer cell lines or in PMN within 24 hr . Our results suggest that increased expression levels of the MRP5 gene are associated with exposure to platinum drugs in lung cancer in vivo and/or the chronic stress response to xenobiotics.

Mol Pharmacol, 2000 Apr, 57(4), 769 - 77
Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant P-glycoprotein with deleted phe(335); Chen GK et al.; In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins . Photoaffinity labeling with {(3)H}cyclosporine and {(3)H}azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp . Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/Dx5 (Dx5) cells with wild-type P-gp . Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells . Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells . Verapamil significantly reversed the {(3)H}daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on {(3)H}daunorubicin accumulation in the mutant DxP cells . Verapamil was not transported by cells expressing either mutant or wild-type P-gp . Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp . In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s) . Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp . In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity . The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.

Mol Pharmacol, 2000 Apr, 57(4), 760 - 8
Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions; Bakos E et al.; The human multidrug resistance protein MRP1 and its homolog, MRP2, are both suggested as being involved in cancer drug resistance and the transport of organic anions . We expressed MRP1 and MRP2 in Spodoptera frugiperda ovarian cells and compared their ATP-dependent transport properties and vanadate-sensitive ATPase activities in isolated membrane vesicles . Both MRP1 and MRP2 actively transported leukotriene C(4) and N-ethylmaleimide glutathione (NEM-GS), although the relative affinity of MRP2 for these substrates was found to be significantly lower than that of MRP1 . Methotrexate was actively transported by both proteins, although more efficiently by MRP2 . ATP-dependent NEM-GS transport by MRP1 and MRP2 was variably modulated by organic anions . Probenecid and furosemide inhibited, whereas under certain conditions sulfinpyrazone, penicillin G, and indomethacin greatly stimulated, MRP2-mediated NEM-GS uptake . Vanadate-sensitive ATPase activity in isolated membranes containing MRP1 or MRP2 was significantly stimulated by NEM-GS and reduced GS, although these compounds acted only at higher concentrations in MRP2 . ATP hydrolysis by MRP2 was also effectively stimulated by methotrexate . Probenecid, sulfinpyrazone, indomethacin, furosemide, and penicillin G all significantly increased MRP2-ATPase activity, whereas these compounds acted more as ATPase inhibitors on MRP1 . These results indicate that MRP1 is a more efficient transporter of glutathione conjugates and free glutathione than MRP2, whereas several anions are preferred substrates for MRP2 . Our data suggest that MRP2 may be responsible for the active secretion of pharmacologically relevant organic anions, such as diuretics and antibiotics, and indicate different modulation possibilities for MRP1 or MRP2 in drug-resistant tumor cells.

Mol Pharmacol, 2000 Apr, 57(4), 732 - 7
Rifampicin is not an activator of glucocorticoid receptor; Herr AS et al.; Rifampicin, an antibiotic widely used in tuberculosis therapy, is known to exert psychotropic side effects in some patients . Recently, rifampicin has been reported to activate the glucocorticoid receptor (GR) in human hepatocytes . Because there is evidence that increased levels of glucocorticoids may induce cognitive impairment, sometimes culminating in depression, the side effects of rifampicin may result from GR activation in central nerve cells . Therefore, we used reporter gene assays to determine whether rifampicin displays glucocorticoid-like effects in human neuroblastoma SK-N-MC cells or mouse hippocampal HT22 cells . Rifampicin was unable to elicit any detectable transactivation of GR in both cell types, whereas cortisol or dexamethasone led to a potent transcriptional response . Rifampicin was also inactive in the same HepG2 cell line that was originally used to demonstrate the effect of rifampicin on GR . Moreover, rifampicin was unable to compete with dexamethasone for binding to GR . Finally, by blocking the multidrug resistance P-glycoprotein transporter (a xenobiotic extrusion pump) with verapamil or cyclosporin A, we excluded the possibility that the lack of effect by rifampicin was due to its export from the cell . Our results establish that rifampicin does not activate GR, and rule out the hypothesis that the psychotropic side effects of rifampicin treatment are a consequence of GR activation.

Biochemistry, 2000 Mar 28, 39(12), 3424 - 32
Activation of the human P-glycoprotein ATPase by trypsin; Nuti SL et al.; The human MDR1 gene product, P-glycoprotein (Pgp), a tandemly duplicated molecule containing two putative ATP- and perhaps two drug-binding sites, is responsible for multidrug resistance in tumors . In this report, we characterized the effects of trypsinization of Pgp on its ATPase function . Incubation of Pgp-containing membranes with trypsin at a ratio of 1000:1 (w/w) resulted in a gradual increase in the basal- and the drug-stimulated ATPase activities of Pgp in a time-dependent manner . The maximal basal-, verapamil-, and vinblastine-stimulated ATPase activities of the trypsinized Pgp were approximately 1.8-, 1.5-, and 1.75-fold higher than the activities of the native Pgp, respectively . Increased basal- and drug-stimulated ATPase activities of the Pgp were also observed when the ratio of membrane protein to trypsin in the incubation mixtures was raised to 10:1 (w/w) . Immunoblotting analysis of Pgp tryptic digests using Pgp-specific NH(2)11, C219, and C494 antibodies together revealed the degradation of full-length Pgp and formation of at least eight peptides migrating in the 36-60 kDa range . Immunoprecipitation reactions using NH(2)11 and C494 antibodies have suggested that the peptides originating from the NH(2) half of Pgp are in strong association with the COOH half of the peptide . These findings suggest that while Pgp fragments together exhibit the ATPase functional characteristics, Pgp possesses a cleavage activation site or region, and its cleavage leads to the activation of basal ATPase function of Pgp.

Schweiz Med Wochenschr, 2000 Feb 26, 130(8), 282 - 90
{Tuberculosis in Switzerland--a millenium problem?}; Karrer W; In Switzerland a family physician sees a case of tuberculosis only every 4-6 years . While tuberculosis does not seem to be a major medical challenge in western countries, it is a real problem in the developing world . Nevertheless, special situations do arise in the western hemisphere where the standard therapy needs to be changed . HIV-positive patients are at greater risk of contracting tuberculosis after exposure, and therapy must be adjusted to the patient's immunological status . Multidrug-resistant tuberculosis is a major challenge to the attending physician, and takes us back to the time before antitubercular drugs existed . Standard medication is ineffective and we have to fall back on alternative drugs . Compliance of patient and physician are crucial for effective treatment, as in the case of all long-term therapy . Medical staff, and especially hospital personnel in emergency and tuberculosis departments, are at high risk of infection . Younger staff are no longer immunised by vaccination or earlier infection as they were a few decades ago . Multidrug-resistant bacteria pose a real threat for these groups and effective protection against transmission is important . Discovering a new vaccine which provides adequate immunisation is the only way to tackle the problem of tuberculosis worldwide . New drugs must also be developed to treat those already suffering from the disease.

Horm Res, 1999, 52(4), 192 - 9
Influence of IGF-I and cell density on MDR1 expression in the T-lymphoblastoid cell line CCRF-CEM; Schwarze CP et al.; The debate about a direct or indirect effect of GH and IGF-I on the recurrence of malignancy, especially in the case of rhGH therapy in patients with leukemia, is still going on . Recent studies suggested that IGF-I plays a role in drug resistance during anticancer therapy . This resistance to diverse cytotoxic drugs, named multidrug-resistance (MDR), is mainly due to high levels of P-glycoprotein (P-gp) . The gene encoding this membrane-associated transporter protein was named MDR1, and increased levels of P-gp are linked to enhanced MDR1 mRNA expression . Our aim was to investigate a possible effect of rhIGF-I on MDR1 gene expression in vitro . We cultured the T-lymphoblastoid cell line CCRF-CEM with different rhIGF-I concentrations (0, 5, 20 and 50 ng/ml) in serum-free medium for 3 days . CCRF-CEM cells are drug-sensitive and express MDR1 at low levels . MDR1 mRNA expression was measured by semiquantitative RT-PCR using a competitive assay with a heterologous DNA construct . In addition, GAPDH mRNA was amplified as an internal control for RNA integrity . P-gp activity was determined by a flow cytometric assay measuring rhodamine 123 accumulation . Furthermore, cell proliferation was monitored in all experiments . Our data do not support an effect of rhIGF-I on MDR1 mRNA expression, P-gp activity or cell proliferation in the CCRF-CEM cell line . MDR1 mRNA levels were inversely correlated to cell density with high significance (p < 0.0001) . In conclusion, multidrug resistance linked to P-gp is not induced by IGF-I in CCRF-CEM cells . At high density, CCRF-CEM cells downregulate MDR1 gene expression . Our experimental model provides a very useful tool for monitoring the influence of growth factors on multidrug resistance in vitro .

Hum Gene Ther, 2000 Mar 1, 11(4), 555 - 65
Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers; Hafkemeyer P et al.; Cancers are frequently chemoresistant because of overexpression of P-glycoprotein . Two different approaches to improve cancer treatment are currently being investigated in clinical trials: inhibition of P-glycoprotein function by reversing agents, and alleviation of leukocytopenia by MDR1 gene transfer to normal bone marrow of patients . We report here that retroviral vectors encoding a mutant P-glycoprotein (MDR1-F983A) protect hematopoietic cells from anticancer drugs even in the presence of trans-(E)-flupentixol, an inhibitor of P-glycoprotein . Transfer of either mutant or wild-type MDR1 to K562 erythroleukemia cells or primary murine bone marrow resulted in reduced accumulation of daunomycin and vinblastine because of increased drug efflux.trans-(E)-Flupentixol at concentrations up to 10 microM failed to reverse drug efflux mediated by the product of the mutant MDR1 while wild-type P-glycoprotein was inhibited . In the presence of 2 microM trans-(E)-flupentixol chemoresistance to daunomycin was circumvented only in K562 cells transduced with wild-type, but not with mutant, MDR1 . Moreover, drug resistance of KB-8-5 epidermoid cancer cells, which express the wild-type MDR1 gene at levels comparable to clinical specimens from multidrug-resistant cancers, was fully overcome in the presence of trans-(E)-flupentixol . Vectors expressing mutant P-glycoprotein may help improve chemotherapy by allowing safe dose intensification under conditions in which multidrug-resistant cancers are rendered drug sensitive by reversing agents.

Gene, 2000 Jan 25, 242(1-2), 167 - 73
Detailed structural analysis on both human MRP5 and mouse mrp5 transcripts; Suzuki T et al.; The multidrug-resistant phenotype in tumor cells is attributed in part to anti-cancer drug efflux transporters such as the MRP family . The amino-terminal structure of MRP5 has not been refined . To determine the amino-terminal structure of a major transcript of the MRP5 gene, we performed primer extension analysis to determine a major transcriptional start site of this gene and compared the structure of human MRP5 and that of mouse mrp5 . We successfully determined the structures of human MRP5 and mouse mrp5 . Estimated amino acid sequences are 1437 and 1436 amino acids for human MRP5 and mouse mrp5 respectively, and were highly conserved (94.1%) . We further showed that our previously identified SMRP mRNA was a splicing variant of the MRP5 gene, which was expressed in various human tissues, suggesting that a short form of MRP5 protein encoded by the SMRP mRNA may have a physiological role.

Jpn J Clin Oncol, 1999 Nov, 29(11), 527 - 34
Expression pattern of chemoresistance-related genes in human malignant brain tumors: a working knowledge for proper selection of anticancer drugs; Nagane M et al.; BACKGROUND: In addition to traditional modalities such as surgical intervention and radiotherapy, chemotherapy is a common therapeutic method for human malignant brain tumors . However, the effectiveness of chemotherapy is frequently hampered by cancer cell chemoresistance, resulting in an unsatisfactory outcome . To overcome this disadvantage, the proper selection of efficacious anticancer agents is required . METHODS: The expression levels of chemoresistance-related genes, MGMT, mdr1, MRP, MTIIA and GST-pi, in 28 surgical specimens of human brain tumors and in 10 human glioma cell lines were examined by Northern blot analysis . In addition, the SD10 values of human glioma cell lines against ACNU, CDDP, ADM and VP16 were estimated by a cell survival assay . RESULTS: The expression levels of each of the chemoresistance-related genes, except MRP, were generally higher in brain tumors than those in non-neoplastic brain tissues . MGMT expression correlated exclusively with ACNU resistance in all glioma cell lines examined (p = 0.0002) . The transcriptional level of mdr1 in the tumor cells correlated with the SD10 values of VCR (p = 0.04) and ADM (p = 0.034) . In contrast, the expression levels of MTIIA and GST-pi did not correlate with resistance to any of the drugs tested . A correlation of MRP mRNA expression with multidrug resistance was not apparent in the 10 cell lines tested . CONCLUSIONS: The data indicate that knowledge of the expression levels of MGMT and mdr1 may be particularly useful for a more rational selection of drugs which are not influenced by these resistance genes and which have improved efficacy against human brain tumors.

Leukemia, 2000 Mar, 14(3), 467 - 73
Novel mechanisms of drug resistance in leukemia; Ross DD; A key issue in the treatment of acute leukemia is the development of resistance to chemotherapeutic drugs . Several mechanisms may account for this phenomenon, including failure of the cell to undergo apoptosis in response to chemotherapy, or failure of the drug to reach and/or affect its intracellular target . This review focuses on the latter mechanism, and on intracellular drug transport resistance mechanisms in particular . Expression of the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) has generally been reported to correlate with prognosis in acute myeloid leukemia (AML) . Additionally, but more controversial, expression of the ABC transporter multidrug resistance protein (MRP) and the vault-transporter lung resistance protein (LRP) have been correlated with outcome in AML . Despite these findings, functional efflux assays indicate the presence of non-Pgp, non-MRP transporters in AML . Recently, a novel ABC transporter, breast cancer resistance protein (BCRP) was cloned and sequenced in our laboratory . Transfection and overexpression of BCRP in drug-sensitive cells confers drug-resistance to the cells . BCRP is a half-transporter, and may homodimerize or form heterodimers (with a yet unknown half-transporter) to produce an active transport complex . Relatively high expression of BCRP mRNA is observed in approximately 30% of AML cases, suggesting a potential role for this new transporter in drug resistance in leukemia.

J Pharm Biomed Anal, 2000 Mar, 22(2), 307 - 14
LC determination of indinavir in biological matrices with electrochemical detection; Fizzano MR et al.; A high performance liquid chromatographic (HPLC) method with electrochemical detection for the quantification of Indinavir in cell culture is described . The sample pre-treatment involved a protein precipitation procedure using acetonitrile . Chromatography was carried out on a base-deactivated reversed-phase column with an isocratic mobile phase . The method was validated with regard to specificity, linearity, limits of detection and quantitation, precision and accuracy, recovery and ruggedness . The proposed HPLC assay was utilised to directly evaluate the capability of P-glycoprotein expressing multidrug resistant cells in mediating the transport and efflux of protease inhibitor (PI) Indinavir, a basic compound in AIDS care.

Int J Oncol, 2000 Apr, 16(4), 783 - 98
Lipids: A key role in multidrug resistance? (Review); Pallares-Trujillo J et al.; Among tumoral resistances, multidrug resistance (MDR) is characterized as cross-resistance to a variety of structurally and functionally unrelated drugs such as vinca alkaloids, colchicine, and anthracyclines . Decreased drug cellular influx and increased cellular ability for drug extrusion are the main mechanisms involved in MDR . Two plasma membrane proteins, p-glycoprotein (p-gp) and the multidrug resistance-associated protein (MRP), act as ATP-dependent cellular efflux . Furthermore, protein kinase C (PKC) is also central to MDR . The present study reviews the role of cholesterol and other lipids in the reduction of drug influx and drug binding to cellular membranes . The study also examines the effect of lipid composition on p-gp activity . Concerning the role of PKC in MDR, two phospholipases involved in diacylglycerol (DG) production increase in MDR cells . These are phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C and phosphatidylethanolamine-specific phospholipase D . A positive feedback mechanism for DG production which includes these phospholipases, a phosphatidylcholine-specific phospholipase C and a phosphatidylcholine-specific phospholipase A2 has also been suggested . The hypothesis of exocytic involvement in MDR is reviewed, and some lipid changes found in MDR cells are interpreted according to those fusogenic properties normally involved in exocytic transport . Also, the possible role of lipid mediators, such as phosphatidic acid and platelet-activating factor, is examined.

Proc Natl Acad Sci U S A, 2000 Mar 14, 97(6), 2515 - 20
Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein; Sauna ZE et al.; P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells . The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle . We show that the K(d) (412 nM) of the substrate analogue {(125)I}iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM) . Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of {(125)I}iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold . To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis . Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding . These results suggest that the release of nucleotide is necessary for reactivation but not sufficient . The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion . These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule . Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.

Proc Natl Acad Sci U S A, 2000 Mar 28, 97(7), 3473 - 8
Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo; Hoffmeyer S et al.; To evaluate whether alterations in the multidrug-resistance (MDR)-1 gene correlate with intestinal MDR-1 expression and uptake of orally administered P-glycoprotein (PGP) substrates, we analyzed the MDR-1 sequence in 21 volunteers whose PGP expression and function in the duodenum had been determined by Western blots and quantitative immunohistology (n = 21) or by plasma concentrations after orally administered digoxin (n = 8 + 14) . We observed a significant correlation of a polymorphism in exon 26 (C3435T) of MDR-1 with expression levels and function of MDR-1 . Individuals homozygous for this polymorphism had significantly lower duodenal MDR-1 expression and the highest digoxin plasma levels . Homozygosity for this variant was observed in 24% of our sample population (n = 188) . This polymorphism is expected to affect the absorption and tissue concentrations of numerous other substrates of MDR-1.

Toxicol Lett, 2000 Apr 3, 114(1-3), 155 - 62
Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance; Sadzuka Y et al.; Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity . Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells . Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux . It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent . We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy . In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors . Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.

Chest, 2000 Mar, 117(3), 738 - 43
Epidemiology and ethnic distribution of multidrug-resistant tuberculosis in southern Israel, 1992-1997: the impact of immigration; Gilad J et al.; STUDY OBJECTIVES: To assess the incidence of tuberculosis in the native and immigrant populations of southern Israel in the period between 1992 and 1997, and to study the prevalence of drug resistance overall and among these subpopulations in the region in order to create guidelines for empirical antituberculous treatment in this region . DESIGN: A retrospective population-based study . SETTING: The southern district of the country and its tertiary-care hospital . PATIENTS: All new culture-proven tuberculosis cases diagnosed in adults residing in the Negev region during the study period . Patients were classified into four groups according to ethnic origin and immigration date . RESULTS: During the study period, 249 new cases involving 249 patients were recorded . Immigrants from the former Soviet Union (IFSU) were significantly younger and of male gender, and the incidence among this group rose sharply . IFSU had higher rates of resistance to any drug or drug combination . Isoniazid resistance rates were 16% overall and 32% among IFSU . Resistance to any drug was observed in 29% overall and 50% of isolates among IFSU . Multidrug-resistant tuberculosis was observed in 8.5% and 17%, respectively . CONCLUSIONS: The population of southern Israel carries very high rates of drug-resistant tuberculosis, mandating quadruple empiric treatment . IFSU should be regarded as having multidrug-resistant tuberculosis until proven otherwise, and empiric therapy with at least five drugs should be considered . This report demonstrates the influence of immigration on the incidence of tuberculosis, and the great value of local surveillance of population-specific resistance rates in an immigrant society, in order to optimize drug treatment and prevent the dissemination of resistant strains.

Mol Microbiol, 2000 Mar, 35(5), 1255 - 63
The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility; Egner R et al.; We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition . In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10 . A T1364F mutation leads to a reduction in Pdr5p-mediated azole and rhodamine 6G resistance . Like S1360F, the T1364F and T1364A mutants were nearly non-responsive to FK506 inhibition . Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p-S1360A is hypersensitive to FK506 inhibition when compared with either wild-type Pdr5p or the non-responsive S1360F variant . Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents . This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change . These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.

Am J Physiol Gastrointest Liver Physiol, 2000 Mar, 278(3), G438 - 46
Characterization of inducible nature of MRP3 in rat liver; Ogawa K et al.; We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct . In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active {Na(+)-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)} and primary active {P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6} transporters . alpha-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment . Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments . Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase . However, the MRP3 protein level was not affected by bilirubin administration . Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or alpha-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.

Int J Cancer, 2000 Mar 15, 85(6), 882 - 8
Changes in phospholipase D isoform activity and expression in multidrug-resistant human cancer cells; Fiucci G et al.; Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy and is often associated with elevated expression of drug transporters such as P-glycoprotein (P-gp) in the cancer cells . MDR is, however, accompanied by additional biochemical changes including modifications of membrane composition and properties . We have shown that MDR is associated with a massive up-regulation of caveolin expression and an elevated surface density of caveolae . We report that phospholipase D (PLD), a constituent enzyme of caveolae and detergent-insoluble glycolipid-rich membranes (DIGs), is up-regulated in human MDR cancer cells . Lysates of HT-29-MDR human colon adenocarcinoma cells, MCF-7 AdrR human breast adenocarcinoma cells and the corresponding parental drug-sensitive cells, were fractionated on discontinuous sucrose density gradients . PLD activity was found to be enriched in low density fractions that contain DIGs and caveolar membranes, and the activity in these fractions was 4- to 6-fold higher in the MDR cells compared with the parental drug- sensitive cells . Utilizing specific antibodies to PLD1 and PLD2, the distribution of PLD isoforms along the gradient was determined and the PLD localized in DIGs and caveolar membranes has been identified as PLD2 . Northern blot analysis of PLD1 and PLD2 mRNA levels has indicated that PLD2 mRNA is elevated in both HT-29-MDR and MCF-7 AdrR cells . PLD1 mRNA levels were either unchanged or reduced in the MDR cells . Finally, in vivo experiments have confirmed previous results showing that activation of PLD by phorbol esters is markedly potentiated in the MDR cells . We conclude that MDR is accompanied by an increase in PLD2 activity in DIGs and caveolar membranes .

Eur J Cancer, 2000 Feb, 36(3), 428 - 34
Regulation of cellular glutathione modulates nuclear accumulation of daunorubicin in human MCF7 cells overexpressing multidrug resistance associated protein; Benderra Z et al.; Multidrug resistance (MDR) is frequently associated with the overexpression of P-glycoprotein (Pgp) and/or multidrug resistance associated protein (MRP1), both members of the ABC superfamily of transporters . Pgp and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from tumour cells . Glutathione (GSH) has been considered to play an important role in the MRP1-mediated MDR . In our study, we examined the effects of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, on the nuclear accumulation of daunorubicin (DNR), in etoposide (VP16) and doxorubicin (ADR) resistant MCF7 cell lines, overexpressing respectively MRP1 (MCF7/VP) and Pgp (MCF7/ADR) . The study of DNR transport was carried out using scanning confocal microspectrofluorometry . This technique allows the determination of the nuclear accumulation of anthracyclines in single living tumour cells . Treatment of MCF7/VP cells with BSO increased the sensitivity of these cells to DNR whilst the cytotoxicity of the drug in MCF7/ADR cells remained unchanged . In MCF7 resistant cells treated with BSO, their GSH level decreased as observed by confocal microscopy . DNR nuclear accumulation in MCF7/VP cells was increased by BSO whereas in MCF7/ADR cells BSO was unable to significantly increase the DNR nuclear accumulation . These data suggest a requirement for GSH in MRP1-mediated resistance whilst the nuclear efflux of GSH conjugates is probably not the primary mechanism of Pgp-mediated MDR . Finally, BSO might be a useful agent in clinical assays for facilitating detection of MRP1 expression.

FEBS Lett, 2000 Mar 3, 469(1), 47 - 51
The effect of glutathione on the ATPase activity of MRP1 in its natural membranes; Hooijberg JH et al.; The transport mechanism by which the multidrug resistance protein 1 (MRP1) effluxes cytotoxic agents out of cells is still not completely understood . However, the cellular antioxidant glutathione (GSH) has been shown to have an important role in MRP1-mediated drug transport . In this study we show that GSH stimulates the ATPase activity of MRP1 in a natural plasma membrane environment . This stimulation was dose-dependent up to 5 mM . The MRP1 substrates vincristine and daunorubicin do not induce MRP1 ATPase activity . In addition, the effect of GSH on the MRP1 ATPase activity is not increased by daunorubicin or by vincristine . In contrast, a GSH conjugate of daunorubicin (WP811) does induce the ATPase activity of MRP1 . In the presence of GSH the effect of WP811 was not significantly increased . Finally, (iso)flavonoid-induced MRP1 ATPase activity is not synergistically increased by the presence of GSH . In conclusion, we show that GSH has no apparent influence on the ATPase reaction induced by several MRP1 substrates and/or modulators . The subclasses of molecules had different effects on the MRP1 ATPase activity, which supports the existence of different drug binding sites.

Ann Trop Med Parasitol, 1999 Sep, 93(6), 569 - 87
The chemotherapy of rodent malaria . LVII . Drug combinations to impede the selection of drug resistance, Part 1: Which model is appropriate?
Peters W.
The principle has finally been accepted that, whenever possible, antimalarial drugs should be deployed in appropriate combinations in endemic areas, in order to minimize the inevitability that monotherapy will, probably sooner than later, select populations of drug-resistant parasites . Which laboratory models can predict the combinations of old or novel compounds that are likely to be of practical value in minimizing this risk? Very few relevant data on the use of Plasmodium falciparum in vitro have been published . Most research has been carried out with one or other strain of chloroquine-sensitive P . berghei or with chloroquine-resistant P . yoelii ssp . NS in mice . The two most widely used procedures to select for resistance are the 'serial technique' (ST), in which drug selection pressure in vivo is gradually increased, and the '2% relapse technique' (2%RT), in which a single, large drug dose is applied at the time of each passage . Both procedures have been used to demonstrate the ability of pairs of drugs (e.g . sulfadoxine with pyrimethamine, cycloguanil with dapsone, pyrimethamine or sulphonamides with chloroquine, mepacrine or mefloquine) or triple combinations (e.g . sulfadoxine-pyrimethamine with chloroquine, mefloquine or pyronaridine) to delay the development of resistance . The relative merits of the ST and 2%RT are discussed and the data obtained by these procedures are compared with the results of deploying drug combinations in man, especially against multidrug-resistant P . falciparum . It is concluded that, even though the rodent malaria models are imperfect, no better alternatives are available at present with which to predict the value of antimalarial combinations for the protection of the individual components . The 2%RT is considered to be the procedure of first choice.

Hepatology, 2000 Mar, 31(3), 684 - 93
Zonal down-regulation and redistribution of the multidrug resistance protein 2 during bile duct ligation in rat liver; Paulusma CC et al.; We have studied regulation of the multidrug resistance protein 2 (mrp2) during bile duct ligation (BDL) in the rat . In hepatocytes isolated after 16, 48, and 72 hours of BDL, mrp2-mediated dinitrophenyl-glutathione (DNP-GS) transport was decreased to 65%, 33%, and 33% of control values, respectively . The impaired mrp2-mediated transport coincided with strongly decreased mrp2 protein levels, without any significant changes in mrp2 RNA levels . Restoration of bile flow after a 48-hour BDL period resulted in a slow recovery of mrp2-mediated transport and protein levels . Immunohistochemical detection of the protein in livers of rats undergoing BDL showed strongly reduced mrp2 staining after 48 hours, which was initiated in the periportal areas of the liver lobule and progressed toward the pericentral areas after 96 hours . Immunofluorescent detection of mrp2 in livers of rats undergoing 48 hours of BDL revealed decreased staining accompanied by intracellular localization of the protein in pericanalicular vesicular structures . Within this intracellular compartment, mrp2 colocalized with the bile salt transporter (bsep) and was still active as shown by vesicular accumulation of the fluorescent organic anion glutathione-bimane (GS-B) . We conclude that down-regulation of mrp2 during BDL-induced obstructive cholestasis is mainly posttranscriptionally regulated . We propose that this down-regulation is caused by endocytosis of apical transporters followed up by increased breakdown of mrp2, probably in lysosomes . This breakdown of mrp2 is more severe in the periportal areas of the liver lobule.

Biol Pharm Bull, 2000 Jan, 23(1), 112 - 5
Reversal of acquired resistance to doxorubicin in K562 human leukemia cells by astemizole; Ishikawa M et al.; This study demonstrates that astemizole, a non-sedating anti-histaminergic drug with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR) . Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 microM in a dose-dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration . Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytofluorographic efflux experiments with doxorubicin . Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner . Moreover, it also inhibits the photolabeling of P-glycoprotein by {3H}azidopine in a dose-dependent manner . These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.

Cancer Res, 2000 Feb 15, 60(4), 1104 - 10
The Mr 193,000 vault protein is up-regulated in multidrug-resistant cancer cell lines; Schroeijers AB et al.; Vaults are 13 megadalton ribonucleoprotein particles composed largely of the major vault protein (MVP) and two high molecular weight proteins, p240 and p193, and a small vault RNA (vRNA) . Increased levels of MVP expression, vault-associated vRNA, and vaults have been linked directly to multidrug resistance (MDR) . To further define the putative role of vaults in MDR, we produced monoclonal antibodies against the Mr 193,000 vault protein and studied its expression levels in various multidrug-resistant cell lines . We find that, like MVP, p193 mRNA and protein levels are increased in various multidrug-resistant cell lines . Subcellular fractionation of vault particles revealed that vault-associated p193 levels are increased in multidrug-resistant cells as compared with the parental, drug-sensitive cells . Furthermore, protein analysis of postnuclear supernatants and co-immunoprecipitation studies show that drug-sensitive MVP-transfected tumor cells lack this up-regulation in vault-associated p193 . Our observations indicate that vault formation is limited not only by the expression of the MVP but also by the expression or assembly of at least one of the other vault proteins.

Cancer Res, 2000 Feb 15, 60(4), 842 - 6
IDN5109, a taxane with oral bioavailability and potent antitumor activity; Nicoletti MI et al.; IDN5109 is a new taxane, derived from 14beta-hydroxy-10-deacetylbaccatin III, selected for its lack of cross-resistance in tumor cell lines expressing the multidrug resistant phenotype . Because, unlike paclitaxel, IDN5109 is a poor substrate for P-glycoprotein, we hypothesized that IDN5109 given p.o . could improve bioavailability compared with paclitaxel . Here, we studied the p.o . and i.v . pharmacokinetics of IDN5109 together with its antitumor activity . Using a high-performance liquid chromatography method, the bioavailability of IDN5109 was determined to be 48% after oral delivery . IDN5109 given p.o . was highly active against the two human ovarian carcinoma xenografts 1A9 and HOC18 (90-100% tumor regressions) and showed significant activity on the paclitaxel-resistant MNB-PTX1 xenograft (10% tumor regressions) . The p.o . administration was as active as the i.v . route at doses reflecting the pharmacokinetic data . IDN5109 is the first taxane with good oral bioavailability and potent antitumor activity and represents a potential candidate for clinical investigation.

Planta Med, 2000 Feb, 66(1), 74 - 6
Potentiating effect of obacunone from Dictamnus dasycarpus on cytotoxicity of microtuble inhibitors, vincristine, vinblastine and taxol; Jung H et al.; The limonoid triterpene, obacunone, was found to enhance the cytotoxicity of vincristine against L1210 cells by approximately 10-fold . Further, it was found that the cytotoxicity of other microtubule inhibitors such as vinblastine and taxol in drug-sensitive KB-3-1 cells as well as in multidrug-resistant KB-V1 cells was enhanced greatly in the presence of obacunone . On the other hand, there was no remarkable potentiating effect of obacunone on the cytotoxicity of other antineoplastic drugs such as adriamycin, cisplatin or 5-fluorouracil . From these results, it is implied that the potentiating action of obacunone may be limited to microtubule inhibitors.

Planta Med, 2000 Feb, 66(1), 30 - 4
Antiplasmodial activity of Cryptolepis sanguinolenta alkaloids from leaves and roots; Paulo A et al.; The roots of Cryptolepis sanguinolenta have been investigated for their chemical composition since 1931 but so far no studies on the leaves have been reported although they are used in traditional medicine in Guinea-Bissau . Two new alkaloids identified as cryptolepinoic acid (1) and methyl cryptolepinoate (2) and the known alkaloids cryptolepine (4), hydroxycryptolepine (5/5a) and quindoline (6), were isolated from the ethanolic and chlorophormic leaf extracts . Aqueous and ethanolic extracts of the leaves and roots and seven alkaloids isolated from those extracts were tested in vitro against Plasmodium falciparum K1 (multidrug-resistant strain) and T996 (chloroquine-sensitive clone) . All the extracts were shown to give 90% inhibition of P . falciparum K1 growth at concentrations < 23 micrograms/ml . Cryptolepine (4) was the most active alkaloid tested with IC50 values (0.23 microM to K1; 0.059 microM to T996) comparable with chloroquine (0.26 microM to K1; 0.019 microM to T996) . The indolobenzazepine alkaloid cryptoheptine (7) was the second most active with IC50 values of 0.8 microM (K1) and 1.2 microM (T996) . Cryptolepinoic acid (1) showed no significant activity while its ethyl ester derivative 3 was active against P . falciparum K1 (IC50 = 3.7 microM) . All the indoloquinoline alkaloids showed cross-resistance with chloroquine but not the indolobenzazepine alkaloid 7 . It was noticed that alkaloids with weakly basic characteristics were active whereas other structurally related alkaloids with different acid-base profiles were inactive . These observations are in agreement with the antimalarial mechanism of action for quinolines.

Chem Pharm Bull (Tokyo), 2000 Jan, 48(1), 150 - 3
Design, synthesis and biological activity of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo- hexopyranosyl)daunomycinone; Aligiannis N et al.; The synthesis and pharmacological evaluation of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo-hexopyrano syl) daunomycinone are described . Their cytotoxic activity was evaluated against normal and resistant cell lines . Both compounds exhibited activity against the adriamycin resistant cell line KB-A1 . These results support the hypothesis that the increased lipophilicity of the sugar part of anthracyclines is associated with their ability to overcome multidrug resistance (MDR).

Cancer Lett, 2000 Mar 31, 150(2), 147 - 53
Demonstration of MDR1 P-glycoprotein isoform expression in benign and malignant human prostate cells by isoform-specific monoclonal antibodies; Kawai K et al.; Prostate cancers are resistant to many anticancer agents at the time of presentation . P-glycoprotein (P-gp) is believed to mediate multidrug resistance phenotype . To elucidate the possible role of P-gp in such an intrinsic drug resistance of prostate cancers, its expression was examined immunohisochemically using two P-gp isoform-specific monoclonal antibodies (mAbs) with the paraffin embedded prostate samples derived from five nonmalignant and 30 untreated prostate cancer patients . In all of five normal prostate tissues, P-gp was consistently detected with both mAbs in the epithelial cells, especially at their apical site, and the level of expression was higher in the inner zone than in outer zone . On the other hand, tumor cells expressed P-gp heterogeneously in distribution and intensity; in 25 of 30 malignant cases P-gp expression was clearly demonstrated, whereas its expression was only faintly detected in other cases . However, the staining intensities for P-gp in prostate cancer cells were generally lower than in normal prostate epithelial cells . Thus, not only normal prostate epithelial cells but prostate cancer cells express at least MDR1 P-gp isoform . These results suggest that P-gp expression might play some role in intrinsic drug resistance of prostate cancer cells to many cytotoxic drugs as well as in relative resistance of the inner zone cells to the prostate carcinogenesis.

Biochem Pharmacol, 2000 May 1, 59(9), 1123 - 32
Pleiotropic resistance to diverse antimalarials in actinomycin D-resistant Plasmodium falciparum; Abrahem A et al.; The development and spread of multidrug-resistant Plasmodium falciparum are major health concerns . The molecular mechanisms of multidrug resistance, including resistance to many quinoline-based antimalarials, are largely unknown . In this study, we report on the isolation and partial characterization of actinomycin D (actD)-resistant P . falciparum (3D7(R)/actD2.3) from a chloroquine-susceptible strain, 3D7 . The stepwise selection of an actD-resistant clone (3D7(R)/actD2.3) led to the isolation and cloning of P . falciparum that grew in the presence of 2 ng/mL of actD . The parental isolate (3D7) did not grow in the presence of a 10-fold lower drug concentration (0.2 ng/mL) . The latter estimate of parasite growth was determined by direct counting of parasites in infected red blood cells . Estimates of drug resistance levels to actD, using a {(3)H}hypoxanthine uptake and incorporation method, showed a 3-fold difference in the IC(50) between 3D7 and 3D7(R)/actD2.3 . Interestingly, 3D7(R)/actD2.3 P . falciparum parasites were less sensitive to several antimalarials (chloroquine, mefloquine, quinidine, and artemisinin) and to the mitochondrial specific dye Rhodamine 123 . Drug transport studies using {(3)H}actD showed that 3D7(R)/actD2.3 accumulated less drug than 3D7 . Moreover, the accumulation of {(3)H}actD was energy dependent . To determine if Pfmdr1 expression, previously implicated in drug resistance to certain antimalarials, mediated the resistance phenotype of 3D7(R)/actD2.3, Pfmdr1 levels in 3D7 and 3D7(R)/actD2.3 were compared by Southern and northern blot analyses . Our results revealed no differences in Pfmdr1 copy number or mRNA levels between 3D7 and 3D7(R)/actD2.3 . Furthermore, comparison of Pfmdr1 sequences between 3D7 and 3D7(R)/actD2.3 showed no differences . In addition, verapamil, which reverses P-glycoprotein-mediated drug resistance in mammalian cells, did not reverse the resistance of 3D7(R)/actD2.3 to actD or chloroquine . Taken together, the findings of this study demonstrated that in vitro selection of P . falciparum for resistance to actD leads to decreased sensitivity to diverse drugs and that this pleiotropic drug resistance is associated with reduced drug accumulation not mediated by Pfmdr1.

Gen Physiol Biophys, 1999 Oct, 18 Spec No, 147 - 54
Detection of apoptosis in a heterogenous cell population using flow cytometry; Sedlak J et al.; Apoptosis induced in human leukemic cells (promyelocytic human leukemic cells HL-60, multidrug-resistant subline HL-60/VCR) and human ovarian carcinoma cells (A2780 and multidrug-resistant subline A2780/ADR) in vitro was detected by flow cytometric analysis or DNA electrophoresis . The cytofluorometric techniques utilized, i . e . detection of phosphatidylserine exposed at the outer surface of the plasma membrane, identification of cells with "sub-G0" DNA content or increased light side scatter (cell internal structure) correlated with the electrophoretic determination of DNA fragmentation ("DNA ladder") . Detection of the 34 kDa mitochondrial protein recognized by the monoclonal antibody Apo2.7 yielded elevated percentages of apoptotic cells, suggesting that this technique detecting both early and late apoptosis in digitonin-fixed cells might not be restricted to the specific detection of programmed cell death.

Clin Lab Med, 2000 Mar, 20(1), 119 - 37
The acute erythroleukemias; Mazzella FM et al.; Acute erythroleukemia is an aggressive leukemia derived from a multipotential stem cell . Three subtypes have been described: (1) M6a with greater than or equal to 30% blasts of the nonerythrocytic component, (2) M6b with greater than or equal to 30% pronormoblasts of the erythrocytic elements, and (3) M6c with greater than or equal to 30% blasts and greater than or equal to 30% pronormoblasts by the aforementioned exclusion criteria . The poor prognosis associated with this disorder positively correlates with a high pronormoblast:myeloblast ratio; unfavorable cytogenetic aberrations; a high proliferative index; and the presence of P-glycoprotein expression (multidrug resistance phenotype) . Chemotherapeutic regimens directed toward these specific parameters should be devised in order to improve the characteristically poor outcome of this patient population.

Biochemistry (Mosc), 2000 Jan, 65(1), 95 - 106
Cellular mechanisms of multidrug resistance of tumor cells; Stavrovskaya AA; Multidrug resistance (MDR) is the protection of a tumor cell population against numerous drugs differing in chemical structure and mechanisms of influence on the cells . MDR is one of the major causes of failures of chemotherapy of human malignancies . Recent studies show that the molecular mechanisms of MDR are numerous . Cellular drug resistance is mediated by different mechanisms operating at different steps of the cytotoxic action of the drug from a decrease of drug accumulation in the cell to the abrogation of apoptosis induced by the chemical substance . Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating . The most investigated mechanisms with known clinical significance are: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of the glutathione detoxification system; c) alterations of the genes and the proteins involved into the control of apoptosis (especially p53 and Bcl-2).

FASEB J, 2000 Mar, 14(3), 511 - 5
P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane; Raviv Y et al.; The multidrug resistance gene product P-glycoprotein confers drug resistance to tumor cells by acting as a transporter that blocks the entry into the cell of a great variety of drugs and hydrophobic peptides . In this study we find that in drug-resistant cells, the insertion of the influenza virus fusion protein (hemagglutinin-2) into the plasma membrane is blocked and that the fusion of the viral envelope with the plasma membrane of these cells is impaired . Multidrug-resistant cells display significant resistance to infection by envelope viruses that invade cells by fusion with the plasma membrane, but not to infection by pH-dependent viruses that penetrate cells by fusion with endocytic vesicles . These observations suggest that multidrug resistance phenomena may protect cells from infection by a large group of disease-causing viruses that includes human immunodeficiency virus, herpes simplex virus, and some cancer-inducing retroviruses.

Bioorg Med Chem Lett, 2000 Feb 7, 10(3), 203 - 7
Preparation of 5-(2,6-dideoxy-2-fluoro-alpha-L-talopyranosyloxy)-6-hydroxynap htho{2,3- f}quinoline-7,12-dione (FT-Alz), a new-type, potentially antitumor substance with various biological activities; Tsuchiya T et al.; The title compound (6), its structure being imaginatively created, has been prepared through coupling of alizarine blue (2), a classical dye, and 3,4-di-O-acetyl-2,6-dideoxy-2-fluoro-alpha-L-talopyranosyl bromide (3) . Compound 6 has considerably higher and different antitumor activity from that of doxorubicin or its analogue (10), and, further, has properties to reverse multidrug resistance (by P-glycoprotein), to inhibit topoisomerase II, and to induce apoptosis.

Anticancer Res, 1999 Nov-Dec, 19(6B), 5229 - 33
Effects of flavonoids on the growth and cell cycle of cancer cells; Choi SU et al.; In this study, we investigated the cytotoxicities of flavone (F01), 3-hydroxyflavone (F02), 6- hydroxyflavone (F03), 7-hydroxyflavone (F04), 3,6-dihydroxyflavone (F05), 5,7-dihydroxyflavone (F06) and 5,6,7-trihydroxyflavone (F07) to human cancer cells including P- glycoprotein (Pgp)-expressing HCT15 cells and its multidrug resistant subline, HCT15/CL02 cells . We also examined the effects of those flavonoids on the cell cycle of these cancer cells . HCT15/CL02 cells did not reveal resistance to all the flavonoids tested in comparison with HCT15 cells . In cell cycle analysis, all the flavonoids tested, except F01 and F04, reduced the G0/G1 population of SF295 cells at growth inhibitory concentrations, and increased G2/M (F02, F03 and F06) or S (F05 and F07) populations . In addition, F02 and F03 decreased the G2/M and G0/G1 population, and increased the S and G2/M population in HCT15 cells, respectively . Meanwhile, in HCT15/CL02 cells, F02 and F03 decreased the G0/G1 populations and increased the S population . In conclusion, we deemed that the flavonoids tested had diverse cytotoxic mechanisms, and exerted their cell growth inhibitory or killing activity by distinctive ways in different cells.

Anticancer Res, 1999 Nov-Dec, 19(6B), 5159 - 62
Acquisition of multidrug resistance in osteosarcomas, analyzed by doxorubicin binding assay, and histologic response to chemotherapy; Kusuzaki K et al.; BACKGROUND: The mechanism by which multidrug resistance is acquired in human osteosarcomas remains unclear . In this study, we analyzed the changes in doxorubicin (DOX) binding ability (%DB), showing chemosensitivity before and after chemotherapy with DOX and cisplatin (CDDP), and evaluated the histologic response of human osteosarcomas to chemotherapy . MATERIALS AND METHODS: Eight osteosarcomas were analyzed . %DB by the DOX binding assay was measured in fresh tumor tissues at biopsy and at resection after preoperative chemotherapy . The histologic response to chemotherapy was also evaluated . RESULTS: Changes in %DB before and after chemotherapy were classified into 4 patterns; A: high (> 80%) to low (< 80%), B: high to high, C: low to low, D: low to high . The two tumors, classified as type A and B were responders (> 90% necrosis), whereas the 6 tumors of type C and D were classified as non-responders (< 90% necrosis) . Based on these data, we speculated that the fraction of cell populations with different chemosensitivities to DOX and CDDP varied and found that type B tumors have no population of resistant cells, whereas type C tumors have a large fraction of resistant cells . CONCLUSION: We concluded that osteosarcomas are composed of mixed cell populations with different chemosensitivities to anticancer agents and also that survival of multidrug resistant cell populations may be the most important mechanism in acquisition of multidrug resistance of osteosarcomas after chemotherapy.

Anticancer Res, 1999 Nov-Dec, 19(6B), 5111 - 5
Multidrug resistance due to impaired DNA cleavage in a VP-16-resistant human leukemia cell line; Fukushima T et al.; We established a VP-16-resistant line of human leukemia cells, K562/VP-H2, derived from K562 cells . K562/VP-H2 cells were 44-fold more resistant to VP-16 than were K562 cells . K562/VP-H2 cells were also resistant to doxorubicin, daunorubicin and mitoxantrone, but showed little or no resistance to vincristine, aclarubicin, idarubicin, idarubicinol, cytosine arabinoside, cis-platinum or camptothecin . K562/VP-H2 cells did not over-express P-glycoprotein or multidrug resistance protein, and showed intracellular accumulation of VP-16 similar to that in K562 cells . While the expressions of topoisomerase II-alpha gene and topoisomerase II-beta gene, or catalytic activity in nuclear extract of K562/VP-H2 cells were similar to that of K562 cells, the VP-16 induced DNA cleavage was reduced in K562/VP-H2 cells compared to K562 cells, suggesting that the reduction of topoisomerase II-mediated DNA cleavage through qualitative alteration of topoisomerase II may be the main mechanism of acquired multidrug resistance for K562/VP-H2 cells . The K562/VP-H2 cell line is an interesting model for studying resistance to antileukemia drugs targeting topoisomerase II.

Anticancer Res, 1999 Nov-Dec, 19(6B), 5043 - 9
Expression of the multidrug resistance protein (MRP1) in breast cancer; Filipits M et al.; BACKGROUND: The multidrug resistance protein (MRP1) is expressed in human breast carcinomas but its clinical significance remains unclear . The aim of the present study was to determine the clinical significance of MRP1 in breast cancer patients . MATERIALS AND METHODS: MRP1 expression of primary carcinomas from 100 breast cancer patients was immunohistochemically determined by means of the monoclonal antibodies QCRL-1/QCRL-3 . RESULTS: MRP1 was negative in 20 (20%) and positive in 80 (80%) breast carcinomas . MRP1 expression was more frequent in both estrogen receptor-negative carcinomas and progesterone receptor-negative carcinomas (p = 0.1 in both cases), but was independent of tumor size and lymph node involvement . Patients with MRP1-negative carcinomas had prolongations of overall survival (p = 0.01 for death due to any cause, p = 0.04 for breast cancer-related death) and disease-free survival (p = 0.07) as compared to those with MRP1-positive carcinomas . Also in subsets of patients (negative lymph nodes; positive lymph nodes; positive estrogen receptor; T1/T2 tumors), overall survival was longer for patients with MRP1-negative carcinomas . In univariate Cox regression analyses, MRP1 positivity was associated with relative risks of 4.9 (95% CI 1.2-20.6; p = 0.03) for death due to any cause, 6.4 (95% CI 0.9-48.0; p = 0.07) for breast cancer-related death and 3.5 (95% CI 0.8-14.9; p = 0.09) for relapse . In multivariate Cox regression analyses, MRP1 positivity had relative risks of 5.1 (95% CI 1.2-21.7; p = 0.03) for death due to any cause, 6.5 (95% CI 0.8-50.1; p = 0.07) for breast cancer-related death and 3.4 (95% CI 0.8-15.1; p = 0.1) for relapse . CONCLUSIONS: Our results suggest that MRP1 might be an important factor in breast cancer indicating excellent prognosis for patients with MRP1-negative carcinomas.

Semin Oncol, 2000 Feb, 27(1 Suppl 1), 8 - 13
Ifosfamide-based drug combinations: preclinical evaluation of drug interactions and translation into the clinic; Vanhoefer U et al.; Ifosfamide is an alkylating antineoplastic agent with documented activity against a variety of solid tumor types, most notably lung cancer, testicular cancer, and sarcoma . Ifosfamide has been included in various drug combination protocols, usually on an empirical basis . To gather more insight into the mechanisms that underlie these drug interactions and to develop guidelines for further improvement of clinical combination protocols, we performed a broad preclinical evaluation program of ifosfamide-based combination regimens using isobologram analysis of drug interactions . In established cisplatin-sensitive and cisplatin-refractory ovarian carcinoma cell lines, a schedule-dependent drug interaction between paclitaxel and activated hydroperoxy-ifosfamide (4-OOH-IF) could be demonstrated . When both drugs were given for 2 hours, simultaneous exposure or the sequence of paclitaxel followed by 4-OOH-IF were additive or synergistic . In contrast, application of 4-OOH-IF before paclitaxel resulted in pronounced antagonism . Based on the sequence-dependent synergistic interactions a phase I trial was initiated with paclitaxel given on day 1 and ifosfamide given on days 2 to 5 in patients with cisplatin-refractory ovarian cancer . Four dose levels were evaluated in 18 patients . The maximum tolerated dose was paclitaxel 175 mg/m2 on day 1 and ifosfamide 2,000 mg/m2 on days 2 to 5, with central nervous system toxicity and nephrotoxicity being dose-limiting . The recommended dose for further evaluation of this combination was paclitaxel 175 mg/m2 on day 1 and ifosfamide 1,500 mg/m2 on days 2 to 5 . Although all patients were heavily pretreated with multiple agents, nine of 18 patients achieved an objective response . Ifosfamide also has been shown to reduce cellular glutathione content; thus, a series of experiments evaluated the ability of activated cyclophosphamide or ifosfamide to deplete cellular glutathione in vitro . It was demonstrated that glutathione depletion is dose- and time-dependent, with 4-OOH-IF leading to a more pronounced suppression of cellular glutathione compared with 4-OOH-Cy . The decrease in cellular glutathione content was maximal at 2 hours after drug treatment; however, cellular glutathione levels returned to normal within 24 hours . When 4-OOH-IF was combined in vitro with cisplatin, schedule-dependent interactions again became obvious . The highest antitumor activity was seen when both drugs were given concurrently; sequential application with 4-OOH-IF given before cisplatin resulted in antagonism . Since adequate glutathione levels are necessary for multidrug resistance protein (MRP) function, glutathione depletion might lead to reversal of MRP-mediated drug resistance . Preliminary data showed that 4-OOH-IF significantly decreases glutathione concentrations in MRP-expressing human HT1080/DR4 sarcoma cells, leading to maximum steady-state reduction after a 90-min exposure to 4-OOH-IF . Taken together the data reported here demonstrate that in vitro ifosfamide may potentiate the antitumor activity of a variety of cytotoxic agents and therefore merits further clinical evaluation in drug combinations (eg, taxanes, anthracyclines).

Cell Mol Neurobiol, 2000 Apr, 20(2), 231 - 53
Determinants of passive drug entry into the central nervous system; Habgood MD et al.; 1 . The blood-brain barriers restrict the passive diffusion of many drugs into the brain and constitute a significant obstacle in the pharmacological treatment of central nervous system diseases and disorders . The degree of restriction they impose is variable, with some lipid-insoluble drugs effectively excluded from the brain, while many lipid-soluble drugs do not appear to be subject to any restriction . 2 . The ease with which any particular drug diffuses across the blood-brain barrier is determined largely by the number and strength of intermolecular forces "holding" it to surrounding water molecules . By quantifying the molecular features that contribute to these forces, it is possible to predict the in vivo blood-brain barrier permeability of a drug from its molecular structure . Dipolarity, polarizability, and hydrogen bonding ability are factors that appear to reduce permeability, whereas molecular volume (size) and molar refraction are associated with increased permeability . 3 . Increasing the passive entry of "restricted" drugs into the central nervous system can be achieved by disrupting the blood-brain barrier (increased paracellular diffusion) or by modifying the structure of "restricted" drugs to temporarily or permanently increase their lipid solubility (increased transcellular permeability) . 4 . Competitive inhibition of outwardly directed active efflux mechanisms (P-glycoprotein and MRP, the multidrug resistance-related protein) can also significantly increase the accumulation of certain drugs within the central nervous system.

Cell Mol Neurobiol, 2000 Apr, 20(2), 165 - 81
Brain microvascular P-glycoprotein and a revised model of multidrug resistance in brain; Golden PL et al.; 1 . P-Glycoprotein is a 170-kDa transmembrane glycoprotein active efflux system that confers multidrug resistance in tumors, as well as normal tissues including brain . 2 . The classical model of multidrug resistance in brain places the expression of P-glycoprotein at the luminal membrane of the brain microvascular endothelial cell . However, recent studies have been performed with human brain microvessels and double-labeling confocal microscopy using (a) the MRK16 antibody to human P-glycoprotein, (b) an antiserum to glial fibrillary acidic protein (GFAP), an astrocyte foot process marker, or (c) an antiserum to the GLUT1 glucose transporter, a brain endothelial plasma membrane marker . These results provide evidence for a revised model of P-glycoprotein function at the brain microvasculature . In human brain capillaries, there is colocalization of immunoreactive P-glycoprotein with astrocytic GFAP but not with endothelial GLUT1 glucose transporter . 3 . In the revised model of multidrug resistance in brain, P-glycoprotein is hypothesized to function at the plasma membrane of astrocyte foot processes . These astrocyte foot processes invest the brain microvascular endothelium but are located behind the blood-brain barrier in vivo, which is formed by the brain capillary endothelial plasma membrane . 4 . In the classical model, an inhibition of endothelial P-glycoprotein would result in both an increase in the blood-brain barrier permeability to a given drug substrate of P-glycoprotein and an increase in the brain volume of distribution (VD) of the drug . However, in the revised model of P-glycoprotein function in brain, which positions this protein transporter at the astrocyte foot process, an inhibition of P-glycoprotein would result in no increase in blood-brain barrier permeability, per se, but only an increase in the VD in brain of P-glycoprotein substrates.

J Pediatr Hematol Oncol, 2000 Jan-Feb, 22(1), 45 - 9
Increased expression of lung resistance-related protein and multidrug resistance-associated protein messenger RNA in childhood acute lymphoblastic leukemia; Ogretmen B et al.; Immunophenotype might be an important indicator for multidrug resistance (MDR) profiles in childhood acute lymphoblastic leukemia (ALL) . The authors analyzed the messenger RNA (mRNA) levels of MDR1, multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP) by reverse transcriptase-polymerase chain reaction (RT-PCR) in childhood pre-B ALL, T-cell ALL, and acute nonlymphoblastic leukemia (ANLL) . Results showed that MRP and LRP, but not MDR1, mRNAs are overexpressed, particularly in children with pre-B ALL compared with T-cell ALL and ANLL tested . In addition, the MRP and LRP mRNA expression levels in initial diagnosis and first relapse samples of one patient with pre-B ALL were similar . Consequently, these preliminary results suggest that the expression of these MDR-related genes in childhood ALL might be regulated differently in a lineage dependent manner.

J Lab Clin Med, 2000 Feb, 135(2), 199 - 209
Competitive reverse transcription-polymerase chain reaction assay for quantification of human multidrug resistance 1 (MDR1) gene expression in fresh leukemic cells; Kobayashi H et al.; We have analyzed MDR1 gene expression in 69 clinical samples obtained from 64 patients with leukemic hematologic malignancies by using a competitive reverse transcription-polymerase chain reaction assay with a heterologous competitor RNA . To exclude a false-positive result caused by concomitant normal lymphocytes that physiologically express MDR1, in samples we determined a cut-off value of 8 amol MDR1 transcript per microgram of RNA by simultaneous measurement of rhodamine 123 dye efflux either in lymphocyte or gated leukemic cell populations . Consequently, 23 of 69 samples were concluded to be MDR1-positive in leukemic cells per se . The MDR1 expression rate was significantly correlated with factors such as a history of preceding chemotherapy, elder age of the patient, and certain disease types (eg, leukemia progressed from myelodysplastic syndrome) . Moreover, the complete response rate after chemotherapy was significantly higher in MDR1-negative patients than in MDR1-positive patients (52% vs 17%, respectively; P = .01) . The assay established will enable the quantification of MDR1 gene expression in blood samples from patients with leukemic hematologic malignancies and will be applicable to clinical laboratories as a routine test.

Hum Cell, 1999 Sep, 12(3), 115 - 23
{Gene therapy using anticancer drug-resistance genes}; Sugimoto Y; Myelosuppression is a major dose-limiting factor in cancer chemotherapy . Introduction of drug-resistance genes into bone marrow cells of cancer patients has been proposed to overcome this limitation . In theory, any gene whose expression protects cells against the toxic effects of chemotherapy should be useful in vivo for this purpose . Among such genes, human multidrug-resistance gene (MDR1) has been studied most extensively for this purpose, and clinical trials of drug-resistance gene therapy have been started in the US for cancer patients who undergo high-dose chemotherapy with autologous hematopoietic stem cell transplantation . In Japan, our clinical protocol of MDR1 gene therapy "A clinical study of drug-resistance gene therapy to improve the efficacy and safety of chemotherapy against breast cancer" has been submitted to the government . To improve the efficacy and safety of this drug-resistance gene therapy, we have constructed a series of MDR1-bicistronic retrovirus vectors using a retrovirus backbone of Harvey murine sarcoma virus and internal ribosome entry site (IRES) from picornavirus to co-express a second gene with the MDR1 gene . MDR1-MGMT bicistronic vectors can be used to protect bone marrow cells of cancer patients from combination chemotherapy with MDR1-related anticancer agents and nitrosoureas . In addition, MDR1-bicistronic retrovirus vectors can be designed to use the MDR1 gene as an in vivo selectable marker to enrich the transduced cells which express therapeutic genes, if disease is curable by the expression of a single-peptide gene in any types of bone marrow cells or peripheral blood cells.

Hum Cell, 1999 Sep, 12(3), 95 - 102
{Mechanisms for resistance to anticancer agents and the reversal of the resistance}; Akiyama S et al.; MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps . Lung resistance related protein (LRP) is also supposed to be involved in MDR . The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated . cMOAT is homologous to MRP1 and supposed to be involved in drug resistance . Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin . The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT . Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol . Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype . The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells . ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells . The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody . Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme . The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells . These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.

Gene Ther, 2000 Feb, 7(4), 348 - 58
Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice; Licht T et al.; The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo . Appropriate selection strategies, however, need to be established . To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h . Selection significantly increased chemoresistance of clonogenic progenitor cells . In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses . If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells . Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so . Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells . Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells . This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system . Gene Therapy (2000) 7, 348-358.

J Clin Oncol, 2000 Mar, 18(5), 1124 - 34
Phase I study of paclitaxel in combination with a multidrug resistance modulator, PSC 833 (Valspodar), in refractory malignancies; Fracasso PM et al.; PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of paclitaxel when given with PSC 833 (valspodar) to patients with refractory solid tumors . PATIENTS AND METHODS: Patients were initially treated with paclitaxel 175 mg/m(2) continuous intravenous infusion (CIVI) over 3 hours . Subsequently, 29 hours of treatment with CIVI PSC 833 was started 2 hours before paclitaxel treatment was initiated . In this combination, the starting dose of paclitaxel was 52.5 mg/m(2) . Paclitaxel doses were escalated by 17.5 mg/m(2) increments for four subsequent cohorts . Each cohort consisted of three patients with the exception of the last cohort, which consisted of six patients . Data for the pharmacokinetics of paclitaxel with and without concurrent PSC 833 administration were obtained . RESULTS: All 18 patients completed at least one course of concurrent treatment (median, two courses; range, one to six) and were evaluable for toxicity . The MTD for paclitaxel with PSC 833 was 122.5 mg/m(2) . Neutropenia was the DLT . All patients had PSC 833 blood concentrations greater than 1, 000 ng/mL before, during, and 24 hours after the paclitaxel infusion . PSC 833 produced small increases in the paclitaxel peak plasma concentrations and areas under the concentration-time curve . However, PSC 833 greatly prolonged the terminal phase of paclitaxel, resulting in plasma paclitaxel concentrations of more than 0.05 micromol/L for much longer than expected . As a result, myelosuppression was comparable to that produced by full-dose paclitaxel given without PSC 833 . Of the 16 patients who were assessable for response, one patient experienced a partial response and an additional nine patients experienced disease stabilization after paclitaxel treatment alone . CONCLUSION: Treatment with paclitaxel 122.5 mg/m(2) as a 3-hour CIVI concurrent with a 29-hour CIVI of PSC 833 results in acceptable toxicity . The addition of PSC 833 alters the pharmacokinetics of paclitaxel, which explains the enhanced neutropenia experienced by patients treated with this drug combination.

Int J Tuberc Lung Dis, 2000 Feb, 4(2), 115 - 7
Anti-tuberculosis drug resistance in two clinics in Ecuador; Mertz BL et al.; SETTING: Two private hospitals, one in the capital city and one in the eastern rainforest of Ecuador . OBJECTIVE: To document the prevalence of anti-tuberculosis drug resistance in Ecuador in patients who had not received prior treatment and in those who had . DESIGN: Drug resistance was determined using the proportion method with solid medium on the first isolate of Mycobacterium tuberculosis from all patients who attended the two hospitals between 1989 and 1996 . Documentation of prior treatment was obtained by patient interview . RESULTS: Resistance was identified in 39 of 161 patients (24%) who had had no prior treatment . Resistance was 14.2% to isoniazid, 11.8% to rifampin and 8.7% to both (multidrug-resistant tuberculosis) . Among 60 patients who had received prior treatment, 18 (30%) were resistant to isoniazid, and 14 (23.3%) to rifampin, while multidrug resistance was seen in 10 (16.7%) . CONCLUSION: In these populations the prevalence of resistance both in patients with no prior treatment and in patients with prior treatment was ominously high . The initial treatment regimens and patient management in Ecuador should be re-evaluated in an effort to prevent further increases in drug resistance.

Int J Tuberc Lung Dis, 2000 Feb, 4(2), 108 - 14
Using treatment failure under effective directly observed short-course chemotherapy programs to identify patients with multidrug-resistant tuberculosis; Becerra MC et al.; SETTING: Public ambulatory care centers in three districts of northern metropolitan Lima, Peru . OBJECTIVE: To document drug resistance patterns of isolates of Mycobacterium tuberculosis from patients identified as treatment failures under a model tuberculosis (TB) control program based on directly observed, short-course chemotherapy (DOT-SCC) . DESIGN: Case series . RESULTS: In a referred, consecutive sample of 173 patients identified as treatment failures on DOT-SCC, 160 (92.5%) had culture-positive TB . Of those 160, 150 (93.8%) had active, pulmonary multidrug-resistant TB (MDR-TB, resistance to at least isoniazid {INH} and rifampicin {RIF}) . Sixty of the 150 (40.0%) had isolates resistant to at least INH, RIF, ethambutol (EMB) and pyrazinamide (PZA), the initial first-line empiric treatment regimen used locally . Forty-four (29.3%) had isolates resistant to at least INH, RIF, EMB, PZA and streptomycin (SM), the first retreatment regimen . This series of patients had isolates resistant to a mean of 4.5 of the ten drugs tested . The local profile of multidrug resistance is very different from that obtained from national data from Peru . CONCLUSION: In this setting, treatment failure on DOT-SCC is strongly predictive of active MDR-TB . Because of existing local drug resistance patterns in northern Lima, 89.3% of MDR-TB patients identified as treatment failures will receive ineffective therapy with two or fewer secondary TB drugs if they are given the five-drug empiric retreatment regimen endorsed by the World Health Organization . Further short-course chemotherapy for these patients would only serve to amplify ominous existing drug resistance patterns.

Mol Pharmacol, 2000 Mar, 57(3), 634 - 41
Transport function and hepatocellular localization of mrp6 in rat liver; Madon J et al.; The multidrug resistance-associated proteins (Mrps) constitute a family of cellular export pumps of the ATP-binding cassette transporter superfamily and play an important role in hepatobiliary excretion . We investigated the transport function and subcellular localization of mrp6, a novel member of the mrp family, in rat liver . Transport studies in vesicles isolated from mrp6 expressing Sf9 cells identified the anionic cyclopentapeptide and endothelin receptor antagonist BQ-123 as a substrate of mrp6 (K(m) approximately 17 microM) . Besides BQ-123, which is also a substrate of mrp2 (K(m) approximately 124 microM), no other common substrates were found for mrp2, mrp6, and the canalicular bile salt export pump Bsep . The cyclic peptides endothelin I and Arg(8)-vasopressin were transported by mrp2 but not by mrp6 . Using a polyclonal antiserum raised against a C-terminal peptide, mrp6 was found to be localized at the lateral and, to a lesser extent, at the canalicular plasma membrane of hepatocytes . The limited overlap of the substrate specificity with the canalicular export pumps mrp2 and Bsep indicates that mrp6 does not play a major role in canalicular organic anion excretion . However, its dual localization at the lateral and canalicular plasma membrane suggests that mrp6 might fulfill a "housekeeping" transport function involved in the regulation of paracellular and/or transcellular solute movement from blood into bile.

J Biol Chem, 2000 Mar 3, 275(9), 6246 - 51
Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin . Role of p-glycoprotein in glycolipid synthesis; Lala P et al.; Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E . coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT . The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines . P-gp (but not MRP) inhibitors, e.g . ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity . Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA . In MDR1-MDCK cells, synthesis of fluorescent N-{7-(4-nitrobenzo-2-oxa-1,3-diazole)}-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA . We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3) . These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic . Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described . The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells . However, the partial drug resistance of MDCK-I cells precludes their being the parental cell . We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.

Eur J Biochem, 2000 Mar, 267(5), 1347 - 58
Characterization of the 5'-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene; Stockel B et al.; The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes . MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane . MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired . We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively . The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression . Basal promoter activity of MRP3 was only 4% of that measured for MRP2 . At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein . Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity . The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3 . Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse . The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.

Drugs Aging, 1999, 15 Suppl 1, 1 - 10
A brief history of pneumococcal vaccines; Austrian R; Attempts to control pneumococcal infection by vaccination, undertaken initially in 1911, have gone through 3 phases during the subsequent 8 decades . Initially, vaccines of killed pneumococcal cells prepared in a variety of ways were used in epidemic settings with inconclusive results, although administered to approximately 1 million recipients . The discovery that adults injected with small amounts of purified capsular polysaccharide developed antibodies to the homologous capsular type led to the trial of a tetravalent vaccine that showed conclusively its ability to prevent infection by the types represented in it . With the advent of penicillin and other effective antipneumococcal drugs, interest in prophylaxis waned . Interest in vaccination was revived only after demonstration that some segments of the population remained at high risk of death if infected and after the emergence of multidrug-resistant pneumococci . Infants and young children, among whom the incidence of pneumococcal infection is high, respond poorly to purified bacterial polysaccharides but develop satisfactory responses to bacterial polysaccharides when these are linked chemically to a protein . The early results of trials with such polysaccharide protein conjugate vaccines give promise that control of a significant portion of pneumococcal infection in the paediatric population will soon be feasible.

J Appl Microbiol, 1999 Dec, 87(6), 939 - 948
Cluster organization of the genes of Streptomyces pristinaespiralis involved in pristinamycin biosynthesis and resistance elucidated by pulsed-field gel electrophoresis; Bamas-Jacques N et al.; Streptomyces pristinaespiralis synthesizes pristinamycin, a member of the streptogramin antibiotic family which consists of a mixture of two types of chemically unrelated compounds named pristinamycins I and pristinamycins II . In order to estimate the size of the Strep . pristinaespiralis chromosome and to elucidate the organization of the pristinamycin biosynthetic and resistance genes already identified, it was decided to use the pulsed-field gel electrophoresis technique . Results indicate that the Strep . pristinaespiralis chromosome is linear and about 7580 kb, as previously shown for several other Streptomyces species . By hybridization, it could be shown that the biosynthetic and resistance genes for pristinamycins I and pristinamycins II, except for the multidrug resistance gene ptr, are interspersed and seem to be organized as a single large cluster, covering less than 200 kb corresponding to 2.6% of the total size of the chromosome . The consequences and significance of such a genetic organization are discussed.

Biochem Pharmacol, 2000 Mar 15, 59(6), 665 - 72
Effects of sodium deoxycholate and sodium caprate on the transport of epirubicin in human intestinal epithelial Caco-2 cell layers and everted gut sacs of rats; Lo YL et al.; The effects of sodium deoxycholate (Deo-Na), a bile salt, and sodium caprate (Cap-Na), a fatty acid, on the transport of epirubicin were investigated in both the human colon adenocarcinoma (Caco-2) cell line and the everted gut sacs of the rat jejunum and ileum . The possible use of these two potent absorption enhancers as multidrug resistance (MDR) reversing agents also was examined . Epirubicin uptake experiments using a flow cytometer showed that Deo-Na and Cap-Na significantly increased the accumulation of epirubicin in Caco-2 cells . These two enhancers significantly increased apical to basolateral absorption of epirubicin across Caco-2 monolayers and mucosal to serosal absorption of epirubicin in the rat jejunum and ileum . Moreover, the addition of Deo-Na or Cap-Na significantly reduced the basolateral to apical efflux of epirubicin across Caco-2 monolayers . The co-presence of verapamil, one typical P-glycoprotein (P-gp) substrate, and Deo-Na or Cap-Na demonstrated further reduction of epirubicin efflux . The study suggests that inhibition of P-gp or other transporter proteins located in the intestines may be involved, at least partially, in the reduction of epirubicin efflux . In conclusion, the therapeutic efficacy of epirubicin may be improved by the use of such low toxicity excipients as absorption enhancers and MDR modulators in formulations.

Toxicology, 2000 Jan 3, 142(2), 127 - 34
Differential sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic metals; Vernhet L et al.; The human multidrug-resistance protein (MRP1), known to mediate cellular efflux of a wide range of xenobiotics, including anticancer drugs, has also been shown to transport antimony, thereby conferring resistance to this heavy metal . The aim of the present study was to investigate whether other cytotoxic metals could be handled by MRPI using MRP1-overexpressing lung tumor GLC4/Sb30 cells . Such cells were found to be 3.4-, 12.7- and 16.3-fold more resistant than parental GLC4 cells to mercuric ion, arsenite and arsenate, respectively, whereas they remained sensitive to other cytotoxic metals tested such as copper, chromium, cobalt or aluminium . MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells to mercuric ions and arsenic while it did not significantly alter sensitivity of GLC4 cells to metals . Arsenate-treated GLC4/Sb30 cells were found to poorly accumulate arsenic through increased MK571-inhibitable efflux of the metal . Arsenate, however, failed to alter MRP1-mediated transport of known MRP1 substrates such as calcein and vincristine . In conclusion, these findings demonstrated that MRP1 likely handled some, but not all, cytotoxic metals such as arsenic and mercuric ions in addition to antimony, therefore resulting in reduced toxicity of these compounds towards MRP1-overexpressing cells.

Cytometry, 2000 Mar 1, 39(3), 195 - 202
Flow cytometric evaluation of fas expression in relation to response and resistance to anthracyclines in leukemic cells; Labroille G et al.; BACKGROUND: Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis . The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport . Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways . METHODS: Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry . Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated . Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist . RESULTS: We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin . The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11 . After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis . Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line . CONCLUSIONS: The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis . On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway .

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Sep, 29(3), 269 - 71
{Detecting expression of the multidrug resistance gene product (P170) in human tumor tissues and cells by flow cytometry}; Kan B et al.; We have detected the human multidrug resistance gene (MDR1) product P170 in 29 solid tumor samples and K562 cell line through indirectimmunoflourence staining by flow cytometry using mouse monoclonal antibody (McAb) . The results showed that the expression of P170 was detected in 18 samples, the positive ratio being 62.1%; the expression was not detected in 11 samples, the negative ratio being 37.9%; and 99.9% of K562 cells expressed P170 . In 16 of the 18 positive samples, the percent ratio of tumor cells for expression of P170 was less than 30%; in the other 2, more than 30% . This indicated that the positive ratio of P170 of most tumor samples was high, but their percent ratio of P170 was low . Thus it provided a parameter for reference in evaluating the efficacy of clinical antitumor treatments.

Hua Xi Yi Ke Da Xue Xue Bao, 1998 Mar, 29(1), 16 - 20
{Reversal of cancer multidrug resistance by Chinese medicine Ams-11, Fw-13 and Tul-17}; Li D et al.; The aim of this project was to find some kinds of Chinese materia medica as effective agents for in vitro reversal of cancer multidrug resistance . Based on the present authors' previous researches, thirty-two kinds of Chinese medicine as research meterials were selected and examined . Using cell growth inhibit assay, the authors found that three of them--Ams-11, Fw-13 and Tul-17 in the doses free from cytotoxity could enhance the sensitivity of multidrug resistant cells to anti-cancer drugs in a dose-dependent way . It seemed that these three kinds of Chinese medicine might be potential effective reversal agents.

Br J Pharmacol, 2000 Feb, 129(4), 687 - 94
Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors; Krautheim A et al.; Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT . Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8 - 10 times the initially lethal concentration) . These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis . Biochemical markers, e.g . cytochrome c release from mitochondria and increase of caspase-3-like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c . In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non-resistant HIT cells . In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase-3-like activity and DNA fragmentation was comparable in HIT and HIT100R cells . Thus, no cross-resistance between these phosphatase inhibitors seemed to exist . Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid . Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely . Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells . The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.

Zhonghua Fu Chan Ke Za Zhi, 1998 May, 33(5), 287 - 9
{Study on multidrug resistant gene (MDR1) expression between neoplastic cells and peripheral blood lymphocytes in ovarian carcinoma}; Yin G et al.; OBJECTIVE: To explore the correlation of MDR1 (P-glycoprotein, P170) gene expression P170 contents among neoplastic tissues, peripheral blood lymphocytes (PBL) and ascites cancerous cells (ACC) during chemotherapy . METHODS: Content of P170 in cancer tissue, ACC and PBL was quantiated dynamicly by using flow cytometric-immunologic method in 48 cases suffered from ovarian carcinoma . RESULTS: Cancer tissue showed positive P170 in 25% of patients . During chemotherapy, P170 content in PBL and ACC were statistically increased . A significant correlation of P170 was found between PBL and ACC (0.01 < P < 0.05) . CONCLUSION: The results indicated that multidrug resistance was produced gradually during chemotherapy . Content of P170 in PBL could probably be used as an indirect index of multidrug resistance for recurrent carcinoma or residual tumor.

Antivir Ther, 1999, 4(2), 125 - 7
Different outcome in the first two patients with an HIV-1 multinucleoside drug-resistant T69SSS insertion in Spain; Briones C et al.; A novel multidrug-resistance mechanism has been described in human immunodeficiency virus type 1 (HIV-1), which involves the insertion of 6 bp between codons 69 and 70 in the reverse transcriptase (RT) gene . Herein, we report the first two patients in Spain carrying viral populations with the 69-SS insert coupled to the T69S mutation . Both patients were selected because of the lack of signal at positions 69/70 in the LiPA RT test despite being reactive to the remaining probes on the LiPA strip . The presence of the T69SSS complex was confirmed by sequence analysis . A common feature for both subjects was their past history with zidovudine monotherapy and zidovudine plus either didanosine or zalcitabine later on in the presence of persistent virus replication . Remarkably, the introduction of triple therapy in patient 1 soon after the emergence of the insert-containing viral strain produced its total displacement, which correlated with a sustained suppression in viral load.

Antivir Ther, 1999, 4(2), 123 - 4
Efficiency of drug resistance genotypic tests in specimens with low HIV viral load; Gomez-Cano M et al.; The early recognition of resistance to antiretroviral agents could allow a rapid switch in therapy and therefore avoid the accumulation of mutations and reduce the risk of cross-resistance . However, the efficiency of genotypic tests in specimens with low viral load (VL) is severely compromised since human immunodeficiency virus (HIV) RNA in these samples often goes unrecognized . The frequency of results provided by a line probe assay (LiPA, Murex), a commercially available drug resistance test and a home-made point mutation assay (PMA) for recognizing the codon 151 multidrug-resistance mutation was examined in 664 plasma samples stratified with respect to VL values . Overall, 421 (63%) samples could be interpreted by both LiPA and PMA . The sensitivity decreased as plasma VL lowered: 89% for samples with VL > 10,000 HIV RNA copies/ml, 77% for those with VL between 500 and 10,000 HIV RNA copies/ml and 37% for specimens with VL < 500 HIV RNA copies/ml . A good agreement existed comparing the sensitivity of the home-made PMA and LiPA . Although the former tends to produce more results, the difference did not achieve statistical significance . Our results support that new, more sensitive, HIV RNA extraction methods need to be implemented for the rapid recognition of drug-resistant mutants in patients experiencing an early rebound in plasma viraemia.

J Biol Chem, 2000 Feb 25, 275(8), 5253 - 6
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis; Loo TW et al.; Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s) . To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking . In SDS gels, the cross-linked product migrated with a slower mobility than the native protein . The cross-linked products were not detected in the presence of dithiothreitol . Cross-linking was observed in 12 of 125 mutants . The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6 . In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking . Cross-linking was inhibited in the presence of ATP plus vanadate . These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide . Based on these results, we propose a model for the arrangement of the TM segments.

Cytometry, 2000 Feb 15, 42(1), 50 - 60
Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis; Boutonnat J et al.; BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy . Commonly-used DNA analysis methods cannot study both parameters simultaneously . A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively . METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance . RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity . Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug . CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients .

Ann Oncol, 1999, 10 Suppl 6, 149 - 53
New approaches in cancer treatment; Sikic BI; Major advances in cellular biology, genetics, pharmacology and immunology in the past decade are beginning to be translated into progress in cancer treatment . This progress is manifested by new cytotoxic drugs which have recently entered clinical practice (taxanes, topoisomerase I inhibitors, gemcitabine, vinorelbine, new purines), as well as the efficacy of monoclonal antibody therapies against the CD-20 antigen of B-cell lymphomas and the Her2/neu oncogene in breast cancer . Several new drugs in development are targeted at reversal or prevention of the multidrug resistance mechanism caused by expression of the MDR1 gene (P-glycoprotein) . Tumour angiogenesis as a target is being studied in several early clinical trials . As with many other biological therapies, the evaluation of these compounds and their integration with standard therapies presents a major challenge to clinical investigators . The emerging field of genomics and gene expression micro-arrays will provide enormous information about the biology of cancers . This technology offers great opportunities for the discovery of new therapeutic targets, which should provide a basis for the design and evaluation of many new agents in the coming decade.

Ann Oncol, 1999, 10 Suppl 6, 53 - 9
Modulation of multidrug resistance (MDR) in hematological malignancies; Covelli A; The term multidrug resistance (MDR) describes the observation that tumour cell lines can become cross-resistant to several structurally unrelated chemotherapeutic agents after exposure to a single cytotoxic drug . In hematological malignancies, MDR is most often associated with overexpression of P-gp, a 170-kd transmembrane glycoprotein encoded by the human MDRI gene . Indeed, P-gp expression has been correlated with drug sensitivity and clinical outcome in several studies in acute myelogenous leukemia (AML), multiple myeloma (MM), and malignant lymphomas (NHL) . A large number of compounds 'off the shelf' have been investigated for their ability to reverse the P-gp mediated MDR . However, most of these agents produced severe toxic effects at doses required to effectively block P-gp function, and modulation of P-gp in normal tissues can affect the pharmacokinetics and, thus, the toxicity of the associated chemotherapeutic agents . Phase I/IIa trials with third generation MDR modulators, such as valspodar, show that these new agents can be safely administered in combination with different chemotherapy regiments after dose adjustments of cytotoxic drugs that a P-gp substrates . Moreover, MDR reversal by valspodar has been demonstrated in the patients with AML, multiple myeloma, and non-Hodgkin's lymphoma . The definition of the clinical benefits of using MDR modulators in haematological malignancies and their full extent awaits the conclusion of the ongoing randomized phase III trials with valspondar in either newly diagnosed or resistant relapsed AML patients, and in multiple myeloma patients who have failed front-line treatment.

Am J Trop Med Hyg, 1999 Dec, 61(6), 964 - 7
No evidence of cardiotoxicity during antimalarial treatment with artemether-lumefantrine; van Vugt M et al.; Artemether-lumefantrine is a new fixed antimalarial combination effective against multidrug-resistant falciparum malaria . A prospective electrocardiographic study was conducted in 150 patients receiving artemetherlumefantrine and 50 treated with artesunate-mefloquine . There was no evidence for clinically significant changes in the electrocardiographic intervals and in particular no relationship between plasma concentrations of lumefantrine and QTc prolongation . Artemether-lumefantrine does not have significant cardiac effects at therapeutic doses.

Eur J Cancer, 1999 Oct, 35(10), 1541 - 5
Inhibition of P-glycoprotein activity and reversal of multidrug resistance in vitro by rosemary extract; Plouzek CA et al.; The transmembrane transport pump P-glycoprotein (Pgp) causes the efflux of chemotherapeutic agents from cells and is believed to be an important mechanism in multidrug resistance (MDR) in mammary tumours . In the present study we demonstrate that an extract of the common dietary herb rosemary (Rosemarinus officinalis Labiatae), increases the intracellular accumulation of commonly used chemotherapeutic agents, including doxorubicin (DOX) and vinblastine (VIN), in drug-resistant MCF-7 human breast cancer cells which express Pgp . Rosemary extract (RE) inhibits the efflux of DOX and VIN, which are known to be substrates of Pgp, but does not affect accumulation or efflux of DOX in wild type MCF-7 cells, which lack Pgp . Treatment of drug-resistant cells with RE increases their sensitivity to DOX, which is consistent with an increased intracellular accumulation of the drug . RE blocks the binding of the VIN analogue azidopine to Pgp . Thus, it appears that RE directly inhibits Pgp activity by inhibiting the binding of drugs to Pgp.

Eur J Cancer, 1999 Oct, 35(10), 1534 - 40
Effect of gamma-linolenic acid on cellular uptake of structurally related anthracyclines in human drug sensitive and multidrug resistant bladder and breast cancer cell lines; Davies CL et al.; This study investigated the effect on drug uptake in multidrug resistant cells by the incorporation of the essential fatty acid gamma-linolenic acid (GLA) . The cell lines used were the MCF-7/R resistant human breast cancer and MGH-U1/R bladder cancer . Uptake of drug (doxorubicin, epirubicin, mitoxantrone and idarubicin) after the incorporation of GLA was investigated quantitatively by flow cytometry and qualitatively by confocal microscopy . There was no observable overall increase in drug uptake due to GLA incorporation into the cells as shown by flow cytometry . However, an increase in uptake of the chemotherapeutic agent idarubicin was observed in GLA-treated resistant cells compared with untreated cells using the confocal microscope . This overall increase in cellular drug uptake was not accompanied by a change in cellular drug distribution . Only one drug, mitoxantrone, displayed a change in intracellular drug distribution due to GLA incorporation into MCF-7/R cells . This suggests that essential fatty acid incorporation into the cellular membranes of some resistant cells may cause a shift in the intracellular distribution of certain chemotherapeutic drugs.

Oncol Rep, 2000 Mar-Apr, 7(2), 257 - 60
In vivo chemosensitivity of human malignant cystosarcoma phyllodes xenografts; Ueyama Y et al.; Malignant cystosarcoma phyllodes (MCSP) is a rare breast tumor . Chemotherapeutic regimens for treatment of MCSP have not been established . We previously established an MCSP xenograft line MC-3-JCK . In this study, we established a new MCSP xenograft line, MC-10-JCK, by serial transplantation in nude mice . We studied the chemosensitivity of these two MCSP tumor xenografts to anticancer drugs in vivo . We also examined the expression of multidrug resistance-related proteins such as p-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) by immunohistochemical analysis . These two xenografts were sensitive to doxorubicin, vincristine and cyclophosphamide in vivo . Immunohistochemically, clinical specimens and xenografts were negative for Pgp and MRP expression . These results are consistent with the chemosensitivity of human MCSP to lipophilic anticancer compounds.

J Infect Dis, 2000 Feb, 181(2), 713 - 6
Selection of multiresistant hepatitis B virus during sequential nucleoside-analogue therapy; Mutimer D et al.; Hepatitis B virus (HBV) drug resistance to lamivudine is always accompanied by mutations in the viral polymerase gene at position 550, termed group 1 (M550V with L526M) or group 2 (M550I) mutations . The latter mutation has not been associated with famciclovir resistance . Thus, the addition of famciclovir to lamivudine therapy in persons with group 2 lamivudine resistance may lead to virus suppression . The effect of lamivudine/famciclovir combination therapy on HBV infection was monitored in 5 lamivudine-resistant patients by quantitative polymerase chain reaction and polymerase gene sequencing of serum virus . No patients treated with combination therapy had a decline in HBV load >1 log10 . Continual evolution of the viral polymerase was detected in association with virologic resistance to both drugs . Cloning experiments identified the preexistence of these multidrug-resistant virus variants as minority species prior to addition of famciclovir therapy . HBV resistance to lamivudine monotherapy is associated with a complex mixture of variants that limit the efficacy of second-line nucleoside-analogue therapy . First-line potent combination therapy may reduce the emergence of HBV drug resistance.

Blood, 2000 Feb 15, 95(4), 1237 - 48
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice; Schiedlmeier B et al.; Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes . We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene . Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed . The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice . SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% {mean +/- SE}) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01) . A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice . On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene . Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation . P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol . (Blood . 2000;95:1237-1248)

Ann Rheum Dis, 2000 Feb, 59(2), 146 - 8
Effects of cyclosporin at various concentrations on dexamethasone intracellular uptake in multidrug resistant cells; Maillefert JF et al.; BACKGROUND: The multidrug resistance phenomenon results from the expression of P-glycoprotein (P-gp), a drug-efflux pump . Corticosteroids are substrates for P-gp, whose function can be inhibited by cyclosporin . This study evaluates the ability of cyclosporin to modulate dexamethasone uptake in multidrug resistant cells . METHODS: The K 562 cell line, which does not express P-gp and a P-gp expressing clone, K562/ADM, were used . Cells were incubated with H3-dexamethasone in the absence or presence of cyclosporin at various concentrations . Then, cells were washed, lysed, and radioactivity was measured . RESULTS: The uptake of dexamethasone alone was higher in sensitive than in resistant cells . Addition of cyclosporin induced a dose dependent increase of dexamethasone uptake in resistant cells, whereas the drug did not influence dexamethasone uptake in parental cells . CONCLUSION: Cyclosporin, at therapeutic concentrations induces a moderate, but significant increase in dexamethasone accumulation in multidrug resistant cells . Thus, cyclosporin might increase the intestinal absorption of corticosteroids or their accumulation in mononuclear cells, or both, thereby increasing their therapeutic efficacy.

Jpn J Cancer Res, 1999 Dec, 90(12), 1380 - 6
Interaction of docetaxel ("Taxotere") with human P-glycoprotein; Shirakawa K et al.; The interaction of docetaxel ("Taxotere") with P-glycoprotein (P-gp) was examined using porcine kidney epithelial LLC-PK1 and LLC-GA5-COL150 cells, overexpressing human P-gp selectively on the apical plasma membrane by transfection of human MDR1 cDNA into the LLC-PK1 cells . The basal-to-apical transport of {14C}docetaxel in LLC-GA5-COL150 cells significantly exceeded that in LLC-PK1 cells, but the apical-to-basal transport was decreased in LLC-GA5-COL150 cells . The intracellular accumulation after its basal or apical application to LLC-GA5-COL150 cells was 4- to 20-fold lower than that of LLC-PK1 cells . Multidrug resistance (MDR) modulators, i.e., cyclosporin A and SDZ PSC 833, inhibited the basal-to-apical transport and increased the apical-to-basal transport of {14C}docetaxel in LLC-GA5-COL150 cells, but verapamil affected only apical-to-basal transport . The intracellular accumulation after basal or apical application to LLC-GA5-COL150 cells was also increased by these three MDR modulators . These observations demonstrated that docetaxel is a substrate for human P-gp, suggesting that docetaxel-drug interactions occur via P-gp . The inhibition of {14C}docetaxel transport by the MDR modulators, as well as daunorubicin and vinblastine, was also found in LLC-PK1 cells, which endogenously express P-gp at lower levels, and concentrations showing similar levels of inhibition were lower than those in the case of LLC-GA5-COL150 cells . These observations indicate that it is necessary to consider the pharmacokinetic and pharmacodynamic interactions of docetaxel via P-gp.

J Cancer Res Clin Oncol, 2000 Feb, 126(2), 111 - 6
In vitro effect of multidrug resistance modifiers on idarubicinol efflux in blasts of acute myeloid leukemia; Schroder JK et al.; Recent results show that the intracellular uptake pattern of idarubicin (IDA) in multidrug-resistant (MDR) cells is nearly identical to that seen in the drug-sensitive parent cell line, whereas the MDR cells have minimal daunorubicin (DNR) uptake compared with the drug-sensitive parent cells . It is known that the major metabolite of IDA, idarubicinol (IDA-OL), has nearly the same cytotoxicity as IDA, while the cytotoxicity of daunorubicinol (DNR-OL) is about 1/30th of that of DNR . We examined the effect of the MDR modifiers verapamil and dexniguldipine on the efflux of IDA, DNR and their hydroxylated metabolites IDA-OL and DNR-OL in blast populations of acute myeloid leukemia (AML), in the MDR-negative cell line CEM-CCRF and in their MDR-positive counterpart (CEM-VBL) . All patients with relapsed or persistent AML had been pretreated with IDA and cytosine arabinoside . The efflux of the anthracyclines was estimated by flow cytometry . A total of 36 patients with AML were investigated; 18 out of 36 AML blast populations showed an efflux of DNR, DNR-OL and IDA-OL . The efflux of DNR, DNR-OL and particularly IDA-OL could be reversed by 10 microM verapamil or 1 microM dexniguldipine . For IDA we found an effusion of 40 +/- 11% in all blast populations which could not be significantly inhibited by the modulators . Similar results for IDA were found in the MDR-positive cell line (CEM-VBL 100) and in their MDR-negative counterpart (CEM-CCRF) . The incubation of CEM-CCRF cells with DNR, DNR-OL, IDA-OL and especially IDA led to MDR induction as determined by reverse transcription/polymerase chain reaction analysis with MDR-specific primer and by cellular efflux studies . We conclude that the outcome of chemotherapy with idarubicin is influenced by MDR, although IDA is not essentially MDR-dependent itself, but because IDA-OL is actively involved in multidrug resistance . Further investigations should consider the question of whether the combination of IDA and MDR modifiers can enhance the serum level of the active metabolite IDA-OL and can reverse the MDR pattern in cells treated with IDA.

Cancer Chemother Pharmacol, 2000, 45(3), 219 - 30
Modulation of adriamycin cytotoxicity and transport in drug-sensitive and multidrug-resistant Chinese hamster ovary cells by hyperthermia and cyclosporin A; Larrivee B et al.; PURPOSE: Chemosensitizers such as cyclosporin A can increase intracellular accumulation of chemotherapeutic agents such as Adriamycin in certain multidrug-resistant (MDR) cell lines with overexpression of P-glycoprotein . It is likely that, when combined with cyclosporin A, hyperthermia could increase membrane permeability to Adriamycin and enhance its cytotoxic effects . The ability of both hyperthermia and cyclosporin A to modulate the cytotoxicity, transport and subcellular distribution pattern of Adriamycin was studied in a pleiotropic MDR Chinese hamster ovary cell line (CH(R)C5) and in the drug-sensitive parent line (AuxB1) . METHODS: Adriamycin cytotoxicity was evaluated by clonogenic cell survival, drug transport using {(14)C}-labeled Adriamycin and intracellular drug distribution by fluorescence microscopy . RESULTS: Adriamycin cytotoxicity was increased in both drug-sensitive and MDR cells by cyclosporin A (5 microM) alone, and by hyperthermia alone (41-43 degrees C) only in sensitive cells . However, when cyclosporin A and 42 degrees C hyperthermia were used in combination, a large increase in drug cytotoxicity occurred in both cell lines . This effect increased with time and was temperature-dependent . The increase in Adriamycin cytotoxicity caused by cyclosporin A and hyperthermia was accompanied by alterations in membrane permeability to the drug . Cyclosporin A increased {(14)C}Adriamycin uptake, while drug efflux decreased, for both AuxB1 and CH(R)C5 cells and nuclei . For AuxB1 cells only, drug distribution studies showed that cyclosporin A promoted an increase in both nuclear and cytoplasmic drug accumulation . Hyperthermia, combined with cyclosporin A, increased {(14)C}Adriamycin uptake . This effect was seen as an increase in intensity of nuclear and cytosolic drug fluorescence in both cell lines . Cyclosporin A alone diminished drug efflux and caused Adriamycin to remain firmly bound in the nucleus of AuxB1 cells, while it remained primarily in the cytoplasm of CH(R)C5 cells . CONCLUSIONS: Hyperthermia alone had little effect on Adriamycin cytotoxicity and transport in MDR cells, in contrast to drug-sensitive cells . This suggests that P-glycoprotein is fully functional in these MDR cells . Our findings suggest that cyclosporin A and hyperthermia could be beneficial by increasing intracellular drug accumulation, thus improving the effectiveness of Adriamycin against both drug-sensitive and MDR cells within a localized target region.

Cancer Chemother Pharmacol, 2000, 45(3), 207 - 12
Effect of P-glycoprotein modulation with cyclosporin A on cerebrospinal fluid penetration of doxorubicin in non-human primates; Warren KE et al.; PURPOSE: P-glycoprotein (Pgp) is a transmembrane drug efflux pump that is expressed in multidrug-resistant cancer cells and in a variety of normal tissues, including brain capillary endothelial cells which comprise the blood-brain barrier . We studied the effects of the Pgp inhibitor, cyclosporin A (CsA), on the cerebrospinal fluid (CSF) penetration of the Pgp substrate, doxorubicin, in non-human primates . METHODS: The animals received doxorubicin alone (2.0 mg/kg i.v . over 60 min) or doxorubicin (1 mg/kg i.v . over 60 min) and CsA (loading dose 4.0 mg/kg i.v . over 2 h, followed by continuous infusion of 12 mg/kg per day over 48 h) . Plasma and CSF were collected over 48 h and the doxorubicin concentration was measured by reverse-phase high-pressure liquid chromatography (HPLC) with fluorescence detection (detection limit 5 nM) . A two-compartment model was fitted to the plasma concentration-time data . RESULTS: Pgp was demonstrated to be present in the epithelium of the choroid plexus by immunohistochemical methods, indicating that CSF drug penetration could be used as a surrogate for blood-brain barrier penetration . Steady state whole blood CsA concentrations, which were measured with a fluorescence-polarization immunoassay (TDX) that detects both CsA and its metabolites, ranged from 551-1315 microg/l at 24 h . The clearance of doxorubicin in four animals was reduced by 34%, 38%, 45% and 49% when given with CsA . The doxorubicin concentration in the CSF was <5 nM in all animals, both after doxorubicin alone and doxorubicin with CsA . CONCLUSIONS: The Pgp inhibitor, CsA, at a concentration that alters systemic clearance of doxorubicin, does not appear to significantly increase the CSF penetration of doxorubicin.

Brain Res, 2000 Jan 3, 852(1), 116 - 26
Disruption of mdr1a p-glycoprotein gene results in dysfunction of blood-inner ear barrier in mice; Zhang ZJ et al.; P-glycoprotein (p-gp), a drug transporter in multidrug-resistant cancer cells, is a transmembrane protein encoded by mdr1a, mdr1b and mdr2 genes in mice . In our previous report, high level p-gp was immunohistochemically detected in capillary endothelial cells of the guinea pig inner ear, supporting a possible role as an extrusion pump in the blood-inner ear barrier (BIB) . We investigated the functional involvement of p-gp in the inner ear using mdr1a gene knock-out mice {mdr1a(-/-) mice} . Pharmacokinetic analyses showed that mdr1a(-/-) mice displayed obviously increased accumulations of the p-gp-transported drugs doxorubicin (adriamycin, ADM) and vinblastine in the inner ear tissues compared with those in mdr1a(+/+) mice . Subsequent functional studies using auditory-evoked brainstem responses showed hearing impairment only in mdr1a(-/-) mice after administering these drugs . Furthermore, inhibition of p-gp function by co-administration of cyclosporin A (CsA) with doxorubicin (ADM) in mdr1a(+/+) mice resulted in increased accumulation of ADM in inner ear tissues and hearing impairment similar to that noted in mdr1a(-/-) mice . We conclude that mdr1a p-gp, which acts as an efflux pump in the inner ear, prevents ototoxicity induced by p-gp substrate drugs and contributes to a new functional mechanism in the BIB.

Nurs Times, 1999 Sep 1-7, 95(35), 56, 59 - 60
Infection control . A long time coming; Hateley P et al.; This article presents the case of a doctor who developed multidrug-resistant tuberculosis in his right lung . Development of the disease was attributed to treatment errors and resulted in surgical intervention to effect a cure . The isolation and management of this patient spanned a total of 12 months . Infection control interventions to minimise the effects of sensory deprivation, given the length of stay of the patient, appear to have been satisfactory, with no treatment for any clinical depression required . The availability of negative pressure ventilation and the then controversial use of masks prevented any nosocomial transmission of MDR TB . Use of masks resulted in a two-tier system of infection control . It was difficult to make such a decision in the absence of any published UK guidelines . Guidelines have subsequently been published.

Biochem Pharmacol, 2000 Mar 1, 59(5), 467 - 70
Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells; Bock KW et al.; Treatment of Caco-2 cells with the antioxidants quercetin or t-butylhydroquinone led to induced protein levels of UDP-glucuronosyltransferase UGT1A6 (ca . 3-fold over controls) and of the apical conjugate export pump multidrug resistance protein 2 (MRP2; 1.9-fold over controls) . In contrast to UGT1A6, MRP2 (symbol ABCC2) was not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin . Immunocytochemistry demonstrated that MRP2 was only expressed at the brush border domain of Caco-2 cell monolayers . The results indicate that UGT1A6 and MRP2 are coordinately induced by antioxidants, facilitating chemoprotection against phenolic toxins and excretion of conjugates into the intestinal lumen.

Bioorg Med Chem, 1999 Dec, 7(12), 2691 - 5
Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of P-glycoprotein-mediated multidrug resistance; Bois F et al.; A series of 4-alkoxy-2',4',6'-trihydroxychalcones have been synthesized and evaluated for their ability to inhibit P-glycoprotein-mediated multidrug resistance (MDR) by direct binding to a purified protein domain containing an ATP-binding site and a modulator-interacting region . The introduction of hydrophobic alkoxy groups at position 4 led to much more active compounds as compared to the parent chalcone . The binding affinity increased as a function of the chain length, up to the octyloxy derivative for which a K(D) of 20 nM was obtained.

Folia Med (Plovdiv), 1998, 40(3), 51 - 7
Quantitative analysis of the expression of apoptosis-related genes; Markova V; BACKGROUND: The development and subsequent introduction of current molecular biology techniques allows the elucidation of some of the mechanisms of the programmed cell death (apoptosis) . The overexpression of the genes directly (B-cell leukemia/lymphoma-2, BCL-2) or indirectly (multidrug resistance gene-1, MDR1) involved in the suppression of apoptosis, as well as the low-level expression of their antagonists (BCL-2 associated X-protein, BAX) result in longer survival and the development of drug resistance in malignant cells . AIM: The objective of this study was to design our own appropriate primers and to synthesize internal RNA standards and this way to evaluate the rate of expression of the MDR1, BCL-2 and BAX genes using a competitive reverse transcription and polymerase chain reaction (cRT-PCR) . MATERIAL AND METHODS: Peripheral blood or bone marrow aspirates from 5 patients and 3 healthy controls were used for RNA extraction . Competitive RT-PCR was employed utilizing the constructed specific primers and synthesized internal RNA standards . Following electrophoresis, the PCR products stained with ethidium bromide were evaluated on the "Gel Doc 1000TM" video image system . RESULTS: The constructed primers specific for each of the three genes and synthesized internal RNA standards with a suitable deletion allow receiving the yield of well-separated competitive amplicons . The measured band's intensity of light and the calculated standard/patient ratio were employed in the analysis of the MDR1, BCL-2 and BAX gene expression . CONCLUSIONS: Competitive RT-PCR is a non-radioactive and comparatively rapid molecular biology method suitable for everyday laboratory practice . The possibility for simultaneous evaluation of the expression of several genes could help in the elucidation of some of the mechanisms underlying the prolonged survival of malignant cells.

Cancer Lett, 1999 Nov 15, 146(2), 117 - 26
Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR; de Bruin M et al.; The emergence of several newly identified members of the ABC transporter family has necessitated the development of antagonists that are able to inhibit more than one transporter . We assessed the ability of the chemosensitizer GF120918 to function as a multispecific antagonist using cytotoxicity assays, rhodamine and calcein efflux assays, and confocal microscopy in cell lines expressing different multidrug resistance transporters . At a concentration of 1 microM in cytotoxicity assays, GF120918 was able to sensitize both S1-B1-20, a subline expressing P-glycoprotein (Pgp), and S1-M1-80, a subline expressing a newly identified mitoxantrone transporter, MXR . GF120918 was ineffective in sensitizing MRP-overexpressing MCF-7 VP-16 cells to etoposide as determined by cytotoxicity studies . In flow cytometry experiments, rhodamine 123 efflux in S1-B1-20 cells was decreased at GF120918 concentrations as low as 25-50 nM, with 250 nM giving complete inhibition of rhodamine efflux . Complete inhibition of rhodamine efflux in mitoxantrone-resistant S1-M1-80 cells required 10 microM . Examination of intracellular mitoxantrone accumulation by confocal microscopy confirmed higher levels of mitoxantrone in S1-B1-20 and S1-M1-80 cells when incubated in the presence of GF120918 than when incubated with mitoxantrone alone . Thus, GF120918 appears to fit the paradigm of a multispecific blocker and is able to block rhodamine and mitoxantrone efflux by the newly identified mitoxantrone transporter . Further studies of this compound should be pursued to determine its feasibility for use in the clinic.

Mutat Res, 1999 Dec 16, 431(1), 25 - 30
Adaptive response towards adriamycin in vitro: circumvention with verapamil; Anuszewska EL et al.; In an attempt to identify mechanisms of adaptive response to adriamycin (ADR), we have earlier isolated ADR-resistant cell lines CHO/R and ME18/R by short-term pulse exposures of parent cell lines to this drug, followed by single-cell cloning . The results presented in this study have shown that the development of resistance to ADR was accompanied by cross-resistance to vinblastine and methotrexate . The resistance of tested cell lines towards ADR was substantially reversed by verapamil (VPL) at non-toxic concentrations . VPL abolished also the capability of these cell lines to express adaptive response after treatment of the cells with a conditioning dose of ADR . From the results of our study, we conclude that similar characteristics play a role in the mechanism of the phenomenon of adaptive response as in the mechanism of pleiotropic multidrug resistance.

Ann Trop Med Parasitol, 1999 Jun, 93(4), 325 - 9
The chemotherapy of rodent malaria . LVI . Studies on the development of resistance to natural and synthetic endoperoxides; Peters W et al.; Chloroquine-sensitive Plasmodium berghei N and chloroquine-resistant P . yoelii ssp . NS were exposed to selection pressure, in the '2% relapse technique', from artemisinin, artesunate, a bicyclic, synthetic endoperoxide Ro 41-3823 (an analogue of arteflene) or Fenozan B07, a synthetic 1,2,4-trioxane endoperoxide . Whereas resistance against artemisinin did develop to a moderate level in both parasites, only a low level of resistance or none developed to the other compounds, and resistant parasites readily lost resistance once drug-selection pressure was withdrawn . The relevance of these observations and the experience of other investigators are discussed in relation to the possible risk that resistance may be developed in nature once endoperoxides are deployed widely against multidrug-resistant P . falciparum.

Cytometry, 2000 Jan 1, 39(1), 16 - 25
Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis; Bour-Dill C et al.; BACKGROUND: Anthracycline resistance is known to be mediated by P-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs . METHODS: The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction . Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution . Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling . RESULTS: An original image analysis procedure was developed to investigate daunorubicin and organelles co-localization . The reliability of the image analysis was controlled through chromatic shift and intensity linearity measurement using calibrated microbeads . The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated . Although no rational relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells . CONCLUSIONS: In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular vesicles identified as Golgi vesicles and resistance to apoptosis induction .

J Natl Cancer Inst, 2000 Feb 2, 92(3), 217 - 24
Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells; Leoni LM et al.; BACKGROUND: Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death . Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity . In this study, we characterized the activity of this new antimitotic drug toward malignant cells . METHODS: We tested antiproliferative activity with an MTT {i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay . RESULTS/CONCLUSIONS: Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells . Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells . Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation . Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells . IMPLICATIONS: The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Int J Tuberc Lung Dis, 2000 Jan, 4(1), 69 - 75
Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin; McNerney R et al.; OBJECTIVE: To investigate rapid detection of drug-resistant tuberculosis using the genotypic Inno-LiPA Rif TB assay and a novel, low-cost, bacteriophage-based susceptibility assay . DESIGN: The performance of the microwell phage replication assay (MPRA) on 18 isolates from suspected multidrug-resistant tuberculosis patients was compared to the LiPA assay performed directly on sputum specimens . Mutations in the rpoB gene identified by LiPA that confer resistance to rifampicin (RMP) were confirmed by DNA sequencing, while susceptibilities were confirmed by the proportion method and BACTEC . A further 19 isolates undergoing routine screening for both RMP and streptomycin susceptibility were included for comparison . RESULTS: Susceptibility to RMP was determined for 17/18 (94.4%) sputum specimens tested by LiPA . Correlation between MPRA, molecular and conventional methods was 100% for the detection of RMP susceptibility . However, for susceptibility to streptomycin one discrepant result was found: an isolate susceptible to streptomycin by the proportion method was found resistant by MPRA to 2 microg/ml of streptomycin . Similarly, an isolate initially resistant by MPRA upon re-testing was found susceptible in agreement with the conventional method . CONCLUSION: LiPA enables rapid detection of drug-resistant infection, while MPRA offers simple, low-tech testing of drug susceptibilities that may be appropriate for application in low-income countries.

Int J Tuberc Lung Dis, 2000 Jan, 4(1), 61 - 8
Effectiveness of infection control measures in controlling a nosocomial outbreak of multidrug-resistant tuberculosis among HIV patients in Italy; Moro ML et al.; SETTING: Between October 1992 and February 1994, 33 cases of multidrug-resistant tuberculosis (MDR-TB) were diagnosed among patients infected by the human immunodeficiency virus (HIV) and hospitalised in an HIV ward in Milan, Italy . This outbreak was part of a much larger outbreak, begun in another hospital and probably transferred through a patient . OBJECTIVE: To evaluate risk factors for transmission and the effectiveness of infection control measures . DESIGN: 1) Active follow-up of exposed patients, 2) cohort study among HIV-infected patients exposed to MDR-TB cases before and after the implementation of control measures, 3) screening of close contacts of MDR-TB cases, and 4) molecular typing by restriction fragment length polymorphism (RFLP) analysis . RESULTS: The risk of MDR-TB was higher in patients with lower CD4+ lymphocyte percentages and longer duration of exposure . No difference in the daily risk was observed for in-patients vs day-hospital patients or by room distance from an infectious case . Of the 90 patients exposed before the implementation of infection control measures (i.e., October 1992-June 1993) 26 (28.9%) developed MDR-TB, whereas none of the 44 patients exclusively exposed after implementation developed MDR-TB, despite the continuing presence of infectious MDR-TB cases in the ward . CONCLUSION: Simple control measures were effective in significantly reducing nosocomial transmission among patients.

Int J Tuberc Lung Dis, 2000 Jan, 4(1), 18 - 25
Transmission of tuberculosis in an endemic urban setting in Brazil; Ferrazoli L et al.; SETTING: Two out-patient facilities in Sao Paulo, Brazil . OBJECTIVE: To study the transmission pattern of tuberculosis (TB) among human immunodeficiency virus (HIV) infected and uninfected persons in a setting endemic for TB . DESIGN: A prospective study comparing HIV-seropositive and -seronegative TB patients identified consecutively between 1 March 1995 and 1 April 1997 . The patients were stratified according to their Mycobacterium tuberculosis isolate IS6110 RFLP patterns . Risk factors were sought for infection with an RFLP cluster pattern strain, inferred to represent recent transmission . RESULTS: Fifty-eight (38%) of 151 HIV-seropositive patients and 36 (25%) of 142 HIV-seronegative patients were infected with M . tuberculosis isolates that belonged to cluster patterns (OR 1.84, 95% CI 1.08-3.13) . Multidrug-resistant (MDR) strains were isolated from 19 patients, all of whom were HIV seropositive; 12 (63%) of these, and 46 (35%) of 132 drug-susceptible isolates had cluster patterns (OR 3.20, 95% CI 1.08-9.77) . CONCLUSION: In a TB-endemic urban setting in Brazil, the proportion of cases resulting from recent transmission appears to be greater among HIV-seropositive than among HIV-seronegative patients . A large proportion of MDR-TB (63%) cases was caused by strains that had cluster RFLP patterns, suggesting recent transmission of already resistant organisms . This type of knowledge regarding TB transmission may help to improve locally appropriate TB control programs.

Anticancer Res, 1999 Jul-Aug, 19(4B), 3327 - 31
Effect of mitoxantrone liposomes on multidrug-resistant breast cancer cells; Poujol S et al.; A major obstacle in efficacy of breast cancer chemotherapy is the emergence of multidrug resistance . We investigated modulation of multidrug resistance by liposome-encapsulated mitoxantrone in a drug resistant human breast MCF7R cell line and the influence of liposome composition . Neutral high phase-transition temperature and anionic low phase-transition temperature phospholipid liposomes, reduced the resistance factor from 142 to 15 and 38, respectively . The higher cytotoxicity obtained with mitoxantrone-encapsulation was not necessarily related to higher intracellular uptake . Our data suggest that liposomes, according to their lipid composition, may alter the P-glycoprotein function by plasma membrane stabilization and modulate multidrug resistance in human cancer.

Anticancer Res, 1999 Jul-Aug, 19(4B), 3243 - 8
Multidrug resistance-associated protein (MRP) mediated transport of daunomycin and leukotriene C4 (LTC4) in isolated plasma membrane vesicles; Ding GY et al.; Studies have been carried out to examine in vitro drug transport in plasma membrane vesicles isolated from HL60/ADR cells that overexpress MRP . The results demonstrate that glutathione (GSH) enhances transport of daunomycin . A greater increase in transport activity occurs when the reaction is carried out in the presence of both GSH and sodium chloride . Sodium chloride alone has no effect on daunomycin transport . It has also been observed that GSH in the presence of sodium chloride induces a major increase in the transport level of LTC4 . Thus far, no metal ion other than sodium chloride has been found to be active in the drug transport system . Kinetic analysis reveals that GSH in the presence of sodium chloride greatly reduces Km and increases Vmax, for daunomycin . Additional studies show that ATPase activity in isolated plasma membrane from HL60/ADR cells is greatly enhanced in the presence of both GSH and sodium chloride . These results suggest the possibility that GSH and sodium chloride stimulate MRP-mediated transport as a result of increased ATPase activity.

Anticancer Res, 1999 Jul-Aug, 19(4B), 3109 - 23
Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs; Orlowski S et al.; P-glycoprotein (P-gp) is an active, ATP-dependent plasma membrane transporter which is responsible for the expulsion of various cytotoxic drugs with different chemical structures out of resistant (MDR) cells . It is also capable of transporting a number of other amphiphilic molecules, the so-called MDR-reversing agents, which belong to a very broad variety of chemical families . Moreover, P-gp can also play a role in steroid secretion and cellular detoxification by transporting various other substrates . In this review, we address the problem of the multiple recognition by P-gp of such a large number of amphiphilic molecules . This is both (i) from a basic viewpoint in order to discuss the underlying molecular mechanisms explaining how the general rule of substrate-enzyme specificity can be violated, and (ii) from a more applied pharmacological viewpoint to show in detail how the interaction of various drugs with P-gp leads to important consequences in terms of the relative effects of these drugs in the anticancer chemotherapy context, as well as for their pharmacokinetic distributions in the whole organism, rationalizing possible adverse drug reactions . In particular, we will present evidence that, independently of the technique used, the mutual interactions between P-gp transport substrates cannot always be reduced to simple competitive effects.

Anticancer Res, 1999 Jul-Aug, 19(4B), 2885 - 91
The use of liposomal anticancer agents to determine the roles of drug pharmacodistribution and P-glycoprotein (PGP) blockade in overcoming multidrug resistance (MDR); Krishna R et al.; Many attempts to circumvent P-glycoprotein (PGP)-based multidrug resistance (MDR) in cancer chemotherapy have utilized PGP blocking agents (also referred to as MDR modulators), which are co-administered with the anticancer drug . This approach is based on the premise that inhibiting PGP function will result in increased accumulation of many anticancer drugs in the tumor cells and restore full antitumor activity . However, co-administration of MDR modulators with anticancer drugs has often resulted in exacerbated toxicity of the anticancer drugs and limited chemosensitization of MDR tumors . These problems appear to be related to MDR modulator blockade of PGP excretory functions in healthy tissues, such as liver and kidney, which markedly reduces anticancer drug clearance properties . Two consequences of these pharmacokinetic interactions are: 1 . Increased toxicity due to modulator-induced changes in biodistribution properties of the anticancer drug . 2 . Problems interpreting preclinical and clinical data with respect to: a) Are therapeutic improvements due to altered pharmacokinetics or PGP modulation within the tumor cells? And, b) Does decreasing the anticancer drug dose to that which is equitoxic in the absence of the modulator potentially compromise tumor therapy due to decreased anticancer drug levels in the tumor tissue? Although many of the difficulties associated with co-administration of MDR modulators and anticancer drugs are manifested by toxicity effects, it is ultimately the ability to obtain effective antitumor activity against resistant tumors that will determine the utility of chemosensitization approaches . Liposomes appear to be well suited to solve many of the problems noted above that are associated with conventional anticancer drugs and MDR modulators . In view of these considerations, we have hypothesized that inadequate tumor delivery of anticancer agents and selectivity of PGP modulation are primarily responsible for the attenuated therapy of extravascular MDR solid tumors overexpressing PGP . Liposomal carriers have been utilized to provide tumor selective delivery of anticancer agents as well as to circumvent many toxicities associated with these agents by altering the pharmacodistribution properties of encapsulated drugs (1-4) . Given the pharmacokinetic changes induced by the MDR modulators on non-encapsulated doxorubicin (DOX), we proposed that liposomes may limit these effects by virtue of their ability to reduce the exposure of encapsulated DOX to the kidneys and alter clearance of DOX in the liver (5,6) . These tissues appear to be key factors involved in modulator-induced DOX pharmacokinetic changes (7) . In conjunction with these toxicity buffering effects, the effect of PGP blockade on the cellular uptake of DOX in the tumor may be able to be selectively increased using liposomal carriers . This is based on the ability of small liposomes to passively extravasate in tumors (1,2,8,9) as well as their inability to accumulate in healthy susceptible tissues . By studying the toxicity and efficacy properties of liposome encapsulated DOX in combination with the MDR modulator PSC 833 we have been able to demonstrate that two factors play a major role in determining the effectiveness of chemosensitization approaches to overcome MDR; 1) optimizing selective localization of anticancer drug localization in tumor tissue and 2) effective blockade of PGP in tumor cells under conditions that do not compromise anticancer drug accumulation into the tumor . Failure to achieve both of these conditions simultaneously may be expected to result in substantially reduced therapy of MDR tumors.

Eur J Biochem, 2000 Feb, 267(3), 649 - 57
The relative importance of passive and P-glycoprotein mediated anthracycline efflux from multidrug-resistant cells; Wielinga PR et al.; For the four anthracyclines idarubicin, daunorubicin, epirubicin and doxorubicin the passive and active efflux rates in intact multidrug resistant cells were compared . Although highly similar structurally, these anti-tumor agents differ in lipophilicity and membrane permeability (k) . The method we used was based on the continuous measurement of the cellular efflux and determination of the ratio (RVp) of transport rates just before and just after inhibition of the active transport with verapamil (Vp) . Hence, RVp - 1 should reflect the active transport rate relative to the passive transport rate . If cells were single, well-stirred compartments, RVp - 1 should equal Vmax/(k.Km), where Vmax is the maximal pumping rate and Km is the Michaelis constant . However, using the plasma membrane permeabilizing agent digitonin, we found an effective intracellular anthracycline store . Particularly, when the efflux was fast, e.g . with idarubicin or in intensively pumping cells, the intracellular transport began to control the cellular efflux . Under these conditions, k underestimated the true plasma membrane permeability (k0) and RVp - 1 underestimated Vmax/(k.Km) . Based on the effects of digitonin on the efflux rates in pumping and nonpumping cells, we developed an index (RVp,corrected - 1) which should equal Vmax/(k0 . Km) . The term Vmax/(k0.Km) varied substantially between the drugs . It appears that differences in lipophilicity between the drugs do not affect passive efflux and pumping equally . This demonstrates that passive permeation plays a substantial and independent role in determining the drug resistance for these anthracyclines . The methods developed here enable dissection of this role from that of drug pumping and intracellular subcompartmentation.

Br J Haematol, 2000 Jan, 108(1), 90 - 2
Role of P-glycoprotein in all-trans retinoic acid (ATRA) resistance in acute promyelocytic leukaemia cells: analysis of intracellular concentration of ATRA; Takeshita A et al.; We analysed the relationship between all-trans retinoic acid (ATRA) resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukaemia (APL) . There was no difference in the intracellular ATRA accumulation between NB4 cells and an MDR1 cDNA-transduced NB4 subline and between ATRA-resistant NB4 cells (NB4/RA) and an MDR1 cDNA-transduced NB4/RA subline . PSC833, a MDR modifier, did not increase the intracellular accumulation of ATRA or affect the expression of CD11b, the nitroblue tetrazolium (NBT) reduction activity, the proportion of apoptotic cells or the morphology of these four ATRA-treated cell lines . Similar results were obtained in the analysis of APL cells from five patients relapsed after ATRA-induced complete remission.

Br J Haematol, 2000 Jan, 108(1), 48 - 54
P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells; Borg AG et al.; The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP) . An inverse and significant association was found between IDA and Pgp-related efflux activity (r = -0.31, P = 0.01) and also MRP (r = -0.25, P = 0.04) but not with LRP (r = -0.13, P = 0.28) . Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell . Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs . 0.296, P = 0.046) . Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied . The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis . There was a non-significant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16.3 months compared with 21.1 months in patients with high IDA levels) . Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.

Crit Rev Eukaryot Gene Expr, 1999, 9(3-4), 337 - 43
The role of the nuclear matrix in cancer chemotherapy; Muenchen HJ et al.; The nuclear matrix is the site of many nuclear functions including transcription, replication, formation of chromatin loops, and control of DNA supercoiling . It contains various structural and functional components that represent targets for antineoplastic agents . Antimetabolites and topoisomerase II inhibitors interact specifically with matrix-associated enzymes, DNA primase, and DNA topoisomerase II, respectively . Alkylating agents and ionizing radiation interact with nuclear matrix proteins and matrix-associated DNA . Many nuclear functions, including multidrug resistance, and others which lead to cell death, have been shown to be compromised when these anticancer agents interact with the nuclear matrix.

Pflugers Arch, 2000 Jan, 439(3), 356 - 62
Modulation of the mdr-1b gene in the kidney of rats subjected to dehydration or a high-salt diet; Morales MM et al.; The localization of the multidrug resistance gene (mdr-1b) messenger ribonucleic acid (mRNA) along the rat nephron and its regulation was investigated under two different experimental situations: dehydration and high-Na+ diet . The mdr-1b mRNA was detected in glomeruli, proximal tubule segments, cortical and medullary thick ascending limbs, inner medullary collecting ducts and thin limbs of Henle's loop . Using the ribonuclease (RNase) protection assay (RPA), the abundance of mdr-1b mRNA was shown to be 35% less in renal cortex than in medulla . The mdr-1b mRNA expression in dehydrated rats in cortex or medulla did not differ from control . However, after 5 or 14 days on a high-Na+ diet, mdr-1b expression had decreased significantly in both cortex and medulla . There was no change in protein expression in dehydrated rats but a significant decrease occurred in rats fed the high-salt diet, confirming the results obtained with RPA . Our results suggest that the mdr-1b product is involved in extracellular volume regulation in rats.

Anticancer Res, 1999 Sep-Oct, 19(5C), 4413 - 20
Multidrug resistant malignant melanoma with intracranial metastasis responding to immunotherapy; Savas B et al.; Metastatic malignant melanoma (MM) is well known for its poor response to chemotherapy, radiotherapy, and its remarkable susceptibility to interleukin-2 (IL-2) based immunotherapies . MM with brain metastatis in particular, has 4-5 months life expectancy from metastasis to death . Drug efflux pumps such as P-glycoprotein (P-gp), or drug detoxifying mechanisms e.g . glutathion epsilon S-transferase-pi (GST) are some of the possible multidrug resistance (MDR) mechanisms in MM . Here we report the first P-gp+ MDR MM with brain metastasis in the literature, demonstrating a remarkable response to IL-2, interferon-alpha (IFN), 5-fluorouracil (5FU) regimen . A 41-year old man was admitted with multiple inoperable brain lesions . Biopsies from intracranial and dermal lesions revealed MM . Cisplatin, carmustine, dacarbazine, tamoxifen (CCDT) together with external cranial radiotherapy were administered, and partial response in lesions and symptoms was achieved . However, after the third course of CCDT treatment, he was admitted to the emergency ward with dramatically increased intracranial lesions, and recurring dermal lesions . A biopsy from the recurred lesions revealed that MM cells were P-gp+, but GST . Administration of a IL-2, IFN and 5FU regimen achieved a remarkable decline in the brain lesions with almost total disappearance of symptoms . He was well and capable of doing work for 18 months . Dermal lesions had not recurred since the beginning of immunotherapy . In contrast, another 34-year old man who developed brain metastases after CCDT for MM, was negative for P-gp and GST . Cranial radiotherapy was started and the above mentioned IL-2 based regimen was administered . However, no response was observed . These two cases together with previous studies demonstrating the susceptibility of P-gp+ MDR cancer cell lines to IL-2 activated killer (LAK) cells in this report suggest that P-gp+ MDR MM is probably a good candidate for IL-2 based treatments.

Mol Pharmacol, 2000 Feb, 57(2), 308 - 16
Photoaffinity labeling and purification of ZG-16p, a high-affinity dihydropyridine binding protein of rat pancreatic zymogen granule membranes that regulates a K(+)-selective conductance; Braun M et al.; In rat pancreatic zymogen granules (ZG), an ATP-sensitive K(+) conductance and a Cl(-) conductance have been characterized that are inversely regulated by an approximately 65-kDa multidrug resistance P-glycoprotein (mdr1) gene product . In search of a label for purification of this protein, we found that the dihydropyridine derivative (-)-{(3)H}BZDC-DHP, a recently developed high-affinity ligand for Mdr1, binds with similar affinity to ZG membranes (ZGM) (K(d) = 6.2 nM) . Binding was inhibited by nanomolar concentrations of the L-type Ca(2+) channel blockers azidopine and verapamil and by micromolar concentrations of the K(+) channel blockers glibenclamide and quinidine . Inhibition by glibenclamide was noncompetitive . The Mdr1 modulators cyclosporin A and vinblastine did not inhibit binding, which is different from Mdr1 . In addition, only (+/-)-BZDC-DHP, azidopine, and verapamil selectively inhibited the K(+) conductance in ZGs, whereas the Cl(-) conductance was not affected . In photoaffinity labeling experiments, (-)-{(3)H}BZDC-DHP surprisingly specifically and selectively labeled a approximately 19-kDa protein in ZGM with a pharmacological profile identical with the high-affinity binding site but did not label a 65-kDa protein . The 19-kDa protein was purified by ion exchange chromatography and SDS-polyacrylamide gel electrophoresis and sequenced . The sequence obtained corresponds to ZG-16p, a recently cloned ZG protein with no apparent homology to Mdr1 . The identity of the 19-kDa protein was confirmed by immunoprecipitation of (-)-{(3)H}BZDC-DHP-labeled ZGM with an anti-ZG-16p antibody . Furthermore, it is shown that ZG-16p is associated with the ZGM . We propose that ZG-16p, as part of the submembranous granule matrix, regulates the ATP-sensitive K(+) conductance of ZGs.

Gastroenterology, 2000 Feb, 118(2), 279 - 88
High multidrug resistance (P-glycoprotein 170) expression in inflammatory bowel disease patients who fail medical therapy; Farrell RJ et al.; BACKGROUND & AIMS: The multidrug resistance (MDR) gene codes for a drug efflux pump P-glycoprotein 170 (Pgp-170) expressed on the surface of lymphocytes and intestinal epithelial cells . Inflammatory bowel disease (IBD) poorly responsive to medical therapy may relate to MDR expression because glucocorticoids are known Pgp-170 substrates . METHODS: Using flow cytometry, we measured peripheral blood lymphocyte (PBL) MDR in 153 IBD patients and 50 healthy volunteers, and assessed the relationship between PBL, mucosal intraepithelial lymphocyte (IEL), and mucosal epithelial cell (EC) MDR expression in a further 20 IBD patients and 19 controls . RESULTS: Compared with controls, PBL MDR was significantly elevated in patients with Crohn's disease who required bowel resection for failed medical therapy (mean +/- SEM, 26.7 +/- 2.8 vs . 11.9 +/- 1.0; P <0.0001) and patients with ulcerative colitis who required proctocolectomy for failed medical therapy (20.3 +/- 2.5 vs . 11.9 +/- 1.0; P = 0.001) . PBL MDR remained stable over time and was not influenced by disease activity or glucocorticoid therapy . Both PBL and mucosal MDR expression appeared independent of disease activity, and there was a significant correlation between PBL MDR expression and both IEL expression (r = 0.92; P < 0.0001) and EC expression (r = 0.54; P < 0.001) . CONCLUSIONS: PBL and mucosal MDR expression may play an important role in determining the response of IBD patients to glucocorticoid therapy.

Blood, 2000 Feb 1, 95(3), 1014 - 22
Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-x(L); Perkins C et al.; We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As(2)O(3) (0.5 to 2.0 micromol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli . These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-x(L) (HL-60/Bcl-x(L)), MDR (HL-60/VCR), or MRP (HL-60/AR) protein . The growth-inhibitory IC(50) values for As(2)O(3) treatment for 7 days against all these cell types ranged from 0.8 to 1.5 micromol/L . Exposure to 2 micromol/L As(2)O(3) for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells . This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (DeltaPsim) and the increase in reactive oxygen species (ROS) . Treatment with As(2)O(3) (2 micromol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase . Significantly, As(2)O(3)-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-x(L), Bax, Apaf-1, Fas, and FasL . Although As(2)O(3 )treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells . However, in these cells, As(2)O(3 )potently induced hyper-acetylation of the histones H3 and H4 . These findings characterize As(2)O(3) as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli.

Cancer Chemother Pharmacol, 2000, 45(1), 78 - 84
Mechanism of action of the dual topoisomerase-I and -II inhibitor TAS-103 and activity against (multi)drug resistant cells; Minderman H et al.; TAS-103 is a recently developed dual inhibitor of topoisomerase-I (topo-I) and topoisomerase-II (topoII) . TAS-103 has documented cytotoxicity in vitro and antitumor activity against a variety of mouse, rat, and human xenografts in vivo . PURPOSE: To determine TAS-103 activity against (multi)drug resistant cells in vitro and to delineate its mechanism of action . METHODS: TAS-103 was evaluated for activity against three human multidrug-resistant cell lines representing resistance mediated by P-glycoprotein (Pgp)-, multidrug resistance protein (MRP), and lung resistance protein (LRP) as well as one camptothecin-resistant cell line associated with a mutated topo-I enzyme . Drug sensitivity following short (2 h), intermediate (6-8 h) and long term (24 h) exposures were compared . The mechanism of action was studied by evaluating inhibition of topoisomerase-I and -II specific DNA relaxation assays, drug-induced DNA/protein cross-link formation, and competitive DNA intercalation with ethidium bromide . RESULTS: Increasing the exposure time only modestly potentiated TAS-103 cytotoxicity (3-5 fold) demonstrating a lack of strong exposure duration dependency . TAS-103 cytotoxicity was not affected by the presence of any of the drug resistance mechanisms studied . TAS-103 inhibits topo-I and -II activity in DNA relaxation assays, but in our assay system TAS-103 was found to have only a weak ability to induce DNA-protein crosslinks . DNA migration patterns in agarose gel electrophoresis indicate that TAS-103 can interact directly with DNA . Also its ability to displace ethidium bromide which has intercalated into the DNA provides an indication on the nature of drug-DNA interaction . CONCLUSIONS: TAS-103 cytotoxicity is not affected by the presence of Pgp, MRP, LRP or mutations in the CAM binding region of the topo-I enzyme and its growth-inhibitory effect appears to be weakly dependent on exposure duration . The presented evidence suggest that the inhibitory effects of TAS-103 on topo-I and -II may in part be related to its DNA binding rather than primarily through stabilization of topo-I or -II intermediates with DNA through specific binding to the enzymes.

Cancer Res, 2000 Jan 1, 60(1), 47 - 50
Fumitremorgin C reverses multidrug resistance in cells transfected with the breast cancer resistance protein; Rabindran SK et al.; Fumitremorgin C (FTC) is a potent and specific chemosensitizing agent in cell lines selected for resistance to mitoxantrone that do not overexpress P-glycoprotein or multidrug resistance protein . The gene encoding a novel transporter, the breast cancer resistance protein (BCRP), was recently found to be overexpressed in a mitoxantrone-selected human colon cell line, S1-M1-3.2, which was used to identify FTC . Because the drug-selected cell line may contain multiple alterations contributing to the multidrug resistance phenotype, we examined the effect of FTC on MCF-7 cells transfected with the BCRP gene . We report that FTC almost completely reverses resistance mediated by BCRP in vitro and is a pharmacological probe for the expression and molecular action of this transporter.

J Biol Chem, 2000 Jan 28, 275(4), 2905 - 10
ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3); Hirohashi T et al.; We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y . (1999) J . Biol . Chem . 274, 15181-15185) . In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA . The ATP-dependent uptake of {(3)H}taurocholate (TC), {(14)C}glycocholate (GC), {(3)H}taurochenodeoxycholate-3-sulfate (TCDC-S), and {(3)H}taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells . The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC . The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively . At 55 nM {(3)H}E(2)17betaG and 1.2 microM {(3)H}TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively . TC, GC, and TCDC-S inhibited the transport of {(3)H}E(2)17betaG and {(3)H}TC to the same extent with an apparent IC(50) of approximately 10 microM . TLC-S inhibited the uptake of {(3)H}E(2)17betaG and {(3)H}TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM) . Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates . This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts.

Am J Physiol Gastrointest Liver Physiol, 2000 Jan, 278(1), G57 - 63
Bile salt excretion in skate liver is mediated by a functional analog of Bsep/Spgp, the bile salt export pump; Ballatori N et al.; Biliary secretion of bile salts in mammals is mediated in part by the liver-specific ATP-dependent canalicular membrane protein Bsep/Spgp, a member of the ATP-binding cassette superfamily . We examined whether a similar transport activity exists in the liver of the evolutionarily primitive marine fish Raja erinacea, the little skate, which synthesizes mainly sulfated bile alcohols rather than bile salts . Western blot analysis of skate liver plasma membranes using antiserum raised against rat liver Bsep/Spgp demonstrated a dominant protein band with an apparent molecular mass of 210 kDa, a size larger than that in rat liver canalicular membranes, approximately 160 kDa . Immunofluorescent localization with anti-Bsep/Spgp in isolated, polarized skate hepatocyte clusters revealed positive staining of the bile canaliculi, consistent with its selective apical localization in mammalian liver . Functional characterization of putative ATP-dependent canalicular bile salt transport activity was assessed in skate liver plasma membrane vesicles, with {(3)H}taurocholate as the substrate . {(3)H}taurocholate uptake into the vesicles was mediated by ATP-dependent and -independent mechanisms . The ATP-dependent component was saturable, with a Michaelis-Menten constant (K(m)) for taurocholate of 40+/-7 microM and a K(m) for ATP of 0.6+/-0.1 mM, and was competitively inhibited by scymnol sulfate (inhibition constant of 23 microM), the major bile salt in skate bile . ATP-dependent uptake of taurocholate into vesicles was inhibited by known substrates and inhibitors of Bsep/Spgp, including other bile salts and bile salt derivatives, but not by inhibitors of the multidrug resistance protein-1 or the canalicular multidrug resistance-associated protein, indicating a distinct transport mechanism . These findings provide functional and structural evidence for a Bsep/Spgp-like protein in the canalicular membrane of the skate liver . This transporter is expressed early in vertebrate evolution and transports both bile salts and bile alcohols.

Tuber Lung Dis, 1998, 79(1), 31 - 42
Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region; Kurepina NE et al.; SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain . OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M . tuberculosis genome . DESIGN: A total of 722 M . tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes . Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites . RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains . Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins . CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M . tuberculosis clinical strains representing the breadth of species diversity . The mapping data indicate that IS6110 insertion sites are not always random.

Antimicrob Agents Chemother, 2000 Feb, 44(2), 450 - 2
Failure of short-course ceftriaxone chemotherapy for multidrug-resistant typhoid fever in children: a randomized controlled trial in Pakistan; Bhutta ZA et al.; The precise duration of therapy of multidrug-resistant (MDR) typhoid with broad-spectrum cephalosporins is uncertain . We prospectively randomized 57 children with culture-proven MDR typhoid to receive treatment with intravenous ceftriaxone (CRO) (65 mg/kg of body weight/day) for 7 days (short course; n = 29) or 14 days (conventional; n = 28) . The response to therapy, as evaluated by the serial monitoring of the typhoid morbidity score and bacteriological clearance, was comparable between groups . In contrast to the conventional therapy, 14% of the children receiving CRO for 7 days had a confirmed bacteriological relapse within 4 weeks of stopping therapy.

Prostate, 2000 Feb 15, 42(3), 172 - 80
Contrasting effects of aspirin on prostate cancer cells: suppression of proliferation and induction of drug resistance ; Rotem R et al.; BACKGROUND: Aspirin is widely used as a preventive measure against occlusive vascular diseases . Since the age group in which aspirin use has become prevalent is similar to the one presenting with prostate cancer, we decided to examine the potential effects of aspirin on prostate cancer . METHODS: We studied the effects of plasma-attainable concentrations of aspirin (0.5-2 mM) on the human prostate cancer cell lines LNCaP, PC-3, and DU 145, employing cytotoxicity assays and flow cytometric analyses . RESULTS: Incubation with aspirin for 3 days reduced cellular proliferation by up to 35-55% in each cell line studied, but induced a tripling of the percentage of cells expressing P-glycoprotein (an efflux pump conferring multidrug resistance) only in the LNCaP cells . Both effects were dose-dependent . The effect on P-glycoprotein expression was reflected in the induction of resistance against adriamycin cytotoxicity . Furthermore, this protective effect of aspirin was reversed by a specific P-glycoprotein inhibitor, PSC833 . The cellular expression of P-glycoprotein returned to normal within 3 days following the removal of aspirin . Aspirin did not affect the cell cycle distribution of LNCaP cells . CONCLUSIONS: This study suggests that aspirin enhances the ability of androgen-responsive prostate cancer cells to resist chemotherapeutic drugs . These findings could potentially have significant clinical ramifications for prostate cancer patients taking aspirin shortly before or during chemotherapeutic sessions .

Trends Biochem Sci, 2000 Jan, 25(1), 1 - 6
Multiple physiological functions for multidrug transporter P-glycoprotein?
Johnstone RW, Ruefli AA, Smyth MJ.
Multidrug resistance mediated by the drug-efflux protein P-glycoprotein (P-gp) is one mechanism that tumor cells use to escape death induced by chemotherapeutic drugs . Although it is irrefutable that P-gp can efflux xenobiotics out of cells, biological regulatory functions for P-gp in multicellular organisms have yet to be established firmly . Recent observations have challenged the notion that P-gp has evolved merely to efflux xenotoxins out of healthy cells and raised the possibility that P-gp and related transporter molecules might play a fundamental role in regulating cell differentiation, proliferation and survival.

Leukemia, 2000 Jan, 14(1), 68 - 76
Drug resistance factors in acute myeloid leukemia: a comparative analysis; Filipits M et al.; To compare the clinical relevance of drug resistance factors in de novo acute myeloid leukemia (AML), we determined their relationship to both response to induction chemotherapy and survival of the patients in univariate as well as multivariate analyses . The drug resistance factors immunocytochemically studied in 111 patients at the time of diagnosis included the lung resistance protein (LRP), P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and bcl-2 . In the univariate analyses, age (P = 0.005), karyotype (P = 0.03), LRP (P = 0.003), P-gp (P = 0.02) and bcl-2 (P = 0.03) predicted for response to induction chemotherapy, whereas MRP1 had no predictive value . Age (P = 0.05), karyotype (P = 0.05) and LRP (P = 0.03) retained their predictive value in the multivariate logistic regression analyses . With regard to overall survival, age (P = 0 . 008), karyotype (P = 0.006), LRP (P = 0.001) and P-gp (P = 0.01) were of prognostic value in the univariate Cox regression analyses but only age (P = 0.01), karyotype (P = 0.02) and LRP (P = 0.01) retained their prognostic significance in the multivariate analyses . A risk score based on the number of independent prognostic factors allowed division of patients into four groups with different outcome . In these groups, the complete remission rates were 93%, 75%, 47% and 33%, respectively, and median overall survival was 2.4, 1.2, 0.6 and 0.2 years, respectively . Thus, several drug resistance factors did predict outcome in the univariate analyses but LRP was the only drug resistance factor with independent predictive and prognostic significance . The proposed risk score might be useful for risk-adapted treatment in the future . Leukemia (2000) 14, 68-76.

J Biol Chem, 2000 Jan 21, 275(3), 1887 - 96
Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis; Thevenod F et al.; Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis . We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl(2) (10 microM) application (controls: 2.4 +/- 1.6%; 5 h: +5.1 +/- 2.3%, 20 h: +5.7 +/- 2.5%, 48 h: +3.3 +/- 1.0% and 72 h: +2.1 +/- 0.4% above controls), while cell proliferation was not affected . Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells ( approximately 4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein +/- the mdr1 inhibitor PSC833 (0.5 microM) . When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 +/- 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 +/- 1.4%; p < or = 0.001) . Cadmium-induced apoptosis and mdr1 up-regulation depended on ROS, since co-incubation with the ROS scavengers N-acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses . Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, induced by cadmium . The data show that 1) cadmium-mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-kappaB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis . These data suggest that mdr1 up-regulation, at least in part, provides anti-apoptotic protection for PT cells against cadmium-mediated stress.

Clin Nucl Med, 2000 Jan, 25(1), 20 - 3
The effect of P-glycoprotein function inhibition with cyclosporine A on the biodistribution of Tc-99m sestamibi; Kabasakal L et al.; PURPOSE: The failure to cure persons with cancer is caused primarily by the development of drug resistance by overexpression of p-glycoprotein . Diverse groups of drugs have been identified, including cyclosporine A, which can reverse drug resistance by inhibiting P-glycoprotein transport . Tc-99m sestamibi is a substrate for P-glycoprotein . P-glycoprotein is normally expressed in biliary canalicular surfaces of hepatocytes and is responsible for the excretion of cationic metabolites from the liver . The aim of the current study was to evaluate the effect of cyclosporine A on the biological distribution of Tc-99m sestamibi in vivo . METHODS: Five patients with alopecia and two renal transplant patients who were treated with cyclosporine A were selected for the study . All patients were examined before and at least 2 weeks after administration of cyclosporine A . Tc-99m sestamibi scintigraphy was performed by obtaining planar abdominal images at 5, 30, 60, 120, and 180 minutes after injection, and the liver-heart ratios were calculated . RESULTS: Plasma cyclosporine A, bilirubin levels, liver enzymes, and creatinine clearance values were obtained from all patients . In three, the plasma cyclosporine A level was increased to more than 400 pg/dl . The liver-heart ratio was increased significantly after cyclosporine A administration (P < 0.01) . After cyclosporine A administration Tc-99m sestamibi excretion was delayed and the uptake in the liver was increased . The difference was 17% at 5 minutes and 38% at 180 minutes . Liver retention was greatest in patients with cyclosporine A toxicity . CONCLUSIONS: With a limited number of patients, this study suggests that Tc-99m sestamibi excretion from the liver is mediated by P-glycoprotein, and inhibition of P-glycoprotein transport not only delays liver excretion but also increases the liver uptake of Tc-99m sestamibi . Because this observation deserves further investigation, the inhibition of P-glycoprotein function with nontoxic multidrug-resistance reversing agents may be used as an intervention to increase the tumor uptake of Tc-99m sestamibi and to increase the sensitivity of Tc-99m sestamibi tumor imaging.

Eur J Biochem, 2000 Jan, 267(2), 277 - 94
Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane; Ferte J; P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype . It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism . Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp . With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions . It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype . Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.

Clin Cancer Res, 1999 Dec, 5(12), 4097 - 104
Prognostic value of p53, glutathione S-transferase pi, and thymidylate synthase for neoadjuvant cisplatin-based chemotherapy in head and neck cancer; Shiga H et al.; Neoadjuvant cisplatin-based chemotherapy has been widely used in the last decade for organ preservation or unresectable disease in advanced stage head and neck cancer . We examined the expression of a series of tumor markers that have been associated with chemotherapy resistance in pretreatment biopsies from 68 patients who received cisplatin-based neoadjuvant chemotherapy at either of two institutions . Patients received either cisplatin/5-fluorouracil (n = 49) or cisplatin/paclitaxel (n = 19) . Expression of p53, glutathione S-transferase pi (GSTpi), thymidylate synthase (TS), c-erbB2, and multidrug resistance-associated protein was examined by immunohistochemistry . Expression of glutathione synthetase mRNA was measured by in situ hybridization . The overall response rate for cisplatin-based neoadjuvant treatment was 79% . The expression of several of the tumor markers was associated with resistance to neoadjuvant treatment, but none reached statistical significance . Overall survival (OS) was strongly correlated with the absence of p53 expression . The OS at 3 years was 81% in the p53-negative group, whereas it was 30% in the p53-positive group for patients treated with neoadjuvant chemotherapy (P < 0.0001) . Expression of GST pi and TS was also significantly correlated with decreased OS after neoadjuvant treatment . At 3 years, the OS rate was 82% in the low GSTpi score group, compared to 46% in the high GSTpi score group (P = 0.0018) . In the TS-negative group, the 3-year OS rate was 71% compared with 40% in the TS-positive group (P = 0.0071) . We conclude that p53, GSTpi, and TS may be clinically important predictors of survival in patients receiving neoadjuvant chemotherapy for head and neck cancer.

Clin Cancer Res, 1999 Dec, 5(12), 3920 - 7
Anti-CD19 antibodies inhibit the function of the P-gp pump in multidrug-resistant B lymphoma cells; Ghetie MA et al.; After chemotherapy, tumor cells with multidrug resistance (MDR) often emerge . MDR is attributable to the expression of membrane transport proteins that inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs . One such protein is P-glycoprotein (P-gp), which functions as an ATP-dependent active transporter . Recently, an anti-P-gp monoclonal antibody (MAb) that inhibits P-gp has been described . Previous studies from our laboratory using the anti-CD19 B-cell lymphoma-reactive MAb, HD37, have suggested that HD37 may also influence MDR . To test this directly, we used Namalwa/MDR1 cells to study the effect of HD37 on the efflux of rhodamine 123 from these cells . We found that HD37 and three other anti-CD19 MAbs inhibited the efflux of rhodamine 123 from Namalwa/MDR1 cells with approximately 50% of the efficiency of the well-known chemosensitizer, verapamil . In contrast, MAbs against seven other molecules expressed on these cells were ineffective . The inhibitory activity of HD37 did not require an Fc portion; F(ab')2 fragments were effective, but Fab' fragments were not, suggesting that higher avidity binding and/or cross-linking of CD19 are necessary . We could find no evidence that HD37 recognizes a cross-reactive epitope on P-gp, modulates P-gp from the cell surface, or enhances the ATPase activity of membranes from treated cells.

Pharmazie, 1999 Dec, 54(12), 876 - 8
Synthesis and cytotoxicity of novel pyrido{1,2-e}purines on multidrug resistant human MCF7 cells; Pinguet F et al.; The anticancer activity of 4-methylaminopyridol{1,2-e}purine 6a, 4-(piperidin-1-yl)pyrido{1,2-e}purine 7a and their 7-methyl derivatives 6b, 7b was investigated against the human MCF7 cancer cell line in vitro . The sensitive cell line showed a range of sensitivities to 6a, 6b, 7a, 7b (IC50: 1.6 to 7.2 x 10(-4) M) and sensitivity to doxorubicin (IC50: 7.5 x 10(-7) M) . A resistant cell line with the multidrug resistant phenotype was sensitive to these derivatives (IC50: 1.8 to 6.7 x 10(-4) M), doxorubicin (IC50: 5 x 10(-5) M) and drug activity seems to be not affected by MDR resistance . Our data show that 6a, 6b, 7a and 7b appear to exert a low cytotoxicity on sensitive and MDR resistant MCF7 human cancer cell lines.

Biochim Biophys Acta, 2000 Jan 15, 1463(1), 121 - 30
Leukotriene C(4) (LTC(4)) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes; Sundkvist E et al.; The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump . In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter . The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake . Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of {(3)H}cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively . Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx . 25% at 1 mM . The cGMP pump was sensitive to temperature without activity below 20 degrees C . The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5 . Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM . Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM . The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM . EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively . The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4) . However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.

J Orthop Res, 1999 Nov, 17(6), 935 - 40
Multidrug resistance-1 and p-glycoprotein in human chondrosarcoma cell lines: expression correlates with decreased intracellular doxorubicin and in vitro chemoresistance; Wyman JJ et al.; We report on two chondrosarcoma cell lines, FS and AQ, that may be used as models of multidrug resistance in chondrosarcoma . Multidrug resistance-1 expression was assayed with reverse transcription-polymerase chain reaction . Immunostaining for the multidrug resistance-1 product, P-glycoprotein, was performed with the monoclonal antibody C494 . Intracellular levels of doxorubicin were measured by fluorescent emission at 590 nm after 1 hour of incubation with the agent and again after 1, 2, and 4-hour washout periods . Chemosensitivity was assayed by staining micropellet cultures of AQ and FS cells with fluorescein acetate before and after the cells were exposed to varying doses of doxorubicin for 48 hours . Cytotoxicity was assessed by comparison of computer-processed images before and after treatment . The FS cell line was positive for multidrug resistance-1 expression, stained heavily for P-glycoprotein, and had significantly lower intracellular levels of doxorubicin than the AQ cell line, which was negative for multidrug resistance-1 and P-glycoprotein . Chemosensitivity testing showed that the FS cell line was significantly more resistant to doxorubicin than was the AQ cell line at all doses tested . Our results show that multidrug resistance-1 expression in a human chondrosarcoma cell line results in resistance to doxorubicin in vitro.

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