Multidrug resistant

Cancer Res, 2001 Oct 1, 61(19), 7189 - 95
Potent antitumor activity and improved pharmacological profile of ST1481, a novel 7-substituted camptothecin; De Cesare M et al.; Relevant drawbacks of the molecular structure and mechanism of the action of camptothecins are the instability of the E ring lactone and the reversibility of drug-target interaction . Such features are expected to limit the clinical efficacy of conventional camptothecins . In an attempt to overcome these limitations and to improve the pharmacological profile of camptothecins, a novel series of seven modified lipophilic analogues was synthesized based on the hypothesis that lipophilicity could promote a rapid cellular accumulation and stabilization of drug-target interaction . A novel analogue (ST1481) of the series, characterized by a potent antitopoisomerase and cytotoxic activity, was selected for preclinical development . A detailed preclinical study of ST1481 was performed in the H460 non-small cell lung tumor model using oral administration and various treatment schedules . Under all of the conditions, ST1481 exhibited an impressive efficacy in terms of tumor growth inhibition (tumor volume inhibition percentage > 99%), log(10) cell kill, rate of complete responses (including "cures"), and an improvement of the therapeutic index compared with topotecan (used as the reference drug) . The cytotoxic potency was also reflected by the in vivo potency, because the drug activity was observed at doses as low as 0.25 mg/kg with the daily schedule . In contrast to topotecan, no cross-resistance to ST1481 was found in ovarian carcinoma cells overexpressing P-glycoprotein (A2780/DX) . A similar trend in the improvement of activity was also observed in the same tumor model growing in vivo with a 100% rate of complete tumor regressions . A rapid intestinal absorption and good oral bioavailability were supported by in vivo distribution studies, because the peak values of drug accumulation were found from 1 to 2 h after administration . The relevant liver accumulation may account for a marked effect of ST1481 against liver metastases induced by the ovarian carcinoma IGROV-1 . In conclusion, the results support the hypothesis that a potent lipophilic camptothecin with a proper substituent at the position 7 may have therapeutic advantages likely related to a rapid intracellular uptake and tissue distribution, stabilization of the drug-target complex, and good oral bioavailability . Overall, the results support the preclinical interest of ST1481 in terms of efficacy, potency, toxicity profile, and ability to overcome multidrug resistance.

Eur J Nucl Med, 2001 Sep, 28(9), 1341 - 50
The role of 99mTc-MIBI scintigraphy in the assessment of MDR1 overexpression in patients with musculoskeletal sarcomas: comparison with therapy response; Burak Z et al.; The occurrence of multidrug resistance (MDR), which is in part due to the overexpression of P-glycoprotein (Pgp), is a major problem in neoadjuvant therapy of malignant musculoskeletal tumours . The aim of this study was to investigate the role of technetium-99m hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) scintigraphy for functional imaging of the MDR1 phenotype in patients with musculoskeletal sarcomas . We aimed to compare 99mTc-MIBI uptake and washout kinetics with the expression of Pgp and with chemotherapy response . Twenty-five patients (16 males and 9 females, aged between 8 and 65 years) with malignant musculoskeletal tumours were studied . After injection of 555-740 MBq 99mTc-MIBI, dynamic flow images of the involved area were obtained for 3 min, and planar images were acquired at 10 min and 1 h . From the dynamic images, a tumour perfusion index (TPI) was obtained using Patlak-Rutland analysis . Tumour to background (T/B) ratios of both early and delayed images and percent wash-out rate (WR%) of 99mTc-MIBI were calculated . Immunohistochemical analysis of Pgp was performed on biopsy specimens and the degree of expression was graded according to a semiquantitative scoring system, from 0 to 6 . After neoadjuvant therapy, tumour response was assessed by examining the ratio of viable cells and by detecting percent necrosis . Scintigraphic results were compared with Pgp status and therapy response . Irrespective of the Pgp status, all patients showed significant perfusion and 99mTc-MIBI uptake in early images . There was not a significant correlation between T/B ratios of early and delayed images and Pgp expression . We observed a positive correlation between WR% and Pgp status (r=0.61, P<0.01), and the wash-out rate of 99mTc-MIBI was significantly higher in patients with high Pgp expression than in those with a low Pgp score (33% +/- 9% vs 17% +/- 9%) . Therapy response was determined in 21 of 25 patients, and in only 5 of 21 cases was the percent necrosis more than 90% . Neither Pgp expression rate nor WR% was found to show a significant correlation with percent necrosis in the bulk tumour specimens . In conclusion, the initial uptake of 99mTc-MIBI in bone and soft tissue sarcomas did not correlate with Pgp expression . A relationship was found between the wash-out rate of 99mTc-MIBI and the Pgp score, with a significant difference in WR% being observed between patients with high and patients with low Pgp expression.

Biochem Pharmacol, 2001 Sep 1, 62(5), 561 - 7
The absence of stereoselective P-glycoprotein- and multidrug resistance-associated protein-mediated transport of daunorubicin; Loetchutinat C et al.; Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP1) . Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins . Thus, both P-gp and MRP1 proteins are able to transport anthracycline but the role of chirality has not, up to now, been addressed . In this study, we compared the P-gp- and MRP1-mediated efflux of daunorubicin and its enantiomer WP900 in multidrug-resistant cells overexpressing either P-gp (K562/ADR cells) or MRP1 (GLC4/ADR cells) . Using fluorescence techniques, we showed that in both cell lines the presence of the pump yielded a gradient of drug concentration: the intracellular free drug concentration in the cytosol was lower than the extracellular free drug concentration . Our data showed that the gradient of concentration generated by the pump was the same whether DNR or WP900 was used . This means that P-gp on the one hand and MRP1 on the other recognise WP900 as well as DNR and that the chirality of the molecule plays no role.

Neoplasma, 2001, 48(3), 208 - 13
Non-steroidal anti-inflammatory agent ibuprofen-induced apoptosis, cell necrosis and cell cycle alterations in human leukemic cells in vitro; Jakubikova J et al.; The cytotoxic activity of the non-steroidal anti-inflammatory agent ibuprofen to human promyelocytic leukemia cell line HL-60, its multidrug-resistant subline HL-60/VCR (MDR-1 gene coded P-glycoprotein), as well as myeloma U266 and B-lymphoblastoid ARH-77 cell lines was demonstrated with the aid of flow cytometric analysis . Ibuprofen inhibited proliferation and induced apoptosis (detected as sub-G0 nuclei, fluorescein diacetate staining, Annexin-V binding cells and agarose electrophoretic detection of nucleosomal DNA fragmentation) in promyelocytic cells and, to a lesser extent, in U266 and ARH-77 cells.

Neoplasma, 2001, 48(3), 182 - 7
Expression of CD20 on acute lymphoblastic leukemia cells in children; Pituch-Noworolska A et al.; CD20 determinant expressed on B precursors is associated with regulation of proliferation, apoptosis and maturation of these cells . The acute lymphoblastic leukemia "common" type (cALL) based on expression of CD20 is subdivided in type I and II . However, the clinical significance of CD20 expression on cALL and significance of cALL type I and II discernment are not fully elucidated . The association of CD20 expression with the expression of multidrug resistance molecule (MDR), CD34, atypical immunophenotypes of leukemia cells and response to induction therapy were determined in the group of 147 patients with acute lymphoblastic leukemia (ALL) B progenitor type (ALL-proB -14 patients) and common type (cALL-133 patients) . The expression of CD20 on leukemia cells was studied routinely at diagnosis before the therapy . This expression was noted on leukemia cells of 6 ALL-proB patients (42.8%) and 66 cALL patients (49.6%) . The expression of CD20 showed no association with the expression of CD34, CD22 and MDR . The reverse association was observed between CD20 expression and the presence of co-expression of myeloid (CD13, CD33, CD65, CD15) and T lymphoid determinants (CD2, CD5, CD7) on leukemia cells . The effect of induction therapy analyzed as time of blast cells cytoreduction in peripheral blood and time of reaching the complete remission showed the slower clearance of peripheral blood from blast cells associated with expression of CD20 . There was no association of CD20 expression with the time of reaching the hematological remission . The above results suggested a "protective" role of CD20 against co-expression of other determinants (myeloid and lymphoid) and no association with the results of induction therapy.

Planta Med, 2001 Oct, 67(7), 614 - 8
Cytotoxic and anti-inflammatory activity of labdane and cis-clerodane type diterpenes; Demetzos C et al.; Two labdane type diterpenes, labd-13(E)-ene,-8alpha,15-diol (1) and labd-13(E)-ene,-8alpha,15-yl acetate (2) were isolated from the hexane extract of Cistus creticus subsp . eriocephalus (Viv.) Greuter & Burdet leaves, while (+)-19-acetoxy-cis-clerodan-3-en-15-oic acid (3) was isolated from the hexane extract of Cistus monspeliensis L . leaves . The compounds were examined for their in vitro cytostatic and cytotoxic activity against nine human leukemic cell lines, three of which exhibited a multidrug resistant phenotype . They were also evaluated for their anti-inflammatory activity in vivo on the back of hairless mice . The cytostatic and cytotoxic activity of the tested diterpenes followed the order 1>2>3 . Topical application of the diterpenes on barrier disrupted skin did not seem to have a significant contribution to the repair rate of the skin barrier.

J Biol Chem, 2001 Dec 7, 276(49), 46400 - 7
Characterization of drug transport by the human multidrug resistance protein 3 (ABCC3); Zelcer N et al.; We have characterized the substrate specificity and mechanism of transport of the human multidrug resistance-associated protein 3 (MRP3) . A murine fibroblast-like cell line generated from the kidneys of mice that lack Mdr1a/b and Mrp1 was retrovirally transduced with MRP3 cDNA . Stable clones overproducing MRP3 were resistant to the epipodophyllotoxins etoposide and teniposide but not to vincristine, doxorubicin, and cisplatin, drugs suggested to be MRP3 substrates by others . The resistance to etoposide was associated with reduced cellular accumulation and enhanced efflux of this drug and was not affected by depleting cells of glutathione but was inhibited by several common organic anion transport inhibitors . Membrane vesicles from infected insect cells expressing MRP3 mediated ATP-dependent transport of estradiol 17-beta-D-glucuronide, leukotriene C(4), dinitrophenyl S-glutathione but not glutathione itself, and etoposide glucuronide, a major metabolite of etoposide in vivo . The transport of estradiol 17-beta-D-glucuronide by MRP3 was inhibited in a concentration-dependent manner by both etoposide and methotrexate . Even though etoposide glucuronide is an excellent substrate for MRP3, this compound is not involved in the etoposide resistance of our MRP3 cells, as these cells extrude unmodified etoposide rather than etoposide glucuronide.

J Surg Oncol, 2001 Oct, 78(2), 110 - 5
Multidrug resistance gene (MDR-1) expression as a useful prognostic factor in patients with human hepatocellular carcinoma after surgical resection; Kato A et al.; BACKGROUND: Multidrug resistance gene (MDR-1) overexpression has been correlated with tumor aggressiveness and worse prognosis in some human neoplasms . The aim of this study is to evaluate the clinical value of MDR-1 mRNA expression as a prognostic factor after surgical resection in human hepatocellular carcinoma (HCC) . METHODS: MDR-1 mRNA levels in tissue samples from 34 patients with HCC, who underwent surgical resection, were measured by quantitative northern blot analysis . We stratified these patients into two groups according to a ratio of MDR-1 mRNA levels of HCC to nontumorous tissue; MDR-1 mRNA ratio > or = 1.0 and < 1.0 . The overall and disease-free survival rates were analyzed using multivariate regression analysis . RESULTS: The median survival periods were 10.3 and 35.8 months for patients with the MDR-1 mRNA ratio > or = 1.0 and < 1.0, respectively, and the corresponding 5-year survival rates were 33 and 54%, respectively, P < 0.05 . The multivariate analysis revealed that TNM stage and MDR-1 mRNA ratio were independent factors for predicting overall survival after surgical resection . CONCLUSION: This study suggested that the measurement of the MDR-1 mRNA levels in HCC and nontumorous liver tissue might be a useful prognostic factor after surgical resection in patients with HCC .

Pharmacol Ther, 2001 May-Jun, 90(2-3), 261 - 5
Artemisinin and its derivatives: an important new class of antimalarial agents; Balint GA; Artemisinin and its derivatives are a potent new class of antimalarials, originated from Artemisia annua, L . The clinical efficacy of these drugs is characterized by an almost immediate onset and rapid reduction of parasitaemia . Their efficacy is high in such areas as well where multidrug-resistance is rampant, but in these areas, their combination with other (effective) antimalarials (e.g., mefloquine) is highly recommended . In this short review, the chemical structures, pharmacological properties, and clinical uses of artemisinin drugs are discussed.

Ann Nucl Med, 2001 Aug, 15(4), 313 - 9
Usefulness of tc-99m MIBI SPECT in predicting multidrug resistance gene expression levels in non-small cell lung cancer--a preliminary report; Aratani T et al.; In this study we investigated whether Tc-99m hexakis 2-methoxy isobutyl isonitnile (Tc-99m MIBI) single-photon emission computed tomography (SPECT) has a correlation with the multidrug resistance (MDRI) and multidrug resistance-associated protein (MRP1) gene expression levels in non-small cell lung cancer (NSCLC) . Fifteen patients with NSCLC were studied . SPECT images were obtained 15 (early) and 120 (delayed) min after injection of Tc-99m MIBI . We chose only one transverse section and set the region of interest over the tumor and out of the body . The mean counts in the tumor on early and delayed images were corrected by using those in the background and represented as Te and Td, respectively . Resected tumor specimens were frozen with liquid nitrogen and each positive control cell line was cultured . After the total ribonucleic acid (RNA) was extracted from specimens and cell lines, the complimentary deoxyribonucleic acid (cDNA) was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) method . Each product was electrophoresed and fluorointensity was measured . The gene expression level was represented as the ratio of that of the positive control cell line . Te and Td indicated a significant correlation with the MDR1 gene expression level (p = 0.015 and p = 0.022), but not the gene of MRP1 (p = 0.100 and p = 0.145) . In conclusion, Te and Td are useful parameters in predicting the MDRI gene expression level, but not MRPI in NSCLC.

Adv Drug Deliv Rev, 2001 Oct 1, 50 Suppl 1, S13 - 31
Intestinal drug efflux: formulation and food effects; Wagner D et al.; The intestine, primarily regarded as an absorptive organ, is also prepared for the elimination of certain organic acids, bases and neutral compounds depending on their affinity to intestinal carrier systems . Several of the transport systems known to mediate efflux in the major clearing organs--liver and kidney--are also expressed in the intestine . Examples of secretory transporters in the intestine are P-glycoprotein, members of the multidrug resistance associated protein family, breast cancer resistance protein, organic cation transporters and members of the organic anion polypeptide family . In this communication, the P-glycoprotein mediated intestinal secretion of talinolol, a model compound showing metabolic stability, has been investigated in the jejunum, ileum and colon of rat intestine by single-pass perfusion . A model has been developed which demonstrates an increase in carrier-mediated secretion in the order jejunum<ileum<colon . Furthermore, the potency of common excipients in peroral drug products towards inhibition of P-gp mediated secretion has been investigated using a radioligand-binding assay and transport studies in Caco-2 cell monolayers . Finally, evidence is provided which demonstrates that constituents of grapefruit juice not only may influence intestinal drug metabolism, but can also interfere with secretory transport systems, leading to a new and yet undescribed mechanism in drug-food interactions.

Int J Tuberc Lung Dis, 2001 Sep, 5(9), 815 - 23
Surveillance of Mycobacterium tuberculosis drug resistance in Hong Kong, 1986-1999, after the implementation of directly observed treatment; Kam KM et al.; OBJECTIVE: To study changing trends in TB epidemiology with emphasis on drug resistance rates in various age groups from 1986-1999 . DESIGN: Laboratory-based data on drug susceptibility testing against streptomycin (SM), isoniazid (NH), rifampicin (RMP) and ethambutol (EMB) had been collected continuously in a centralised TB laboratory in Hong Kong . Epidemiological parameters such as sex, age and drug resistance rates in new and retreatment cases were measured and analysed for longitudinal trends . RESULTS: Of 48 924 non-duplicate isolates from new TB cases, 7045 (14.4%) were resistant to one or more drugs, 5773 (11.8%) were resistant to SM and/or INH while 881 (1.8%) were multidrug-resistant (MDR-TB) . Of 3857 isolates from retreatment patients, 1176 (30.5%) were resistant to one or more drugs, 616 (16.0%) were resistant to SM and/or {NH, and 467 (12.1%) were MDR-TB . For isolates from new cases, significant declines were observed in the resistance rates against any drug, SM alone, INH alone, SM+INH and INH+RMP . For retreatment isolates, significant declines were also observed in resistance to any drug and INH+RMP . In both new and retreatment cases, isolates from patients aged over 65 years showed significantly lower drug resistance (any drug and INH+RMP) compared with other age groups (16-34 years and 35-65 years) . CONCLUSION: With successful implementation of DOTS over a 14-year period, laboratory-based surveillance data showed significant declines in drug resistance, including MDR-TB . This has occurred amidst demographic changes associated with a generally ageing population as well as highly mobile sectors that are in constant exchange with highly endemic areas.

Ontogenez, 2001 Jul-Aug, 32(4), 295 - 301
{Relationship between the induction of MDR1, a multidrug resistance gene in tumor cells, and apoptosis}; Ktitorova OV et al.; Gene MDR1 coding for P-glycoprotein belongs to a group of genes responsible for cell defense . Overexpression of this gene determines the resistance of tumor cells to a series of chemotherapeutic drugs known as multidrug resistance . Many chemotherapeuticals induce both apoptosis and transcriptional activity of the MDR1 gene in tumor cells . It is not known, however, how these two processes are associated with each other . In order to elucidate a possible link between them, we have studied the sphyngomyelinic pathway of signal transduction . This pathway is activated in response to various stress factors and includes the hydrolysis of sphyngomyelin of cytoplasmic membrane resulting in an accumulation of intracellular ceramide, which activates cascades of enzymatic reactions leading to various cell responses, including apoptosis . C2 ceramide (N-acetyl-D-sphyngosine) and cytosar (1 beta-D-arabinosylcytosine, or ara C) were used to induce the sphyngomyelinic pathway . Their effects on human hemoblastosis cell lines (K562 and H9 cell lines) were examined . C2 ceramide and ara C induced apoptosis in both cell lines over an 18-h incubation . C2 ceramide also induced an increase in the expression of the gene MDR1 in both cell lines, while ara C increased the activity of the gene MDR1 only in H9 cells . The results obtained provide evidence for the contribution of ceramide-mediated signal pathway to the control of MDR1 activity.

J Biol Chem, 2001 Nov 30, 276(48), 44653 - 62 Epub 2001 Sep 25.
The stereoselective targeting of a specific enzyme-substrate complex is the molecular mechanism for the synergic inhibition of HIV-1 reverse transcriptase by (R)-(-)-PPO464: a novel generation of nonnucleoside inhibitors; Maga G et al.; The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (+/-)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3'-azido-3'-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J . Med . Chem . 42, 4462-4470) . The (+/-)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis . Only one enantiomer, (R)-(-)-PPO464, displayed antiviral activity against both the wild type and the K103N mutant HIV-1 RT and was found to interact exclusively with the reaction intermediate formed by RT complexed with both the DNA and the nucleotide substrates . Being the first compound of its class to display this behavior, (R)-(-)-PPO464 is the representative of a novel generation of nonnucleoside inhibitors . (R)-(-)-PPO464 showed significant synergism when tested in combination with other RT inhibitors and efficiently inhibited viral replication when tested against the laboratory strain HIV-1 IIIB or against either wild type or multidrug-resistant clinical isolates . Pharmacokinetic studies in mice and rats showed a more favorable profile for (R)-(-)-PPO464 than for the corresponding racemate . (R)-(-)-PPO464 was also found to easily cross the blood-brain barrier . The coadministration of the HIV-1 protease inhibitor ritonavir increased the bioavailability of (R)-(-)-PPO464, having little effect on its plasma and brain elimination rates.

Jpn J Cancer Res, 2001 Sep, 92(9), 968 - 74
Altered topoisomerase IIalpha and multidrug resistance-associated protein levels during drug selection: adaptations to increasing drug pressure; Matsumoto Y et al.; To understand resistance to topoisomerase II inhibitors, we used four cancer cell lines (ZR-75B, MDA-MB-231, T47D, and MCF-7) and performed a single-step selection process to isolate 50 clones resistant to topoisomerase II inhibitors . Of these, 26 were isolated with VP-16 and 24 with mAMSA . Sixteen of these isolates (four from each cell line; two selected with VP-16 and two with mAMSA) were further exposed to higher drug concentrations . Characterization of the resistant sublines revealed the adaptation that occurs with increasing drug concentration during in-vitro selections . Reduced topoisomerase IIalpha mRNA level was observed in the majority of the initial isolates . This reduction was accompanied by a decrease in topoisomerase II activity . Other isolates showed increased levels of multidrug resistance-associated protein (MRP) . With advancing resistance, MRP expression was increased further, concomitantly with some recovery in topoisomerase IIalpha expression and topoisomerase II activity . In these sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression . These results extend previous studies demonstrating how cellular adaptation to increasing drug pressure utilizes more than one mechanism . Reduced expression of topoisomerase IIalpha occurs early in the selection process . MRP overexpression can occur early or can help to confer high levels of resistance . In the latter case, MRP overexpression allows some recovery of topoisomerase II activity without loss of high drug resistance.

World J Surg, 2001 Jul, 25(7), 927 - 33
Cytotoxic treatment of adrenocortical carcinoma; Ahlman H et al.; Adrenocortical carcinoma (ACC) is a rare, aggressive tumor that is often detected in an advanced stage . Medical treatment with the adrenotoxic drug mitotane has been used for decades, but critical prospective trials on its role in residual disease or as an adjuvant agent after surgical resection are still lacking . The concept of a critical threshold plasma level of the drug must be confirmed in controlled studies . Because individual responsiveness cannot be predicted, the use mitotane is still advised for nonresectable disease . In case of cortisol or other steroid overproduction, several drugs (e.g., ketoconazole or aminoglutethimide) may be used . Chemotherapy with single agents (e.g., doxorubicin or cisplatin) have been disappointing, with low response rates (< 30%) and a short response duration . Part of this refractoriness may be explained by the fact that ACC tumors express the multidrug-resistance gene MDR-1 . Chemotherapy with multiple agents has been tested in smaller series and has resulted in significant side effects . The best results were achieved by the combination of etoposide, doxorubicin, and cisplatin associated with mitotane, achieving a response rate of 54%, including individual complete responses . To be able to make progress in treating advanced ACC disease, adjuvant multicenter trials must be encouraged . When mitotane-based therapies are used, monitored drug levels are mandatory.

Cancer, 2001 Sep 1, 92(5), 1059 - 73
Therapeutic options for acute myelogenous leukemia; Estey EH; BACKGROUND: General therapeutic options for patients with acute myelogenous leukemia (AML) are reviewed and specific new therapies are described . METHODS: Data in this review came from the published literature and the M . D . Anderson Cancer Center's acute leukemia database . RESULTS: Outcome following standard therapy of AML is so variable that is best to speak of a range of outcomes determined by various prognostic factors . Therapy can (and usually does) fail because of treatment-induced mortality or (more usually) resistance to therapy . Performance status and age are the principal predictors of early death, whereas cytogenetics, a history of abnormal blood counts, and MDR1 expression are predictors of resistance . Using this information, physicians can categorize patients into those in whom 1) standard therapy is indicated, 2) either standard or investigational therapy is appropriate, and 3) investigational therapy is indicated . The majority of even newly diagnosed patients belong to Group 3 . The availability of allogeneic or autologous transplantation does not alter this conclusion . Investigational therapies have been developed that are directed against the CD33 surface antigen, the multidrug-resistant MDR1 protein, and other targets . Because of the number of new therapies clinical research in AML should emphasize pilot trials rather than traditionally large Phase III studies . CONCLUSIONS: Most patients with newly diagnosed AML should be offered investigational regimens .

Bioelectromagnetics, 2001 Oct, 22(7), 470 - 8
Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells; Holandino C et al.; Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems . In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy . DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells . In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability . Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied . The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells . These results were confirmed by flow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment . Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed .

Toxicol Sci, 2001 Oct, 63(2), 196 - 207
Assessment of cisplatin-induced nephrotoxicity by microarray technology; Huang Q et al.; Microarrays are a new technology used to study global gene expression and to decipher biological pathways . In the current study, microarrays were used to examine gene expression patterns associated with cisplatin-mediated nephrotoxicity . Sprague-Dawley rats received either single or seven daily ip doses of cisplatin (0.5 or 1 mg/kg/day) or the inactive isomer transplatin (1 or 3 mg/kg/day) . Histopathological evaluation revealed renal proximal tubular necrosis in animals that received cisplatin for 7 days, but no hepatotoxic findings . Microarray analyses were performed using rat specific arrays containing 250 toxicity-related genes . Prominent gene expression changes were observed only in the kidneys of rats that received cisplatin for 7 days . Mechanistically, the gene expression pattern elicited by cisplatin (e.g., Bax upward arrow and SMP-30 downward arrow) suggested the occurrence of apoptosis and the perturbation of intracellular calcium homeostasis . The induction of multidrug resistance genes (MDR1 upward arrow, P-gp upward arrow) and tissue remodeling proteins (clusterin upward arrow, IGFBP-1 upward arrow, and TIMP-1 upward arrow) indicated the development of cisplatin resistance and tissue regeneration . Select gene expression changes were further confirmed by TaqMan analyses . Gene expression changes were not observed in the liver following cisplatin administration . In contrast to these in vivo findings, studies using NRK-52E kidney epithelial cells and clone-9 liver cells suggested that liver cells were more sensitive to cisplatin treatment . The discrepancies between the in vivo and in vitro results suggest that caution should be taken when extrapolating data from in vivo to in vitro systems . Nonetheless, the current study elucidates the biochemical pathways involved in cisplatin toxicity and demonstrates the utility of microarrays in toxicological studies.

J Photochem Photobiol B, 2001 Sep 15, 62(3), 146 - 52
Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells; Teiten MH et al.; Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer . The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy . The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7/DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7 . No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact . The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light (lambda=650 nm), was significantly greater in MCF-7/DXR as compared to MCF-7 cells . A light dose of 2.5 J cm(-2) inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7/DXR . The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity . The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity . Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype.

Chem Biol, 2001 Sep, 8(9), 843 - 55
The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies; Martello LA et al.; BACKGROUND: During the past decade, Taxol has assumed an important role in cancer chemotherapy . The search for novel compounds with a mechanism of action similar to that of Taxol, but with greater efficacy particularly in Taxol-resistant cells, has led to the isolation of new natural products . One such compound, (+)-discodermolide, although structurally distinct from Taxol, has a similar ability to stabilize microtubules . In addition, (+)-discodermolide is active in Taxol-resistant cell lines that overexpress P-glycoprotein, the multidrug-resistant transporter . Interestingly, (+)-discodermolide demonstrates a profound enhancement of the initiation process of microtubule polymerization compared to Taxol . RESULTS: The synthesis of (+)-discodermolide analogs exploiting our highly efficient, triply convergent approach has permitted structure-activity relationship (SAR) studies . Small changes to the (+)-discodermolide structure resulted in a dramatic decrease in the ability of all four discodermolide analogs to initiate tubulin polymerization . Two of the analogs also demonstrated a decrease in total tubulin polymerization, while a change in the olefin geometry at the C8 position produced a significant decrease in cytotoxic activity . CONCLUSIONS: The availability of (+)-discodermolide and the analogs, and the resultant SAR analysis, have permitted an exploration of the similarities and differences between (+)-discodermolide and Taxol . Docking of the X-ray/solution structure of (+)-discodermolide into the Taxol binding site of beta-tubulin revealed two possible binding modes (models I and II) . The preferred pharmacophore model (I), in which the C19 side chain of (+)-discodermolide matches with the C2 benzoyl group of Taxol and the delta-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.

Histochem J, 2001 May, 33(5), 305 - 9
Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy; Ruspantini I et al.; In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997) . The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment . In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica . After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM) . The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water) . P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

Histochem J, 2001 May, 33(5), 259 - 66
Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells; Candussio L et al.; To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein . This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells . When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed . Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations . In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine . In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP . Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil . Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules . RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed . Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles . This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients . In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells . These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane . The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized . Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.

Indian J Pediatr, 2001 Aug, 68(8), 733 - 6
Typhoid vaccines; Aggarwal A et al.; Typhoid fever continues to be a major public health problem in developing countries with about 33 million cases per year . Protective efficacy of traditional acetone/phenol killed vaccines is similar to newer typhoid vaccines (Ty21A and Vi antigen vaccine) but side effects of these newer vaccines are considerably less . Though the mortality is low, typhoid fever causes considerable morbidity and loss of working days . Problems during treatment are increasing due to emergence and spread of multidrug resistant S . typhi . Hence to decrease the incidence of typhoid fever in addition to ensuring safe water supply and excreta disposal a typhoid vaccine needs to be introduced in the National Immunization Schedule.

Mol Pharmacol, 2001 Oct, 60(4), 674 - 80
Activation of phospholipase C induces the expression of the multidrug resistance (MDR1) gene through the Raf-MAPK pathway; Yang JM et al.; Resistance to multiple, unrelated cancer chemotherapeutic drugs can be mediated by P-glycoprotein, the MDR1 gene product . Numerous substances, including chemotherapeutic drugs, heavy metals, growth factors, activated oncogenes, or changes in temperature increase MDR1 gene expression . Because several of these factors regulate cellular function through the activation of phospholipase C (PLC), we postulated that PLC-mediated signaling could be central to regulating the expression of MDR1 . Transfection of NIH 3T3 cells with a pMJ30-PLC-gamma 1 expression vector increased the activity of the MDR1 promoter by 2- to 10-fold . PLC-mediated activation required a region between -106 and -99 of the MDR1 promoter . Treatment of cotransfected cells with platelet-derived growth factor further enhanced the activity of the MDR1 promoter . The stimulatory effect of PLC on the MDR1 promoter was increased by cotransfection with constitutively active v-raf and was blocked by the dominant-negative mutant, c-Raf-C4 . The activity of mitogen-activated protein kinase (MAPK) was also increased in PLC-gamma 1-transfected cells . Furthermore, PD-98059 and U0126, two MAPK inhibitors, blocked PLC-gamma 1-induced expression of MDR1 . The results of Northern blot analysis showed that activation of PLC by heat shock and growth factors increased expression of endogenous MDR1 mRNA in human renal carcinoma cells . These effects were blocked by inhibitors of the PLC-MAPK pathway . In summary, our results indicate for the first time that activation of PLC by a variety of cellular stimuli can regulate the expression of MDR1 and that the transcriptional modulation of MDR1 expression by PLC is mediated by the Raf-MAPK pathway.

Invest New Drugs, 2001, 19(3), 239 - 43
Phase II trial of CI-980 in patients with disseminated malignant melanoma and no prior chemotherapy . A Southwest Oncology Group study; Whitehead RP et al.; Malignant melanoma is increasing in frequency at a rapid rate in the United States . Metastatic disease is chemoresistant with DTIC considered the most active single agent . CI-980 is a synthetic mitotic inhibitor that blocks the assembly of tubulin and microtubules . It has shown cytotoxic activity against a broad spectrum of murine and human tumor cell tines . CI-980 can cross the blood brain barrier, is effective when given orally or parenterally, and is active against multidrug resistant cell lines overexpressing P-glycoprotein . In this trial, patients with disseminated melanoma with measurable disease, SWOG performance status of 0-1, no prior chemotherapy or immunotherapy for metastatic disease, and adequate hepatic and renal function, were enrolled . Treatment with CI-980 was given by 72 h continuous i.v . infusion at a dose of 4.5 mg/m2/day, days 1-3 every 21 days . Twenty-four patients were registered on this study with no patients ineligible . They ranged in age from 33-78 with performance status of 0 in 15 patients and 1 in 9 patients . Nineteen patients had visceral disease with 12 having liver involvement . There were no confirmed responses . The overall response rate was 0% (95% CI 0%-14%) . The median overall survival is eleven months (95% CI 4-14 months) . The most common toxicities were hematologic and consisted of leukopenia/granulocytopenia and anemia, with nausea/vomiting and malaise/fatigue/weakness also frequent . CI-980 administered at this dose and schedule has insufficient activity in the treatment of disseminated malignant melanoma to warrant further investigation.

Invest New Drugs, 2001, 19(3), 211 - 7
Tetramethylpiperidine-substituted phenazines inhibit the proliferation of intrinsically multidrug resistant carcinoma cell lines; van Niekerk E et al.; The effects of nine new tetramethylpiperidine (TMP)-substituted phenazines on the growth of a human esophageal cancer cell line (WHCO3), two human hepatocellular carcinoma cell lines (PLC and HepG2) and three human colon cancer cell lines (CaCo2, COLO 320DM and HT29) were compared to those of clofazimine, B669 and five standard chemotherapeutic agents . The three most active TMP-substituted phenazines against these cell lines were B3962, B4126 and B4125 with mean IC50 values for all the cancer cell lines tested of 0.36, 0.47 and 0.48 microg/ml respectively . B3962 and B4126, but not B4125 were also the most active against a semi-continuous human fibroblast culture (MRC5) . The compound with the highest tumor specificity relative to the fibroblast culture, was B4125 . Importantly, there was minimal variation in sensitivity of the different cell lines, including a multidrug resistant cell line (COLO 320DM) expressing high levels of P-glycoprotein, to the TMP-substituted phenazines . This was not the case with the standard chemotherapeutic agents . The efficacy of compounds such as B4125 against a broad spectrum of multidrug resistant cancer cell lines, together with their relatively high tumor specificity, suggests that these agents may be useful in the treatment of intrinsically resistant cancers such as colon and liver cancer.

Drug Metab Dispos, 2001 Oct, 29(10), 1256 - 62
Efflux of glutathione conjugate of monochlorobimane from striatal and cortical neurons; DeCory HH et al.; Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented . Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG) . The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid . The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations . In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons . Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons . Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.

Hum Gene Ther, 2001 Sep 20, 12(14), 1785 - 96
Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents; Rappa G et al.; We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP) . We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes . In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR) . Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants . Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate . By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml . The correct viral integration in the genome was established by Southern blotting . By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein . The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts . Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs . However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents . We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents . The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.

Biol Pharm Bull, 2001 Sep, 24(9), 1032 - 6
Reversal effects of antifungal drugs on multidrug resistance in MDR1-overexpressing HeLa cells; Iida N et al.; In this study, the antiproliferative effects of vinblastine (VLB), paclitaxel (TXL), doxorubicin (DXR), daunorubicin (DNR) and 5-fluorouracil (5-FU) were assessed in the human cervical carcinoma cell line HeLa-Ohio (HeLa) and Hvr100-6 cells, established by growing the parental HeLa cells in the presence of progressively greater concentrations of VLB in the culture medium . Flow cytometric analysis indicated the induction of MDR1 (P-glycoprotein) in Hvr100-6 cells with no alterations in levels of multidrug resistance-associated protein (MRP) . Resistance to VLB, TXL, DXR and DNR was found in Hvr100-6 cells with relative resistances of ca . 300, 4000, 50 and 200, respectively, whereas no resistance was found to 5-FU . The reversal effects of antifungal drugs, fluconazole, itraconazole, ketoconazole, miconazole and amphotericin B on multidrug resistance were also assessed using Hvr100-6 cells . Itraconazole was found to have potent reversal effect on the resistance to VLB and TXL, but the others had no such effect . This reversal effect of itraconazole was concentration-dependent, with dose modifying factors of 3.2, 10.1 and 435.7 at 0.1, 0.25 and 0.5 microM of itraconazole, respectively . In addition, this reversal effect of itraconazole was explained by the inhibition of accumulation of the anticancer drugs.

Genetika, 2001 Jul, 37(7), 869 - 87
{Phagotherapy in terms of bacteriophage genetics: hopes, perspectives, safety, limitations}; Krylov VN; The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine . In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections . Constructs obtained by gene engineering are increasingly used to change some bacteriophage: properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages . In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics . Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.

Am J Physiol Regul Integr Comp Physiol, 2001 Oct, 281(4), R1119 - 26
Expression and functional activity of P-glycoprotein in cultured hepatocytes from Oncorhynchus mykiss; Sturm A et al.; P-glycoproteins encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the plasma membrane that mediate the extrusion of hydrophobic compounds from the cell . Using cultured isolated rainbow trout hepatocytes, we characterized an mdr1-like transport mechanism of the teleost liver . Immunoblots with the monoclonal antibody C219, which recognizes a conserved epitope of P-glycoproteins, revealed the presence of immunoreactive protein(s) of 165 kDa in trout liver and cultured hepatocytes . In trout liver sections, the immunohistochemistry with C219 stained bile canalicular structures . Compounds known to interfere with mdr1-dependent transport (verapamil, vinblastine, doxorubicin, cyclosporin A, and vanadate) all increased the accumulation of rhodamine 123 by hepatocytes . Verapamil, vinblastine, and cyclosporin A decreased the efflux of rhodamine 123 from hepatocytes preloaded with rhodamine 123 . By contrast, the substrate of the canalicular cation transporter tetraethylammonium and the inhibitor of the multidrug resistance-associated protein MK571 had no effect on rhodamine 123 transport . The results demonstrate the presence of an mdr1-like transport system in the teleost liver and suggest its function in biliary excretion.

Am J Physiol Gastrointest Liver Physiol, 2001 Oct, 281(4), G1034 - 43
Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate; Ito K et al.; Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC) . The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains . For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined . Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates . Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates . These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

Toxicology, 2001 Oct 5, 167(1), 59 - 72
Expression of MRP1 and related transporters in human lung cells in culture; Lehmann T et al.; Multidrug resistance type 1 P-glycoproteins (P-gp) and multidrug resistance associated proteins (MRP) were studied in differentiated primary human lung cells in culture, in comparison with permanent human lung cell lines and primary alveolar type II cells from rat lung . AII cells exhibited low basal levels of mdr1b mRNA, that increased over time and after oxygen radical production induced by paraquat . mRNAs coding for antioxidative enzymes catalase (CAT), maganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase (Cu/Zn-SOD) were not changed . H358, A549, H322 cells expressed low levels of MDR1 mRNA, but the mdr1 substrate rhodamine 123 (Rh 123) was transported out of H358 and H322 cells in a non-invasive, single cell fluorescence assay . The dye efflux could be inhibited by the chemosensitizer, verapamil . Normal human bronchial epithelial cells (NHBEC) expressed immuno-reactive MDR1 P-gp and the MPR protein that was active in the fluorescence assay using the MRP substrate carboxy-dichlorofluorescein (CDF) and MK-571 as an inhibitor . We did observe inter-individual variation of MRP in both the mRNA and the immunoreactive protein in NHBEC culture . Over time (12 weeks) the protein was relatively stable in NHBEC and epithelial cells from peripheral lung (PLC), but the mRNA level was drastically increased when explant cultures were continued (18 weeks).

Toxicology, 2001 Oct 5, 167(1), 47 - 57
Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures; Hirsch-Ernst KI et al.; P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane . Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity . Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells . Mdr1b gene expression increases with time in primary rat hepatocyte culture . In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM) . Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100 . In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect . The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells . These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.

Toxicology, 2001 Oct 5, 167(1), 37 - 46
Regulation of biliary drug efflux pump expression by hormones and xenobiotics; Fardel O et al.; Biliary elimination of endogenous compounds and xenobiotics usually requires carrier-mediated systems allowing movement across the canalicular membrane of hepatocytes . The major systems implicated belong to the ATP binding cassette transporter family: P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), principally mediate the passage into the bile of cationic and anionic compounds, respectively, whereas the bile salt export pump (BSEP) handles biliary acids and also some anticancer drugs . Expression of these canalicular proteins can be altered in response to various hormones and structurally unrelated xenobiotics . Indeed, glucocorticoids up-regulate expression of both MRP2 and BSEP in rat hepatocytes, whereas insulin induces P-gp . P-gp expression is also up-regulated by numerous chemical carcinogens, such as polycyclic aromatic hydrocarbons and 2-acetylaminofluorene and by some anticancer drugs, such as anthracyclins . 2-Acetylaminofluorene also induces MRP2; in addition, expression of this transporter in liver cells is increased in response to various drugs, such as the barbiturate phenobarbital, the chemopreventive agent, oltipraz and the anticancer drug, cisplatin . Most of the chemical inducers acting on canalicular transporter levels are well-known to up-regulate some hepatic drug metabolizing enzymes, suggesting a coordinate regulation of liver detoxifying proteins in response to these compounds.

Toxicology, 2001 Oct 5, 167(1), 25 - 35
Basal expression of the rat, but not of the human, multidrug resistance protein 2 (MRP2) gene is mediated by CBF/NF-Y and Sp1 promoter-binding sites; Kauffmann HM et al.; The most important biliary efflux transporter known so far is the multidrug resistance protein 2 (MRP2) . Previously, we isolated and characterized the 5'-flanking region of the rat mrp2 gene . In the present study, we performed site-directed mutagenesis experiments indicating that both a Y-Box and a GC-Box in the rat mrp2 promoter are essential for the full basal expression of the gene, but have no significant relevance for its inducibility by the chemical carcinogen 2-acetylaminofluorene . Gel mobility shift experiments demonstrated the binding of the transcription factor CBF/NF-Y, but not of EFIA/YB-1, to the Y-Box . Site-directed mutations in the Y-Box decreasing reporter gene activity of a promoter construct prevented the binding of NF-Y . Consequently, NF-Y contributes substantially to the basal expression of the gene . A site-directed mutation in the GC-Box also reduced basal expression and resulted in a reduced complex formation with the transcription factor Sp1 . The corresponding region of the human MRP2 promoter comprises no Sp1 site, but a Y-Box-like element binding YB-1 but not NF-Y, which, however, does not contribute to basal expression . In conclusion, NF-Y and Sp1 binding sites play a decisive role in the basal expression of the rat mrp2 gene, while the human MRP2 gene is regulated differently.

Toxicology, 2001 Oct 5, 167(1), 3 - 23
Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters; Leslie EM et al.; The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells . In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance . Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents . MRP1 also transports unmodified xenobiotics but often requires GSH to do so . Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic . The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed . Finally, MRP homologues have been described in other species including yeast and nematodes . Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.

FEBS Lett, 2001 Sep 7, 505(1), 103 - 8
Differential display analysis of mutants for the transcription factor Pdr1p regulating multidrug resistance in the budding yeast; Miura F et al.; The transcription factor Pdr1p recognizes Pdr1p/Pdr3p-response element (PDRE) to activate genes involved in multidrug resistance of the budding yeast . To identify novel targets of Pdr1p, we compared transcriptomes among the yeast cells bearing wild, disrupted and gain-of-function alleles of PDR1 using a high-throughput fluorescent differential display PCR . Consequently, we identified 20 transcripts apparently regulated by Pdr1p, which are derived from well-known target genes as well as those that have never been described in the context of drug resistance . Intriguingly, among the latter, a previously unrecognized gene bearing a small putative open reading frame preceded by a functional PDRE was found.

Appl Immunohistochem Mol Morphol, 2001 Sep, 9(3), 242 - 9
Intrinsic expression of drug resistance-associated factors in meningiomas; Tews DS et al.; Meningiomas, commonly benign tumors, rarely display aggressive behavior by recurrences and invasion . In addition to surgery, irradiation is beneficial for recurrent, atypical, and malignant meningiomas . The role of chemotherapy, however, remains controversial, although there is evidence that meningiomas respond well to adjuvant chemotherapy . A major obstacle in chemotherapy remains drug resistance with reduced cellular drug accumulation through membrane efflux pumps, drug detoxification, and alterations in drug target specificity . In 84 classic, atypical, and malignant meningiomas, the immunohistochemical expression profile of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), metallothionein, and topoisomerase IIalpha were studied . All types of meningiomas showed constant expression of P-gp, LRP, MRP, and topoisomerase IIalpha; metallothionein was found in 67% of the tumors, especially in atypical and malignant meningiomas . Furthermore, metallothionein . P-gp, LRP, and topoisomerase IIalpha were strongly expressed by normal and neoplastic vessels, which may confer to impaired penetration of therapeutic agents through the blood-brain and blood-tumor barrier . Neither recurrent nor previously irradiated meningiomas revealed any significant difference to primary tumors . These intrinsic drug resistances indicate that successful chemotherapy may require additional inhibition of these factors to be a promising approach in the management of meningiomas.

Southeast Asian J Trop Med Public Health, 2001 Jun, 32(2), 255 - 61
Clinical trial of halofantrine with modified doses for treatment of malaria in the hospital for tropical diseases; Krudsood S et al.; The spread of falciparum malaria resistant to chloroquine all over Southeast Asian continent has led to increasing use of alternative antimalarial drugs . Halofantrine has been shown to be effective against multidrug resistant Plasmodium falciparum . One hundred and twenty falciparum malaria cases were randomly assigned to one of three different halofantrine regimes . Group I (HA1) received 500 mg three times daily for 3 days (total dose: 4,500 mg), group II (HA2) received 500 mg three times daily for the first and the third day (total dose: 3,000 mg) and group III (HA3) received 500 mg three times for one day followed by 500 mg once daily for 7 days (total dose: 4,500 mg) . No significant difference in the cure rate was observed among the three regimes (cure rate: 89%, 73%, 97% respectively) . However, the cure rate was significantly higher in the HA3 group when compared to the HA2 group . There were no overt cardiac problems seen in this study . Thus, halofantrine has high efficacy in the recommended treatment dose of 500 mg three times after meals on the first day followed by 500 mg once a day after a meal for 7 days (total dose: 4,500 mg).

Clin Cancer Res, 2001 Sep, 7(9), 2912 - 22
Expression of beta-tubulin isotypes in human ovarian carcinoma xenografts and in a sub-panel of human cancer cell lines from the NCI-Anticancer Drug Screen: correlation with sensitivity to microtubule active agents; Nicoletti MI et al.; Paclitaxel resistance has been associated with overexpression of P-glycoprotein and alterations involving tubulin . To investigate the clinical relevance of these in vitro resistance mechanisms, we established 12 human ovarian carcinoma xenografts, using samples from patients before the start of therapy or after paclitaxel treatment . These xenografts showed a wide range of sensitivity to paclitaxel, and in 4 of them, very low levels of multidrug resistance-1 expression were detected . Using quantitative PCR and human specific primers, the expression of five beta-tubulin isotypes was determined . HM40 was the predominant, accounting for 84.7-98.7% of all tubulin; expression of the other four isotypes (Hbeta9, Hbeta4, H5beta, and Hbeta2) was also detected but at lower levels . No correlation could be demonstrated between isotype expression and paclitaxel sensitivity in these 12 xenografts . A similar pattern of beta-tubulin isotype expression was observed in a subset of cell lines from the National Cancer Institute-Anticancer Drug Screen . In these cell lines, however, a significant correlation between increased expression of Hbeta4 isotype and resistance to paclitaxel was found . Taken together, these results suggest that altered expression of specific beta-tubulin isotypes may not play a significant role in paclitaxel sensitivity in vivo and argue against a possible significance in a clinical setting.

Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11283 - 8 Epub 2001 Sep 11.
Origin of multidrug resistance in cells with and without multidrug resistance genes: chromosome reassortments catalyzed by aneuploidy; Duesberg P et al.; Cancer cells and aneuploid cell lines can acquire resistance against multiple unrelated chemotherapeutic drugs that are over 3,000-fold those of normal levels and display spontaneous resistances up to 20-fold of normal levels . Two different mechanisms were proposed for this phenotype: (i) classical mutation of drug metabolizing genes or (ii) chromosome reassortments, catalyzed by cancer- and cell line-specific aneuploidy, which generate, via new gene dosage combinations, a plethora of cancer phenotypes, including drug resistance . To distinguish between these mechanisms, we have asked whether three mouse cell lines can become drug resistant, from which two or three genes have been deleted, and on which multidrug resistance is thought to depend: Mdr1a, Mdr1b, and Mrp1 . Because all three lines could acquire multidrug resistance and were aneuploid, whereas diploid mouse cells could not, we conclude that aneuploid cells become drug resistant via specific chromosome assortments, independent of putative resistance genes . We have asked further whether aneuploid drug-resistant Chinese hamster cells revert spontaneously to drug sensitivity in the absence of cytotoxic drugs at the high rates that are typical of chromosome reassortments catalyzed by aneuploidy or at the very low or zero rates (i.e., deletion) of gene mutation . We found that four drug-resistant hamster cell lines reverted to drug sensitivity at rates of about 2-3% per generation, whereas two closely related lines remained resistant under our conditions . Thus, the karyotypic instability generated by aneuploidy emerges as the common source of the various levels of drug resistance of cancer cells: minor spontaneous resistances reflect accidental chromosome assortments, the high selected resistances reflect complex specific assortments, and multidrug resistance reflects new combinations of unselected genes located on the same chromosomes as selected genes.

Br J Haematol, 2001 Sep, 114(3), 581 - 90
Cells from chronic myelogenous leukaemia patients at presentation exhibit multidrug resistance not mediated by either MDR1 or MRP1; Carter A et al.; Tetramethylrosamine (TMR) is excluded from P-glycoprotein (MDR1)-enriched cell lines, but it stains efficiently MDR1-poor parent lines . Application of the TMR resistance assay to cells obtained from chronic myelogenous leukaemia (CML) patients revealed, in all individuals, a significant resistance compared with healthy donors (P < 0.001) . Cells from the same patients at later phases exhibited a further increase in TMR resistance . Doxorubicin was excluded from all cell samples obtained from CML patients at presentation . The resistance to TMR and doxorubicin was energy-dependent, and was not modulated by inhibitors of MDR1 and multidrug-resistance protein-1 (MRP1) . Transcription of mRNAs suspected as relevant to multidrug resistance was assessed using comparative reverse transcription polymerase chain reaction . All cells from the CML patients transcribed high levels of MRP3, MRP4 and MRP5 compared with healthy donors . Low levels of MDR1, MRP1, MRP2, MRP6, lung resistance-related protein and anthracycline resistance-associated protein were equally transcribed in cells from healthy donors and CML patients . These results indicated that neither MDR1 nor MRP1 mediate the resistance in these cells . Our results shed light on a resistance mechanism operative in CML patients, which, together with the resistance to apoptosis, is responsible for the lack of response of CML patients to induction-type protocols used to treat acute myeloid leukaemia patients.

Br J Haematol, 2001 Sep, 114(3), 557 - 65
Cross-resistance to cytosine arabinoside in a multidrug-resistant human promyelocytic cell line selected for resistance to doxorubicin: implications for combination chemotherapy; Mansson E et al.; The pyrimidine analogue cytosine arabinoside (AraC) is one of the most effective drugs used in the treatment of acute leukaemia . Overexpression of the multidrug resistance (MDR-1) gene and its product, P-glycoprotein (P-gp), is associated with cellular resistance to drugs, such as anthracyclines and vinca alkaloids . This resistance can be reversed by cyclosporine analogues or verapamil (ver) . We investigated the in vitro cross-resistance to AraC in a doxorubicin-resistant HL60 cell line, with an elevated expression of the MDR-1 gene . The resistant clone showed an eightfold increased resistance to AraC and a two- to fourfold resistance to the other analogues, as measured by cytotoxicity test . There was no significant increase in the activity of 5'-nucleotidase or in the amount of deoxyribonucleotide pools between cell lines . We could, however, detect a reduction in deoxycytidine kinase (dCK) activity (30%, P = 0.021, using deoxycytidine as substrate) and the level of AraC triphosphates was significantly reduced in the resistant cells (70%, P = 0.009) . When the cells were exposed to cyclosporin A (CsA) or the cyclosporine analogue PSC 833 (PSC) in combination with AraC, there was more extensive apoptosis, as measured by formation of oligonucleosomal DNA fragmentation and caspase-3-like activity, than with exposure to AraC alone . We also found an increased retention of AraC in the resistant cells when incubated with AraC in combination with CsA . Ver in combination with AraC, failed to increase apoptosis for the resistant cell line . Our data suggests that the resistance to AraC for the P-gp-expressing cells is a result of a reduction of dCK activity and an increase in efflux, the latter possibly depending on P-gp . A combination of CsA or PSC with AraC may improve the effect of AraC in vivo.

Biochem Pharmacol, 2001 Sep 15, 62(6), 765 - 72
Effects of miltefosine on various biochemical parameters in a panel of tumor cell lines with different sensitivities; Rybczynska M et al.; We investigated endocytosis activity, uptake of miltefosine (hexadecylphosphocholine), phospholipid and cholesterol content, the cell cycle, and apoptosis in 13 tumor cell lines (MCF7, MCF7/ADR, KB-3-1, KB-8-5, KB-C1, HeLa, HeLa-MDR1-G185, HeLa-MDR1-V185, CCRF/CEM, CCRF/VCR1000, CCRF/ADR5000, HL-60, HL-60/AR) with different sensitivities to treatment with the antitumor phospholipid analogues miltefosine and D-21266 (octadecyl-(N,N-dimethyl-piperidino-4-yl)-phosphate) . In this panel of cell lines, MDR1 (multidrug resistance gene 1)- and MRP1 (multidrug resistance-associated protein)-expressing cells were found to be slightly more resistant to both compounds than sensitive parental cells . No correlation was found between resistance to miltefosine and endocytosis, intracellular concentration of miltefosine, the phospholipid and cholesterol content, induction of apoptosis, or cell cycle alterations in all the cell lines tested . Wild-type p53 containing WMN Burkitt's lymphoma cells and wild type p53-deficient CA46 exhibited similar sensitivities to miltefosine . The low percentage of apoptosis induced in MCF7 cells lacking caspase 3 indicated that caspase 3 seems to play an essential role in miltefosine-induced apoptosis.

Biochem Pharmacol, 2001 Sep 15, 62(6), 733 - 41
Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes; Meng LH et al.; Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a novel anticancer drug candidate . The compound has significant in vitro and in vivo activity against malignant tumor cells and xenografts, especially some human solid tumor models . This anticancer activity of salvicine is associated with its ability to induce tumor cell apoptosis . Salvicine was also found to have a profound cytotoxic effect on multidrug-resistant (MDR) cell lines by down-regulating the expression of MDR-1 mRNA of MDR cells . Salvicine acted as a topoisomerase II (Topo II) poison through its marked enhancement effect on Topo II-mediated DNA double-strand breaks as observed in the DNA cleavage assay . Strong inhibitory activity of salvicine against Topo II was observed in a kDNA decatenation assay, with an approximate IC(50) value of 3 microM . A similar result was obtained by a Topo II-mediated supercoiled DNA relaxation assay . In contrast, no inhibitory activity was observed against the catalytic activity of Topo I . When the effects of salvicine on individual steps of the catalytic cycle of Topo II were dissected, it was found that the mechanism by which salvicine inactivates Topo II is different from that by other anti-Topo II agents . Salvicine greatly promoted Topo II-DNA binding and inhibited pre- and post-strand Topo II-mediated DNA religation without interference with the forward cleavage steps . In addition, salvicine was not a DNA intercalative agent, as demonstrated by DNA unwinding assays . The results of this study indicate that the inhibitory activity of salvicine against Topo II was derived from its ability to stabilize DNA strand breaks through interactions with the enzyme alone or with the DNA-enzyme complex . It is therefore postulated that salvicine acts on Topo by trapping the DNA-Topo II complex, which in turn produces anticancer effects.

Biochem Pharmacol, 2001 Sep 15, 62(6), 693 - 704
MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell lines; Kane SE et al.; The human multidrug resistance-1 gene (MDR1) is a dominant selectable and amplifiable marker in mammalian tissue culture cells . MDR1 is also being investigated as a gene therapy tool, both to protect normal cells against chemotherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes . The success of these strategies will depend on whether MDR1 expression can be sustained at levels high enough to confer a survival advantage on target cells . However, the MDR1 selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug . The current report is a detailed molecular analysis of MDR1 selection stringency compared with the common neo selectable marker . A bicistronic vector encoding MDR1 and neo genes linked through an internal ribosome entry site was transferred into NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDR1 expression) or to G418 (to select for neo expression) . Surviving populations and individual clones of cells were analyzed for expression levels of MDR1 and neo gene products; resistance to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vector DNA . These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expression can be accommodated primarily by gene amplification, even within an individual transduced clone and starting from a single-copy proviral integration event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection . Taken together, these results have important implications for the potential utility of MDR1 as a selectable marker and as a gene therapy tool in hematopoietic cells.

Adv Drug Deliv Rev, 2001 Jul 28, 49(3), 317 - 23
Upregulation of caveolin in multidrug resistant cancer cells: functional implications; Lavie Y et al.; Multidrug resistance (MDR) is a multifactorial process that involves elevated expression of drug transporters as well as additional biochemical changes that contribute to the drug resistant phenotype . Here we review recent results indicating the upregulation of constituents of rafts and caveolae, including glucosylceramide, cholesterol and caveolin-1, in MDR cells . Accordingly, the number of plasma membrane caveolae is greatly increased in MDR cells . The relationship between caveolin and MDR may be linked to the function of caveolin-1 in mediating cholesterol efflux, a pathway that we hypothesized to facilitate the delivery of drugs from intracellular compartments to plasma membrane resident drug transporters . An additional link seems to exist between the upregulation of GlcCer synthase and attenuation of ceramide-mediated apoptotic signaling . These adaptations may promote cell survival during chemotherapy and, hence, would be positively selected during cell exposure to cytotoxic drugs . However, the overexpression of caveolin-1, an oncosuppressive protein, may also reverse or attenuate important aspects of the phenotypic transformation of MDR cells . The molecular mechanisms by which caveolin-1 exerts its effects on cell proliferation, cell survival, and multidrug resistance remain to be fully elucidated.

Photochem Photobiol, 2001 Aug, 74(2), 331 - 8
In vitro photobiological evaluation of Rhodac, a new rhodacyanine photosensitizer; Delaey EM et al.; We have previously shown that the rhodacyanine dye, Rhodac, exhibits a potent photocytotoxic activity in HeLa cells . In this study several aspects of the photobiological activity of Rhodac were further examined . Rhodac displayed no selective cytotoxicity toward several malignant cell lines after photosensitization (3.6 J/cm2), although HeLa cells were found to be the most sensitive . Interestingly, MCF-7/Adr cells, a multidrug-resistant subline, were less sensitive to the antiproliferative effect of photoactivated Rhodac . The subcellular localization, as revealed by confocal laser microscopy, demonstrated that the dye was mainly concentrated in the cytosolic membranes of the perinuclear region . The Rhodac-induced inhibition of HeLa cell proliferation after light exposure was found to be strictly oxygen dependent . In addition, photoactivated Rhodac induced poly(adenosine 5' diphosphate-ribose)polymerase cleavage, caspase-3 activation and apoptosis in HeLa cells . In the current work it was further demonstrated that Rhodac binds specifically to high-density lipoproteins and low-density lipoproteins, while no binding was observed to very low-density and heavy proteins . To sum up, our results show that Rhodac is an interesting and potent photosensitizer . Further in vivo experiments are required to elucidate whether the lipoprotein binding leads to a selective uptake of Rhodac in tumor cells and to address its efficacy in photodynamic therapy.

Science, 2001 Sep 7, 293(5536), 1793 - 800
Structure of MsbA from E . coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters; Chang G et al.; Multidrug resistance (MDR) is a serious medical problem and presents a major challenge to the treatment of disease and the development of novel therapeutics . ABC transporters that are associated with multidrug resistance (MDR-ABC transporters) translocate hydrophobic drugs and lipids from the inner to the outer leaflet of the cell membrane . To better elucidate the structural basis for the "flip-flop" mechanism of substrate movement across the lipid bilayer, we have determined the structure of the lipid flippase MsbA from Escherichia coli by x-ray crystallography to a resolution of 4.5 angstroms . MsbA is organized as a homodimer with each subunit containing six transmembrane alpha-helices and a nucleotide-binding domain . The asymmetric distribution of charged residues lining a central chamber suggests a general mechanism for the translocation of substrate by MsbA and other MDR-ABC transporters . The structure of MsbA can serve as a model for the MDR-ABC transporters that confer multidrug resistance to cancer cells and infectious microorganisms.

Steroids, 2001 Sep, 66(9), 701 - 5
Biliary excretion of tauroursodeoxycholate-3-sulfate in the rat; Akimoto K et al.; Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, multidrug resistance protein 2 . On the other hand, a multiplicity of canalicular organic anion transport has been suggested . Ursodeoxycholic acid, the 7beta-epimer of chenodeoxycholic acid, is clinically used for various hepatobiliary diseases . In our previous study, the contribution of multidrug resistance protein 2 for biliary excretion of taurine-conjugated bile acid sulfates depended on the numbers of hydroxyl residue . Therefore, to further examine the effect of hydrophobicity on the substrate specificity of multidrug resistance protein 2, we examined the effect of bile acid conjugates and organic anions on biliary excretion of tauroursodeoxycholate-3-sulfate, taurine and sulfonate-conjugated ursodeoxycholic acid, in rats . Biliary tauroursodeoxycholate-3-sulfate excretions was markedly delayed in Eisai hyperbilirubinemic rats . Taurolithocholate-3-sulfate inhibited but ursodeoxycholate-3,7-disulfate did not affect biliary tauroursodeoxycholate-3-sulfate excretion . Biliary tauroursodeoxycholate-3-sulfate excretion was inhibited by sulfobromophthalein, but was not inhibited by dibromosulfophthalein and cefpiramide . These findings indicate that tauroursodeoxycholate-3-sulfate is very specific for multidrug resistance protein 2.

Comp Biochem Physiol A Mol Integr Physiol, 2001 Sep, 130(2), 301 - 10
Expression of P-glycoprotein in the chicken; Barnes DM; The multidrug resistance gene product, P-glycoprotein, may act as a defense mechanism against natural and man-made environmental toxins . Like mammals, chickens show high levels of P-glycoprotein expression in the liver, small intestine, and kidney . Expression of P-glycoprotein rapidly increased with age in the liver and kidney reaching a plateau by 2 and 4 days of age, respectively; however, expression of P-glycoprotein in the duodenum did not significantly change with age . Addition of dietary antibiotics (monensin, bacitracin), as models for dietary toxins, altered P-glycoprotein expression . Monensin increased P-glycoprotein expression in the liver and duodenum . Bacitracin reduced P-glycoprotein expression by 45% in the liver, but did not alter expression in the duodenum . Intraperitoneal injection of E . coli lipopolysaccharide, a model for acute inflammation, rapidly increased expression of Pgp protein in the liver ( approximately 2-fold) . Expression then declines to pre-induction levels by 24 h . Similar responses were observed in the spleen and kidney but not the duodenum . These results confirm the presence of an avian P-glycoprotein homologue and suggest that dietary constituents regulate the expression of P-glycoprotein . Changes in P-glycoprotein expression may represent an important physiological response to foods containing toxins and an important component of the acute phase immune response.

Biochem Pharmacol, 2001 Oct 1, 62(7), 811 - 9
Antiproliferative prostaglandins and the MRP/GS-X pump role in cancer immunosuppression and insight into new strategies in cancer gene therapy; Homem de Bittencourt PI Jr et al.; A dramatic complication in late-stage cancer patients is host immunosuppression . Cyclopentenone prostaglandins (CP-PGs) overproduced in cancer may impair the function of the immune system . These agents, if produced at high concentrations, are powerful cytostatic and cytotoxic compounds that may arrest cell proliferation and immune response in cancer . Lymphoid tissues of tumor-bearing animals accumulate large amounts of CP-PGs, whereas the tumor tissue does not . This may be because cancer cells are able to overexpress multidrug resistance-associated protein (Mg(2+)-dependent vanadate-sensitive GS-conjugate export ATPase, MRP/GS-X pump), which extrudes CP-PGs to the extracellular space as glutathione S-conjugates . In contrast, MRP/GS-X pump activity is disproportionately low in lymphocytes . This led us to propose the transfection of lymphocytes with multidrug resistance-associated protein genes (MRP) for further autologous transfusion or direct in vivo delivery to lymphocytes by using adenovirus-retrovirus chimeras in order to restore immune system function in cancer, at least partially . We are currently evaluating MRP-transfected lymphocyte (MTL) therapy, using Walker 256 tumor-bearing rats as a model.

Blood, 2001 Sep 15, 98(6), 1927 - 34
Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF; Senchenkov A et al.; DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia . HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis . After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence . Similar results were obtained in HL-60 cells . Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF . By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation . Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50% . Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM) . Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis . Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM . Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.

J Control Release, 2001 Sep 11, 76(1-2), 1 - 10
Effect of PSC 833 liposomes and Intralipid on the transport of epirubicin in Caco-2 cells and rat intestines; Lo Y et al.; Clinical applications of first-generation multidrug resistance (MDR) modulators, such as cyclosporin A (CsA) have been hampered because of their severe side effects in vivo . Recent investigations have led to the development of a more potent and less toxic modulator, PSC 833, which is a nonimmunosuppressive analogue of CsA . However, adverse pharmacokinetic interactions between anticancer drugs and PSC 833 have resulted in increased toxicity as compared to the individual toxicity . Our study evaluated the MDR reversing effect of PSC 833 in free, liposomal or Intralipid formulations on the uptake and transport of epirubicin in Caco-2 cells and everted gut sacs of rats . The results showed that PSC 833 in free or liposomal formulations significantly enhanced the intracellular accumulation of epirubicin in a dose-related manner in Caco-2 cells . The optimum in enhancement was observed at the concentration of 2 microM PSC 833 . These formulations markedly increased the apical to basolateral absorption of epirubicin in Caco-2 cells and substantially improved the mucosal to serosal absorption of epirubicin in rat jejunum and ileum . PSC 833 in free, liposomal or Intralipid formulations all significantly reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers . However, PSC 833 in liposomes showed greater enhancement than other formulations . In conclusion, PSC 833 and PSC 833 liposomes have the function as MDR reversing agents for the inhibition of intestinal P-glycoprotein . Liposomal preparations of PSC833 may provide a useful alternative dosage form for intravenous administration of PSC 833 to be combined with anticancer drugs to circumvent drug resistance in cancer chemotherapy.

Kidney Int, 2001 Sep, 60(3), 1069 - 76
P-glycoprotein inhibitors stimulate renal phosphate reabsorption in rats; Prie D et al.; BACKGROUND: Dipyridamole (Dip) was previously shown to increase renal phosphate (Pi) reabsorption in humans . However, the mechanism(s) underlying this renal tubular effect is not fully elucidated . It is known that Dip inhibits the activity of the P-glycoprotein (Pgp) multidrug resistance protein 1 (MDR1) expressed on the apical membrane of renal proximal tubular cells where the Na-Pi cotransporter (NPT2) is also expressed . We hypothesized that Dip could increase renal Pi reabsorption by inhibiting Pgp activity . METHODS: To test this hypothesis, the effects of Dip, verapamil (Ver), and cyclosporine A (CsA), three unrelated Pgp inhibitors, were studied on the renal Pi reabsorption in rats . RESULTS: All three drugs decreased the fractional excretion of Pi (FE(Pi)) in a dose-dependent manner within one hour after beginning the drug infusion, without altering the glomerular filtration rate or serum parathyroid hormone concentration . Sodium-dependent Pi uptake but not Na-glucose transport was increased in brush-border membrane vesicles (BBMVs) when comparing treated with untreated rats . Western blot analysis showed that NPT2 protein was increased in BBMVs from treated rats . Dip and Ver had no effect when applied directly to BBMVs prepared from untreated rats . Pretreatment of rats with colchicine prevented the effects of Dip on the FE(Pi) and NPT2 expression in brush-border membranes . CONCLUSIONS: Our results suggest that inhibition of Pgp in the proximal tubule increases Pi uptake and NPT2 translocation to the apical membrane.

Cancer Detect Prev, 2001, 25(4), 394 - 405
Expression pattern of hybrid phenotype in adult acute lymphoblastic leukemia; Nakase K et al.; We examined the expression of hybrid phenotype in 236 adults with acute lymphoblastic leukemia (ALL; 188 B-lineage ALL and 48 T-lineage ALL) . In B-lineage ALL, myeloid antigen (mAg) CD15 was concentrated in CD10-CD20- cases (49%); CD13 (42%); and CD33 (43%) in CD10+CD20- cases . This trend had no correlation with the presence of Ph1 or t(4;11) chromosomal abnormality . T-cell antigen CD2, CD4, and CD7 was seen in four, four, and two cases, respectively, and CD4+ and CD7+ cases commonly expressed CD13 and/or CD33 (CD13/CD33) . In T-lineage ALL, expression of mAg, CD11b (47%), CD13 (38%), CD15 (28%), and CD33 (51%) was restricted to CD3- cases . B-cell antigen CD19 was found in two cases with CD7 solely as T-cell antigen, and these cases possessed CD13/CD33 . CD21 was detected in three cases with CD3 . In whole ALL, CD13/CD33 was associated closely with the presence of stem-cell antigen CD34, and in T-lineage ALL, CD13/CD33 had a significant correlation with additional stem-cell features, such as HLA-DR, multidrug resistance 1 (MDR1) and c-kit gene expression . Our results suggest that immature ALL cells frequently express B+M+, T+M+, and occasionally B+T+M+ phenotype; that B+T+M- phenotype is extremely rare; and that mAg expression in B-lineage ALL is complicated as compared to T-lineage ALL.

J Soc Biol, 2001, 195(1), 39 - 46
{Lineage-switching by pluripotent cells derived from adults}; Dieterlen-Lievre F; When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation . Each primordial germ layer gives rise to a particular set of lineages . Until recently, it was considered that fate switches between germ layers were impossible . In the last two or three years however, a fair number of such switches have been described (Table I), the most spectacular of which entails the differentiation of neural stem cells into various derivatives . This unexpected plasticity opens important prospects for cell therapy . Stem cells, which are the cells that display this plasticity, are defined by the two properties of self renewal and pluripotency . They are set apart during ontogeny and are responsible for maintaining the homeostasis of a tissue . This notion, first established in the case of hematopoietic stem cells was later extended to other fast renewing cells, such as those in the intestinal epithelium or epidermis, and more recently to cells reputedly non-renewable, i.e . neurons . A new strategy has been described, which has the interesting feature that it can be applied to the isolation of stem cells from various lineages . It consists in sorting out cells on the basis of the efflux of Hoechst 33342 dye (Goodell et al., 1996) . When a cell suspension stained with this dye is examined under two distinct wave lengths, a "side population" (SP), characterized by weak fluorescence, can be identified and sorted out . The dye efflux property of these cells is due to the activity of the mdr (multidrug resistance) gene, which encodes a protein responsible for the building of a canal which serves to extrude toxins from the cells . A means of distinguishing a truly multipotent stem cell from a progenitor committed to a specific lineage has been reported . This consists in the expression of the Pax7 gene . Pax7-/- mouse muscles have no satellite cells, i.e . they miss the cells normally responsible for the regeneration of muscle . In contrast they do have an SP population . These SP cells are incapable of differentiating into muscle, but give rise to 10 times more hematopoietic colonies, when cloned in vitro, than SP cells from wild type muscle do . Thus Pax7 appears to be a commitment gene, in the absence of which stem cells cannot become specified to the muscle lineage . As a conclusion, this review emphasizes various features of the recent findings: 1) the unexpected plasticity uncovered in recent years is restricted to the stem cells of each tissue; 2) the switch in phenotype has to be "forced" on these stem cells by drastic experimental conditions enforced in the host: often sublethal irradiation is superimposed on a genetic deficiency . Progress in this field, concerning both conceptual and applied aspects, will require the identification of the factors characterizing the niches which promote integration and fate switches of stem cells, probably a combination of growth factors and intercellular interactions . Finally a key issue, before any therapeutical applications can be considered, is how to control the proliferation of transplanted stem cells in their new environment.

Acta Paediatr, 2001 Aug, 90(8), 943 - 7
Childhood and adolescent tuberculosis in northern Taiwan: an institutional experience during 1994-1999; Wong KS et al.; This study evaluated the clinical characteristics of childhood and adolescent tuberculosis (TB) at the end of the twentieth century in a referral children's hospital in northern Taiwan . The hospital charts were reviewed retrospectively of children/adolescents aged less than 18 y who were seen in a children's hospital in northern Taiwan between 1994 and 1999 and diagnosed with TB . A total of 62 individuals was diagnosed during this period . The patients' demographic data, presenting symptoms, clinical features, bacteriological results, drug susceptibility and tuberculin skin-test results were analysed . Most diagnosed cases lay in one of two main age ranges, younger than 5 y and adolescents . The presenting symptoms of study subjects were typically non-specific . Bone involvement occurred for 21 patients (33.9%) and was the most common extrapulmonary manifestation . Mycobacterium tuberculosis was isolated from 47 patients (75.8%) . Five isolates were resistant to pyrazinamide, three to streptomycin and one to isoniazid, but no multidrug resistant isolates of TB were detected from culture-proven cases . Seventeen of 47 (36.2%) culture-proven patients revealed negative acid-fast staining initially but, subsequently, M . tuberculosis was isolated from various clinical specimens using a standard method at a later date . The tuberculin skin test was positive for 24 of 32 patients (75%) who received such an examination . Conclusion: Extrathoracic TB was more common in children below 5 y of age than their adolescent counterparts, and chiefly involved the peripheral long bones . The potential drug resistance of M . tuberculosis in childhood and adolescent TB did not appear to have been a major problem in northern Taiwan before the year 2000.

Cell Mol Life Sci, 2001 Jul, 58(8), 1113 - 20
Opposite pattern of MDR1 and caveolin-1 gene expression in human atherosclerotic lesions and proliferating human smooth muscle cells; Batetta B et al.; Cholesterol esterification and smooth muscle cell (SMC) proliferation are the crucial events in the development of atherosclerotic lesions . The objective of this study was to analyse cholesterol esterification and the expression of MDR1 (multidrug resistance), ACAT (acyl-CoA:cholesterol acyltransferase) and caveolin-1 genes in atherosclerotic and healthy vascular walls, in SMCs obtained from atherosclerotic lesions and saphenous veins . Results demonstrated higher levels of cholesterol esters, ACAT and MDR1 mRNAs and lower levels of caveolin-1 mRNA in atherosclerotic segments compared to adjacent serial sections of the same artery and the corresponding non-atherosclerotic arteries from cadaveric donors . SMCs isolated from atherosclerotic plaques manifested an increased capacity to esterify cholesterol and to grow at a faster rate than SMCs isolated from saphenous veins . In addition, when SMCs from atherosclerotic plaques were cultured in the presence of progesterone, a potent inhibitor of cholesterol esterification, significant growth suppression was observed . An increase in ACAT and MDR1 expression and a concomitant decrease in caveolin-1 expression were also observed in SMCs isolated from atherosclerotic arteries as early as 12 h after serum stimulation . An opposite pattern was found when SMCs were treated with progesterone . These findings support the idea that cholesterol esterification plays a role both in early atherogenesis and in clinical progression of advanced lesions and raise the possibility that the cholesterol ester pathway might directly modulate the proliferation of SMCs.

J Clin Microbiol, 2001 Sep, 39(9), 3339 - 45
Spread of drug-resistant pulmonary tuberculosis in Estonia; Kruuner A et al.; Restriction fragment length polymorphism (RFLP) analysis of 209 Mycobacterium tuberculosis clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS6110 banding pattern . These strains shared the majority of their IS6110 DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%) . This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains . The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of M . tuberculosis belonged to family A . These strains were confirmed by spoligotyping as members of the Beijing genotype family . The spread of Beijing genotype MDR M . tuberculosis strains was also frequently seen in 1997 to 1999 . The members of this homogenous group of drug-resistant M . tuberculosis strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.

Scand J Infect Dis, 2001, 33(8), 563 - 7
Tuberculosis: trends and the twenty-first century; Arnadottir T; The global burden of tuberculosis is enormous, even if estimates are somewhat uncertain . The forces counteracting control measures, namely demographic factors, drug resistance, HIV, migration, poverty and marginalization, are enormous as well . With accelerated reforms in tuberculosis programs important progress can be made towards the control of tuberculosis early in the 21st century . This is confirmed by studying reports from countries where control measures have been implemented and sustained . Well-functioning programs can make good use of technological progress, such as improved tools for diagnosis and treatment, when these become available at an affordable cost . It is important now to use the opportunity of increased resources in order to reform tuberculosis programs . The biggest impact on global tuberculosis control in the 21st century can be made in Asia . Success in this part of the world depends on political commitment . Elsewhere, the main forces counteracting control measures are HIV in Africa and multidrug resistance in parts of Europe and the former Soviet Union . Here solutions are still on the drawing board . The long time-frame for tuberculosis control when using the currently recommended strategy, the uncertain impact of "improved" tools on this time-frame and the constant threat that political commitment will not be sustained are reasons why field workers look towards new technology in hope of progress in vaccine research . Here, the prospects are uncertain and the forecasted time-frame is long . Skeptics even doubt that an effective vaccine can be developed . However, when predicting progress it is important to realize that it is for the most part unpredictable.

J Tongji Med Univ, 2001, 21(1), 56 - 8
Expression of multidrug-associated protein, P-glycoprotein, P53 and Bcl-2 proteins in bladder cancer and clinical implication; Chen Z et al.; The expression of multidrug resistant proteins in bladder cancer and clinical implication was studied . Expression of multidrug-associated protein (MRP), P-glycoprotein (P-gp), P53 and Bcl-2 proteins were detected by using immunohistochemical method in 40 specimens of bladder transitional cell carcinoma . The results showed that the positive rate of MRP, P-gp, P53 and Bcl-2 was 52.5%, 57.5%, 47.5% and 62.5% respectively . The positive rate of MRP, P-gp, P53 and Bcl-2 in the grade I, II and III of tumors was 46.3%, 38.5%, 38.5%, 23.1%; 52.9%, 39.8%, 47.1%, 76.4%; 60.0%, 80.0%, 60.0%, 90.0% respectively . The positive rate of MRP, P-gp, P53 and Bcl-2 in 24 primary tumor specimens was 37.5%, 41.7%, 33.3%, 45.8% and that in 16 cases in recurrent specimens receiving chemotherapy 75.0%, 81.3%, 68.8%, 87.5% respectively . It was suggested the positive rate of MRP, P-gp, P53 and Bcl-2 was increased with the advance of tumor grade . The positive rate of four proteins in all recurrent cases was significantly increased (P < 0.05) . The expression of MRP, P-gp, P53 and Bcl-2 proteins might be the important factors for chemotherapy failure.

Nucleic Acids Res, 2001 Sep 1, 29(17), 3611 - 20
5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure; Kostenko E et al.; Oligonucleotide conjugates bearing two pyrene residues attached to 5'-phosphate through a phosphoramide bond were synthesised . Fluorescence spectra of the conjugates show a peak typical of monomer emission (lambda(max) 382 nm) and a broad emission peak with lambda(max )476 nm, which indicates the excimer formation between the two pyrene residues . Conjugation of these two pyrene residues to the 5'-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands . A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes . The 5'-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA . The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence . Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.

Cancer Res, 2001 Sep 1, 61(17), 6459 - 66
Multidrug resistance-associated protein 3 is a tumor rejection antigen recognized by HLA-A2402-restricted cytotoxic T lymphocytes; Yamada A et al.; The identification of tumor rejection antigens recognized by CTLs and its application in peptide-based specific immunotherapy against melanomas have been extensively investigated in the past decade . However, only a small number of studies regarding these issues in other epithelial cancers have been reported . In this study, we show that a multidrug resistance-associated protein 3 (MRP3) is a tumor rejection antigen recognized by HLA-A2402-restricted CTLs established from T cells infiltrating into lung adenocarcinoma . MRP3 is expressed in differing quantities in tumor cells of various tissue types and origins . Four dominant MRP3-derived antigenic peptides that are recognized by the CTLs have been identified, each possessing in vitro immunogenicity . Namely, these four peptides (MRP3-503, MRP3-692, MRP3-765, and MRP3-1293) can induce peptide-specific CTLs after in vitro stimulation with these peptides in peripheral blood mononuclear cell cultures of HLA-A24(+) cancer patients, with the CTLs expressing cytotoxicity against HLA-A2402(+) MRP3(+) tumor cells but not against either HLA-A2402(-) or MRP3(-) target cells . The peptide specificity of the cytotoxicity of the CTLs was further confirmed by using peptide-loaded HLA-A24(+) EBV-transformed B cells . Widespread MRP3 expression in various tumor cell lines and tumor tissues at the mRNA level was confirmed . Furthermore, reactivities of the MRP3-peptide-induced CTLs against tumor cells correlated with MRP3 expression in the tumor cells . These results suggest that MRP3 and its derived peptides described in the present paper are potential candidates for cancer vaccines in regard to HLA-A24(+) patients with various tumors, particularly for those tumors that show anticancer drug resistance.

J Biol Chem, 2001 Oct 5, 276(40), 36877 - 80 Epub 2001 Aug 22.
Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers; Loo TW et al.; The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes . There is considerable evidence indicating that residues in transmembrane segments 4-6 and 10-12 form the drug-binding site . We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms . The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold) . 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4-6 and 10-12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 degrees C . The cross-linked product migrated with lower mobility than native P-gp in SDS gels . 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9-25 A . Vinblastine and cyclosporin A inhibited cross-linking . The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is "funnel" shaped, narrow at the cytoplasmic side, at least 9-25 A in the middle, and wider still at the extracellular surface.

Nucl Med Biol, 2001 Aug, 28(6), 735 - 40
Study of tea polyphenol as a reversal agent for carcinoma cell lines' multidrug resistance (study of TP as a MDR reversal agent); Zhu A et al.; The aim of this study was to examine MDR1 expression product P-glycoprotein (Pgp) and study the effect and mechanism of tea polyphenol (TP) in reversion of multidrug resistance (MDR) in carcinoma cell lines . Immunocytochemical method was used for qualitative detection of Pgp . A comparative study of cytotoxicity and multidrug resistance reversion effect was made by MTT assay for tea polyphenol and quinidine in MCF-7 and MCF-7/Adr cell lines . The multidrug resistance reversion effect and mechanism were studied by measuring the uptake of 99mTc-tetrofosmin in the carcinoma cell lines . (1) The Pgp overexpression in MCF-7/Adr cells was found to be strong positive, while the Pgp expression of MCF-7 was negative . (2) Although both tea polyphenol and quinidine could not remarkably change the toxicity of adriamycin to MCF-7, they could improve the sensitivity of MCF-7/Adr to adriamycin . The reversion index of tea polyphenol and quinidine was 3 and 10 respectively . (3) The cellular uptake of 99mTc-tetrofosmin was remarkably lower in MCF-7/Adr than in MCF-7 . The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited a 4, 13, 16 fold increase in the presence of 200, 400 and 500 microg/ml of tea polyphenol respectively . The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited only a 4-fold increase in the presence of 200 microM of quinidine . Immunocytochemistry can detect P-glycoprotein expression level qualitatively . Tea polyphenol is not only an anti-tumor agent, but also a multidrug resistant modulator similar to quinidine . The multidrug resistance reversion mechanism of tea polyphenol seems to be its inhibition of the activity of P-glycoprotein . Tea polyphenol has the advantage of very low toxicity in tumor treatment.

Chem Biol Interact, 2001 Jul 31, 137(1), 1 - 13
Fluorescent verapamil analogue for monitoring acidic intracellular organelles in multidrug resistant and sensitive cells; Mankhetkorn S et al.; Resistance to chemotherapeutic agent is a major cause of treatment failure in patients with cancer . In many cases, the primaly mechanism leading to a multidrug-resistant phenotype is the plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein . However, acidic intracellular organelles seem also to participate in resistance to chemotherapeutic drugs and the determination of the pH of these organelles is of importance . In the present study we have used a new fluorescent derivative of verapamil, 2-2-diphenyl-5-{(methylaminomethyl)anthracene} pentanenitrile (EDP 96), and show that it is an efficient inhibitor of the P-gp-mediated efflux of anthracycline in K562 resistant cells . The fluorescence of EDP 96 is environmental and pH sensitive . EDP 96 is a weak base (pKa=6.0) and its accumulation into K562 cells is accompanied by a significant fluorescence increase due to its entry of the drug into acidic regions in the cells . We have used this properties to develop a new method to accurately determine the pH of acidic organelle.

BMC Cancer . 2001;1(1):10 . Epub 2001 Aug 01.
Long-term cultivation of colorectal carcinoma cells with anti-cancer drugs induces drug resistance and telomere elongation: an in vitro study; Kuranaga N et al.; BACKGROUND: The role of telomerase activation in the expression and/or maintenance of drug resistance is not clearly understood . Therefore, we investigated the relationships, among the telomerase activity, telomere length and the expression of multidrug resistance genes in colorectal cancer cell lines cultivated with anti-cancer drugs . METHODS: LoVo and DLD-1 cells were continuously grown in the presence of both CDDP and 5-FU for up to 100 days . Cell proliferation, telomerase activity, telomere length and the expression of multidrug resistance genes were serially monitored as the PDL increased . RESULTS: The expression of multidrug resistance genes tended to increase as the PDL increased . However, an abnormal aneuploid clone was not detected as far as the cells were monitored by a DNA histogram analysis . Tumor cells showing resistance to anti-cancer drugs revealed a higher cell proliferation rate . The telomere length gradually increased with a progressive PDL . The telomerase activity reached a maximum level at 15 PDL in LoVo cells and at 27 PDL in DLD-1 cells . An increase in the mRNA expression of the telomerase components, especially in hTERT and in hTR, was observed at the same PDLs . CONCLUSIONS: These results suggest that a high telomerase activity and an elongation of telomeres both appear to help maintain and/or increase drug resistance in colorectal cancer cells . Cancer cells with long telomeres and a high proliferative activity may thus be able to better survive exposure to anti-cancer drugs . This is presumably due to an increased chromosome stability and a strong expression of both mdr-1 and MRP genes.

No Shinkei Geka, 2001 Jul, 29(7), 625 - 30
{Chemotherapy for gliomas based on the expression levels of drug resistant genes}; Matsumoto Y et al.; Drug resistance, which often occurs during chemotherapy, is still a great obstacle to the success of human malignancy treatment . Among many possible mechanisms of drug resistance (biological, biochemical, kinetic or pharmacological), both typical and atypical multidrug-resistance (MDR) have been extensively studied . We picked up MDR-1, MXR, MRP1, MRP2, TopoII alpha, MGMT, and GST-pi as drug-resistant gene, based on experimental data and previous reports . Expression of these genes were measured in 14 malignant glioma specimens by reverse transcription polymerase chain reaction assay . We chose anticancer drugs for each patient, based on results of drug resistant gene expression to acquire good response to drugs . Though our follow-up periods are not long enough to analyze the results of our chemotherapy, 78% (7/9) of our glioma patients who were treated with our chemotherapy are free from tumor progression . The assays, which measure the expression of drug resistant genes, are necessary to allow rapid detection of the drug-sensitivity to chemotherapy in malignant glioma patients.

Leukemia, 2001 Sep, 15(9), 1377 - 87
Induction of chemoresistance in HL-60 cells concomitantly causes a resistance to apoptosis and the synthesis of P-glycoprotein; Campone M et al.; The appearance of multidrug-resistant (MDR) proteins or the acquisition of a defective apoptotic programme are major drawbacks in the treatment of cancers since both induce a resistance to classical chemotherapy . However, a link between the two mechanisms has not, as yet, been clearly established . In this study, HL-60 cells cultured in the continual presence of a sub-lethal dose of doxorubicin (dox; HL-60/Dox) were used as a model to study acquired chemoresistance . During the induction of chemoresistance, the appearance of a functional P-glycoprotein (P-gp), in addition to the expression of anti-apoptotic Bcl-2, Bcl-XL and pro-apoptotic Bax proteins was assessed . Parental cells which are sensitive to dox, have no P-gp activity and express Bcl-2 and Bax . After 4 weeks of treatment, a functional P-gp was detected in HL-60/Dox cells . In addition, the synthesis of Bcl-2 appeared to be replaced by Bcl-XL while that of Bax remained unchanged . These cells were also resistant to apoptosis induced by both P-gp and non-P-gp substrates . This inability to induce apoptosis could have resulted from the induction of the expression of the inhibitor of apoptosis protein (XIAP) . Our data show that acquired chemoresistance could involve a parallel induction of P-gp and an impairment of the apoptotic pathway.

Biochim Biophys Acta, 2001 Aug 29, 1533(1), 55 - 65
Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles; Podgorski I et al.; Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites . In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps . The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid . The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG) . The results show that the efflux of 13-OXO-SG is ATP-dependent . In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose . Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil . In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG . Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps . The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.

Lancet, 2001 Aug 11, 358(9280), 445 - 9
Comparison of the effectiveness of WHO short-course chemotherapy and standard Russian antituberculous regimens in Tomsk, western Siberia; Mawer C et al.; BACKGROUND: There has been a resurgence of tuberculosis in Russia in the past decade . Traditional Russian services for treatment of tuberculosis are very different from those in the west . We aimed to compare the effects of WHO short-course chemotherapy with standard Russian antituberculous regimens . METHODS: New tuberculosis patients aged 18 years or older were included in a trial and systematically allocated to traditional Russian tuberculosis treatments or WHO short-course chemotherapy in the two largest tuberculosis diagnostic and treatment centres of Tomsk Oblast, western Siberia . Standard WHO tuberculosis outcomes and rates of sputum conversion were used as primary outcomes . Analyses were by intention-to-treat . FINDINGS: 646 new cases were enrolled into the trial, of which 356 patients were given Russian tuberculosis treatment (155 smear positive) and 290 were given WHO short-course chemotherapy (155 smear positive) . There was no statistical difference between the proportion cured or completing treatment (63% for both groups {difference in proportion=0%, 95% CI -11 to 11%}); or dying (short-course chemotherapy, 8% vs Russian, 11% {difference in proportion=-3%, 95% CI -9 to 4%}) . There was no statistical difference with respect to sputum conversion rate at 6 months (91% vs 85% {difference in proportion=6%, 95% CI -2 to 13%}) . Overall, outcomes were worse among patients with multidrug resistant isolates than non-resistant isolates . INTERPRETATIONS: WHO short-course chemotherapy treatment for tuberculosis can work well in Russia.

FEBS Lett, 2001 Aug 17, 503(2-3), 179 - 84
Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone; Lage H et al.; Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters . Here we demonstrate that a member of the ABC-transporter family, the heterodimer 'transporter associated with antigen processing' (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype . This tumor cell line shows an atypical MDR phenotype in the absence of 'P-glycoprotein' or 'MDR-associated protein' overexpression but with an enforced 'breast cancer resistance protein' expression level . Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents . Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation . Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.

Drug Resist Updat, 2001 Feb, 4(1), 66 - 74
The ABC transporter genes of Plasmodium falciparum and drug resistance; Peel SA; The seminal observations that (a) chloroquine-resistant Plasmodium falciparum strains accumulate less drug than more sensitive parasites, and (b) chloroquine resistance could be modulated in vitro by the classic multidrug-resistance (MDR) modulator verapamil, suggested not only that parasite resistance to multiple drugs may be similar to the MDR phenotype described in mammalian cancer cells, but that homologous proteins may be involved . These findings prompted search for MDR-like genes in the parasite . To date, three full-length ABC transporter genes have been isolated from P . falciparum: two P-glycoprotein-like homologues, pfmdr1 and pfmdr2, and a homologue of the yeast GCN20 gene, pfgcn20.

Clin Infect Dis, 2001 Sep 15, 33(6), e42 - 7 Epub 2001 Aug 06.
Simultaneous infection with multiple strains of Mycobacterium tuberculosis; Braden CR et al.; Drug-susceptible and drug-resistant isolates of Mycobacterium tuberculosis were recovered from 2 patients, 1 with isoniazid-resistant tuberculosis (patient 1) and another with multidrug-resistant tuberculosis (patient 2) . An investigation included patient interviews, record reviews, and genotyping of isolates . Both patients worked in a medical-waste processing plant . Transmission from waste was responsible for at least the multidrug-resistant infection . We found no evidence that specimens were switched or that cross-contamination of cultures occurred . For patient 1, susceptible and isoniazid-resistant isolates, collected 15 days apart, had 21 and 19 restriction fragments containing IS6110, 18 of which were common to both . For patient 2, a single isolate contained both drug-susceptible and multidrug-resistant colonies, demonstrating 10 and 11 different restriction fragments, respectively . These observations indicate that simultaneous infections with multiple strains of M . tuberculosis occur in immunocompetent hosts and may be responsible for conflicting drug-susceptibility results, though the circumstances of infections in these cases may have been unusual.

Acta Cient Venez, 2001, 52(1), 68 - 77
{Pharmacological biomodulation in cancer}; Arvelo F et al.; The discovery of the P-glycoprotein as a mediator of multidrug resistance (MDR) represents one of the most important research accomplishments in antineoplastic pharmacology during the last decade . Demonstration of Pgp in epithelial tissues, untreated and chemotherapeutically pretreated human malignancies, and identification of various agents capable of reversing in vitro resistance has generated enthusiasm for clinical studies throughout the world . This review discusses recent developments of experimental and clinical investigations of MDR reversing agents in cancer.

J Biol Chem, 2001 Oct 26, 276(43), 39990 - 40000 Epub 2001 Aug 16.
Methylation-dependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells; Worm J et al.; The molecular basis of methotrexate resistance was studied in human MDA-MB-231 breast cancer cells, which are inherently defective in methotrexate uptake and lack expression of the reduced folate carrier (RFC) . Transfection of MDA-MB-231 cells with RFC cDNA restored methotrexate uptake and increased methotrexate sensitivity by approximately 50-fold . A CpG island in the promoter region of RFC was found to be methylated in MDA-MB-231 cells, but was unmethylated in RFC expressing, methotrexate-sensitive MCF-7 breast cancer cells . Chromatin immunoprecipitation with antibodies against acetylated histones H3 and H4 showed that the RFC promoter was enriched for acetylated histones on expressed, unmethylated alleles only . Treatment of MDA-MB-231 cells with 5-aza-2'-deoxycytidine restored RFC expression but also led to increased methotrexate efflux and did not reverse methotrexate resistance . This suggests that 5-aza-2'-deoxycytidine up-regulates both methotrexate uptake and some methotrexate-resistance mechanism(s) . Reverse transcription-polymerase chain reaction analysis showed increased expression levels of several ATP-dependent efflux pumps in response to 5-aza-2'-deoxycytidine treatment, including P-glycoprotein and members of the multidrug resistance-associated protein family . Up-regulation of P-glycoprotein in response to 5-aza-2'-deoxycytidine was associated with demethylation of a CpG island in the MDR1 promoter, whereas the mechanism(s) for 5-aza-2'-deoxycytidine-induced up-regulation of multidrug resistance-associated proteins is probably indirect . Dipyridamole inhibited methotrexate efflux and reversed methotrexate resistance in 5-aza-2'-deoxycytidine-treated MDA-MB-231 cells.

Jpn J Cancer Res, 2001 Aug, 92(8), 896 - 903
Anticancer drug-mediated induction of multidrug resistance-associated genes and protein kinase C isozymes in the T-lymphoblastoid cell line CCRF-CEM and in blasts from patients with acute lymphoblastic leukemias; Beck JF et al.; The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear . For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype . Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF-CEM cells treated with diverse anticancer drugs--adriamycin, camptothecin, etoposide or vincristine--at sublethal concentrations for 24 h . MDR1, MRP1, LRP and PKC isozyme alpha, beta(1), beta(2), epsilon, iota, eta, theta, zeta gene expression was determined by cDNA-PCR . We found significant dose-dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes . Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin . PKCzeta was upregulated throughout by each anticancer drug applied in this setting . A series of selected CCRF-CEM-derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line . MDR1 and PKCeta gene expression levels were correlated highly significantly . Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance-associated protein 1 (MRP1), lung cancer resistance-related protein (LRP) and most PKC isozymes, predominantly PKCzeta . Another patient with T-ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCeta and PKCtheta . Furthermore, the camptothecin and etoposide-mediated induction of resistance factors in the CCRF-CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases . However, selective inhibitors of PKC isozymes (bisindolylmaleimide GO 6850, indolocarbazole GO 6976) produced no significant effects here . Therefore, the PKC isozymes eta, theta and zeta are of interest as potential targets to overcome drug resistance in ALL.

Jpn J Cancer Res, 2001 Aug, 92(8), 886 - 95
Reversing effect of agosterol A, a spongean sterol acetate, on multidrug resistance in human carcinoma cells; Aoki S et al.; The effect of agosterol A, a novel polyhydroxylated sterol acetate isolated from a marine sponge, on P-glycoprotein (P-gp)-mediated multidrug-resistant cells (KB-C2) and the multidrug resistance associated protein (MRP1)-mediated multidrug-resistant cells (KB-CV60) was examined . Agosterol A reversed the resistance to colchicine in KB-C2 cells and also the resistance to vincristine in KB-CV60 cells at 3 to 10 microM concentration . Agosterol A at 3 mM increased the vincristine concentration in both KB-C2 cells and KB-CV60 cells to the level in parental KB-3-1 cells . Agosterol A also decreased the efflux of vincristine from both KB-C2 cells and KB-CV60 cells to the level seen in KB-3-1 cells . Agosterol A inhibited the {(3)H}azidopine-photolabeling of P-gp and also inhibited the uptake of {(3)H}S-(2,4-dinitrophenyl)glutathione (DNP-SG) in inside-out membrane vesicles prepared from KB-CV60 cells . We conclude that agosterol A directly inhibited drug efflux through P-gp and/or MRP1.

Cas Lek Cesk, 2001 Jun 5, 140(13), 409 - 10
{Multiresistant tuberculosis in the Czech Republic in 1998 and causes of its occurrence}; Krejbich F et al.; Multidrug resistant tuberculosis (MDR-TB) belongs to the most serious forms of TB . The number of MDR-TB patients represents an indicator of the effectiveness of TB regimentation . The aim of the study was to determine the number of registered MDR-TB patients in CR in 1998 and to identify causes of the resistance . Analysis of clinical documentation of 27 patients with MDR-TB positive sputum was done in 1998 . According to the documentation, this form of tuberculosis was not confirmed in 5 persons . Out of 22 patients with confirmed MDR-TB, 21 were males and one female . In 3 of them we could not reveal whether they had been cured with antituberculotics before MDR-TB developed . Such resistance was then classified as an initial form . In 19 patients we found that resistance developed during treatment . Those cases were classified as secondary forms . In 16 patients the secondary resistance was related to the interruption of treatment, which was done wantonly by the patients in 14 cases . The consistent treatment of the sensitive TB is therefore the best prevention for MDR-TB development . Incidence of MDR-TB cases has not been influenced by immigration from countries with high rate of occurrence of MDR-TB.

J Biol Chem, 2001 Oct 19, 276(42), 38636 - 44 Epub 2001 Aug 15.
Characterization of binding of leukotriene C4 by human multidrug resistance protein 1: evidence of differential interactions with NH2- and COOH-proximal halves of the protein; Qian YM et al.; Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions . The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner . How MRP1 binds and transports such structurally diverse substrates is not known . We have used {(3)H}leukotriene C(4) (LTC(4)), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells . These studies revealed that: (i) LTC(4) labels sites in the NH(2)- and COOH-proximal halves of MRP1, (ii) labeling of the NH(2)-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH(2)-proximal site is preferentially inhibited by S-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14-17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC(4) is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC(4) binding to the NH(2)-proximal site, and (vii) the attenuation of LTC(4) binding to the NH(2)-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2 . These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC(4) binds to one of two sites composed, at least in part, of elements in the NH(2)-proximal half of the protein.

Cancer Res, 2001 Aug 15, 61(16), 6185 - 93
Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines; Keshelava N et al.; Neuroblastomas can acquire a sustained high-level drug resistance during chemotherapy and especially myeloablative chemoradiotherapy . p53 mutations are rare in primary neuroblastomas, but a loss of p53 function could play a role in multidrug resistance . We determined p53 function by measuring induction of p21 and/or MDM2 proteins in response to melphalan (L-PAM) in seven L-PAM-sensitive and 11 L-PAM-resistant neuroblastoma cell lines . p53 was functional in seven/seven drug-sensitive but in only 4/11 drug-resistant cell lines (P = 0.01) . In four of the seven cell lines lacking p53 function, mutations of p53 were detected by the microarray GeneChip p53 Assay and automated sequencing, whereas six cell lines with functional p53 had no evidence of p53 mutations . All of the cell lines with wild-type (wt) p53 showed a strong transactivation of the p53-HBS/CAT reporter gene, whereas the four cell lines with mutant p53 failed to transactivate p53 HBS/CAT . Overexpression of MDM2 protein (relative to p53 functional lines) was seen in two p53-nonfunctional cell lines with wt p53; one showed genomic amplification of MDM2 . Nonfunctional and mutated p53 was detected in a resistant cell line, whereas a sensitive cell line derived from the same patient before treatment had functional and wt p53 . Loss of p53 function was selectively achieved by transduction of human papillomavirus 16 E6 (which degrades p53) into two drug-sensitive neuroblastoma cell lines with intact p53, causing high-level drug resistance to L-PAM, carboplatin, and etoposide . These data obtained with neuroblastoma cell lines suggest that the high-level drug resistance observed in some recurrent neuroblastomas is attributable to p53 mutations and/or a loss of p53 function acquired during chemotherapy . If confirmed in patient tumor samples, these data support development of p53-independent therapies for consolidation and/or salvage of recurrent neuroblastomas.

Cancer Res, 2001 Aug 15, 61(16), 6034 - 7
A novel 7-modified camptothecin analog overcomes breast cancer resistance protein-associated resistance in a mitoxantrone-selected colon carcinoma cell line; Perego P et al.; We selected a mitoxantrone-resistant HT29 colon carcinoma cell line (HT29/MIT) that exhibited a very high degree of resistance to the selecting agent and marked resistance to topotecan and SN38, but limited resistance to doxorubicin . The development of drug resistance was independent of expression of P-glycoprotein or multidrug resistance-associated protein but was associated with high up-regulation of the breast carcinoma resistance protein (BCRP) as shown by Western blot analysis . BCRP overexpression was associated with a reduced intracellular accumulation of topotecan, a known substrate for BCRP . Conversely, a lipophilic 7-modified camptothecin analogue (ST1481) displayed a complete lack of cross-resistance in HT29/MIT cells, suggesting that the drug was not a substrate for BCRP because no defects in intracellular accumulation were found . This conclusion is consistent with the antitumor efficacy of ST1481 against a BCRP-expressing tumor . These results may have therapeutic implications because the antitumor efficacy of ST1481 is in part related to a good bioavailability after oral administration, and the drug is currently under Phase I clinical evaluation.

Microbios, 2001, 106(414), 137 - 45
HIV-1 multi-dideoxynucleoside resistance mutation (Q151M): prevalence, associated resistance mutations and response to antiretroviral salvage treatment; Quiros-Roldan E et al.; The prevalence and clinical implications of the Q151M multidrug-resistance mutation gene (mut) to antiretroviral drugs in the HIV reverse transcriptase (RT) gene have not yet been fully explained . In the present study three out of 350 (0.85%) of HIV-infected patients who underwent a drug-resistance genotyping assay because of therapeutic failure showed the Q151M mut . All these patients had been previously treated with zidovudine in association with didanosine . One such patient failed to respond to all salvage regimens tried and was shown to harbour some of the characteristic mut associated with Q151M (77L and 116Y) . Another two patients partially responded to salvage regimens, both virologically and immunologically, and harboured the M184V mut in the RT gene . The prevalence of Q151M mut in our group was less (0.85%) than in other studies, which ranged from 2 to 19% . The M184V mut seemed to confer some viro-immunological benefit when associated with the Q151M mutation, compared with the latter alone.

J Pharmacol Exp Ther, 2001 Sep, 298(3), 1199 - 205
Multidrug resistance-associated protein-1 functional activity in Calu-3 cells; Hamilton KO et al.; The purpose of this work was to determine whether the in vitro bronchiolar epithelial cell model, Calu-3, possesses efflux pump activity by the multidrug resistance-associated protein-1 (MRP1) . Reverse transcription-polymerase chain reaction demonstrated MRP1 gene expression in Calu-3 cells . Indirect fluorescence studies showed a basolateral membrane localization of MRP1 compared with P-glycoprotein (Pgp) that was found on the apical side of these cells . An increase in the rate of accumulation of the MRP1 substrate calcein was observed following treatment with the organic anion/MRP1 inhibitor indomethacin, the Pgp inhibitors cyclosporin A (CsA) and vinblastine, as well as conditions of energy depletion . Total calcein efflux was significantly decreased with the MRP1 inhibitors probenecid and indomethacin, while total efflux was unchanged following treatment with CsA . In the latter case, however, intracellular calcein levels postefflux were significantly greater . Probenecid and indomethacin increased calcein net secretion 2.4- and 3.5-fold, respectively . The efflux of etoposide, a known substrate for both Pgp and MRP1, was shown to be mainly Pgp-mediated by using the multidrug-resistant inhibitors quinidine (mixed Pgp/MRP1), CsA (Pgp), and MK571 (MRP1) . Together, these data suggest that Calu-3 cells possess MRP1 functional activity that is subordinate to Pgp efflux . We present here kinetic analysis of calcein efflux from Calu-3 cells to support our findings.

J Biol Chem, 2001 Oct 19, 276(42), 38697 - 702 Epub 2001 Aug 14.
Haa1, a protein homologous to the copper-regulated transcription factor Ace1, is a novel transcriptional activator; Keller G et al.; The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1 . The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins . Two main target genes of Haa1 are the multidrug resistance gene YGR138c and the YRO2 homolog to the plasma membrane Hsp30 . Haa1 is localized to the nucleus . Haa1-induced expression of YGR138c and YRO2 appears to be direct . Induction of HAA1 using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes . Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells . Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts . Haa1 does not exhibit any cross-talk with Ace1 . Overexpression of Haa1 does not compensate for cells lacking a functional Ace1 . The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.

Drug Resist Updat, 1999 Jun, 2(3), 188 - 197
Physiology and molecular genetics of multidrug resistance in Entamoeba histolytica; Orozco E et al.; Entamoeba histolytica presents the evolutionarily conserved multidrug-resistance (MDR) phenotype, discovered in mammalian cells . MDR cells overexpress the membrane P-glycoprotein, which excludes unrelated drugs from the cytoplasm . E . histolytica mutants exhibit cross-resistance to unrelated drugs, which are pumped out from the cytoplasm . In drug-resistant trophozoites, the constitutively expressed EhPg1 gene appears to be up-regulated by a C/EBP-like factor and a multiprotein complex that were not found in drug-sensitive trophozoites . The drug-induced EhPgp5 gene, on the other hand, appears to be up-regulated by AP-1 and HOX factors . Here we review the main physiological and molecular facts of the MDR phenotype in E . histolytica .

Tumori, 2001 May-Jun, 87(3), 179 - 86
Immunohistochemical study of 49 cutaneous melanomas: p53, PCNA, Bcl-2 expression and multidrug resistance; Loggini B et al.; AIMS AND BACKGROUND: Thickness and level of invasion are the main morphological elements for an approximate but not sufficiently sensitive prognostic evaluation of cutaneous melanomas . By using immunohistochemical methods it is possible to detect biological markers related to prognosis . We have studied p53, PCNA, Bcl-2 and P-gp expression in 49 primary cutaneous melanomas . MATERIALS: We used the immunophosphatase APAAP immunohistochemical method . The percentage of labeled cells (according to four classes of positivity: <5%; 5-25%; 25-50%; >50%) and the localization of immunoreactivity were expressed for each marker . Statistical analysis was performed to determine the correlations between markers and level or thickness of melanomas . RESULTS: We found a good correlation between p53 expression and melanoma thickness (P <0.005), PCNA and P-gp expression . No relationship was observed between Bcl-2 expression and the different variables considered or other markers . CONCLUSIONS: Our data seem to indicate an unfavorable prognostic role of higher nuclear p53 expression . However, we believe that our results need to be integrated with patients' clinical follow-up and with the study of the expression of these markers in benign melanocytic lesions to gain more accurate information about their prognostic significance.

Chest, 2001 Aug, 120(2), 343 - 8
Treatment experience of multidrug-resistant tuberculosis in Florida, 1994-1997; Narita M et al.; OBJECTIVE: To study the clinical characteristics and results of patients with diagnoses of multidrug-resistant tuberculosis (MDR-TB) in the state of Florida . METHODS: Retrospective chart review of all patients (n = 81) with diagnoses of MDR-TB in Florida between January 1, 1994, and July 31, 1997 . RESULTS: The average number of resistant drugs was 4.8 (range, 2 to 11) . Of 81 patients, 46 patients (57%) completed adequate therapy, 26 patients (32%) died, and 9 patients (11%) never completed a satisfactory course of treatment . Patients who received at least part of their therapy at A . G . Holley State Hospital, a specialized tuberculosis (TB) treatment center, had significantly higher treatment completion rates (79%) than those treated as outpatients alone (48% treatment completion rate, p < 0.001), even after the exclusion of patients who were acutely ill and died within 2 months of diagnosis . CONCLUSION: In Florida, a specialized TB care program for MDR-TB, including at least partial inpatient therapy, yielded higher treatment completion rates compared to outpatient treatment alone.

Antimicrob Agents Chemother, 2001 Sep, 45(9), 2468 - 74
Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator; Perez-Victoria JM et al.; Drug resistance has emerged as a major impediment in the treatment of leishmaniasis . Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents . In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp . The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP . (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively . (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance . (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line . (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP . (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine . Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites . The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.

Eur J Biochem, 2001 Aug, 268(16), 4459 - 67
Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells; Loetchutinat C et al.; Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs . In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR . However, the nature and role of intracellular vesicles in MDR are unclear . We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug-resistant counterpart K562/ADR that overexpresses P-glycoprotein . We used four anthracyclines (all of which are P-glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pKa and lipophilicity as daunorubicin . The intrinsic fluorescence of anthracyclines allowed us to use macro- and micro-spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells . We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3'-amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug-resistant cells . These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrug-resistant K562 cells.

Semin Immunol, 2001 Oct, 13(5), 267 - 74
Dendritic cell migration to lymph nodes: cytokines, chemokines, and lipid mediators; Randolph GJ; Mobilization of dendritic cells into lymphatic vessels requires cytokine stimulation and induction of the chemokine receptor CCR7 . The respective roles of the CCR7 ligands CCL19 and CCL21 in mediating migration are not fully defined, but chemotaxis to CCL19 mediates Langerhans cell exit from the epidermis . Optimal chemotaxis to CCL19 occurs when DCs are triggered with exogenous leukotriene C(4), an eicosanoid transported out of the cell via the ATP binding cassette (ABC) transporter multidrug resistance related protein 1 (MRP1, ABCC1) . Indeed, MRP1 and the related multidrug resistance protein 1 (MDR1, p-glycoprotein, ABCB1) may control the intracellular and extracellular accumulation of key signaling lipids that regulate dendritic cell migration .

Anticancer Res, 2001 May-Jun, 21(3C), 2141 - 7
Cyclooxygenase-2, P-glycoprotein-170 and drug resistance; is chemoprevention against multidrug resistance possible?
Ratnasinghe D, Daschner PJ, Anver MR, Kasprzak BH, Taylor PR, Yeh GC, Tangrea JA.
BACKGROUND: It is generally accepted that P-glycoprotein 170 (MDR1/Pgp170) expression in breast tumors results in poor response to chemotherapy due to its ability to export chemotherapeutic agents . Studies indicate that the use of non-steroidal anti-inflammatory drugs (NSAIDs) may enhance the anti-tumor activity of cancer chemotherapeutic agents and reduce the risk of many cancers . The best known function of NSAIDs is to block the enzyme cyclooxygenase (Cox), the rate limiting enzyme in the conversion of arachidonic acid to prostaglandins . In this study we investigated whether expression of the inducible isoform of Cox (Cox-2) is linked with the multidrug resistance phenotype in breast cancer . METHODS: Expression of Cox-2 and MDR1/Pgp170 was investigated in tumor specimens along with normal epithelium in breast cancer patients using immunohistochemisrty . Expression of Cox-2, MDR1/Pgp170, Protein Kinase C (PKC), and Activator Protein 1 (AP1) were investigated in a series of increasingly resistant human MCF-7 breast cancer cells compared to wild type using immunohistochemistry, Western blots, Northern blots, RT-PCR, and Southern blots . RESULTS: Immunohistochemical analyses of human breast tumor specimens revealed a strong correlation between expression of Cox-2 and MDR1/Pgp170 . In drug resistant cell lines that over-express MDR1/Pgp170 there was also significant up-regulation of Cox-2 expression . In addition, PKC and AP1 subunits c-Jun and c-Fos were also upregulated . We hypothesized that increased prostaglandin production by Cox-2 induces PKC and the expression of transcriptional factor c-Jun, which in turn, induces the expression of MDR1/Pgp170 . CONCLUSION: We propose that pretreatment with selective Cox-2 inhibitors may be useful in the prevention of multidrug resistance in response to cancer chemotherapy and should be further evaluated.

J Biol Chem, 2001 Oct 12, 276(41), 38108 - 14 Epub 2001 Aug 10.
Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity; Ito K et al.; The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes . Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents . MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter . We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein . In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2 . We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated {3H}E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect . In addition, only the most conservatively substituted mutant (W1254Y) transported {3H}leukotriene C4, whereas all other substitutions eliminated transport of this substrate . On the other hand, all substitutions of Trp1254 eliminated transport of {3H}MTX . Finally, we found that sulfinpyrazone stimulated {3H}E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold . In contrast, sulfinpyrazone failed to stimulate {3H}MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants . Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Crit Rev Oncol Hematol, 2001 Sep, 39(3), 275 - 87
Acute myeloid leukemia in the elderly: biology and therapeutic strategies; Pinto A et al.; Age represents one of the most important adverse prognostic factors in acute myeloid leukemia (AML) . The therapeutic results for patients older than 60 years accrued into clinical trials of intensive chemotherapy are largely unsatisfactory (complete remission rates rarely superior to 50-60%; median relapse-free survival usually less than 12 months) . Because only 30-40% of elderly patients are actually entered into these trials, the overall failure of current treatments appear even more disappointing when considered in the context of the whole population of older individuals with AML . This appears primarily due to intrinsic differences in the biology of leukemia itself and to host-related factors (i.e . reduced tolerance to chemotherapy and comorbidity) . AMLs of older subjects display several biological overlaps with secondary AMLs including multilineage involvement, phenotype, unfavorable cytogenetics and elevated activity of multidrug resistance genes . The clinical application of biologically-based prognostic factors may enable to separate patients who may actually benefit from aggressive chemotherapy from those who should be offered attenuated/palliative treatments or enrolled upfront into experimental trials of new drugs or biologic/immunologic treatments . This may hopefully result in a 'risk-adapted' strategy aimed at improving disease free survival and/or quality of life for patients with differing risk profiles.

Radiat Res, 2001 Sep, 156(3), 272 - 82
Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells; Turcotte S et al.; The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay . Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy . Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione . Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells . GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same . GST pi was the most highly expressed isoform in the two cell populations . Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C . This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid . Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C . Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment . Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures . A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume . A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.

Biochem Biophys Res Commun, 2001 Aug 17, 286(2), 406 - 13
Reversal of cisplatin and multidrug resistance by ribozyme-mediated glutathione suppression; Nagata J et al.; gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in intracellular detoxification, especially of anticancer drugs . Increased levels of GSH are commonly found in the drug-resistant human cancer cells . We designed a hammerhead ribozyme against gamma-GCS mRNA (anti-gamma-GCS Rz), which specifically down-regulated gamma-GCS gene expression in the HCT-8 human colon cancer cell line . The aim of this study was to reverse the cisplatin and multidrug resistance for anticancer drugs . The cisplatin-resistant HCT-8 cells (HCT-8DDP cells) overexpressed MRP and MDR1 genes, and showed resistance to not only cisplatin (CDDP), but also doxorubicin (DOX) and etoposide (VP-16) . We transfected a vector expressing anti-gamma-GCS Rz into the HCT-8DDP cells (HCT-8DDP/Rz) . The anti-gamma-GCS Rz significantly suppressed MRP and MDR, and altered anticancer drug resistance . The HCT-8DDP/Rz cells were more sensitive to CDDP, DOX and VP-16 by 1.8-, 4.9-, and 1.5-fold, respectively, compared to HCT-8DDP cells . The anti-gamma-GCS Rz significantly down-regulated gamma-GCS gene expression as well as MRP/MDR1 expression, and reversed resistance to CDDP, DOX and VP-16 . These results suggested that gamma-GCS plays an important role in both cisplatin and multidrug resistance in human cancer cells .

Yao Xue Xue Bao, 1997 Apr, 32(4), 245 - 50
{A comparative study on effect of two bisbenzylisoquinolines, tetrandrine and berbamine, on reversal of multidrug resistance}; Tian H et al.; A comparative study on the effect of two bisbenzylisoquinolines, tetrandrine (TTD) and berbamine (BBM), and verapamil (VRP) on reversing multidrug resistance was reported . TTD, BBM and VRP showed significant activity in reversing adriamycin (ADR) and vincristine (VCR) resistance in acquired resistant MCF-7/Adr and KBv200 cell lines, and the effect was shown to be dose-dependent . TTD, at the concentration of 10 mumol.L-1, completely reversed ADR resistance in MCF-7/adr cells . TTD, BBM and VRP increased intracellular ADR accumulation in MCF-7/adr cells . There is minor difference in structure between TTD and BBM . TTD showed greater activity than VRP in reversing MDR, while BBM showed similar activity to that of VRP . TTD also showed significant activity in vivo in reversing ADR resistance in MDR MCF-7/Adr solid tumor in nude mice.

Yao Xue Xue Bao, 1997 May, 32(5), 321 - 5
{Reversal of multidrug resistance by cyproheptadine in KBV200 cells}; Miao ZH et al.; The cyproheptadine (CYP) reversal of multidrug resistance (MDR) and its mechanism in KBV200 cell line were studies . MTT assay showed that CYP 15.0 mumol.L-1 could reverse vincristine, adriamycin (ADR) and etoposide resistance in KBV200 cells by a factor of 5.5, 2.0 and 1.9, respectively . CYP appeared to have no influence on the cytotoxicity of 5-fluorouracil (5-FU) and melphalan (MEL) in the cells . These results indicate that CYP is a MDR reversing agent . CYP 15.0 mumol.L-1 increased the ADR accumulation in KBV200 cells from 0.68 +/- 0.03 microgram/10(6) cells to 1.36 +/- 0.08 micrograms/10(6) cells (P < 0.01) . CYP 15.0 mumol.L-1 was shown to obviously increase rhodamine 123 (R123) accumulation in and decrease its efflux from the cells . There was no change in PGP dying intensity under immunocytochemical assay and in mdr1 RNA level through slot blot analysis in the KBV200 cells exposed continuously to CYP 15.0 mumol.L-1 for 72 h . These results suggest that CYP acts by inhibiting the pumping function of PGP.

Cancer Gene Ther, 2001 Jun, 8(6), 440 - 9
The FBMD-1 stroma cell line secretes a unique moiety which can increase retroviral transduction of lineage-committed and primitive human peripheral blood progenitor cells; Buss EC et al.; Peripheral blood progenitor cells are a prime target for gene therapy approaches . As recent data point to the relevance of soluble stroma factors for the efficient transduction of progenitor cells, we tested the stroma-conditioned medium (SCM) of the two cell lines FBMD-1 and L88/5 as well as desulfated and O-sulfated heparin (HS dS and HS OS) for their effect on transduction of peripheral blood progenitor cells . We transduced CD34+ cells of nine tumor patients with the retroviral SF-MDR vector containing the human multidrug resistance 1 (MDR1) gene under serum-free conditions on the fibronectin fragment CH-296 with or without SCM . Provirus-specific polymerase chain reaction showed a median 1.6-fold higher integration rate of the transgene into committed progenitor cells for the group with added FBMD-1 SCM (P=.008) . This was maintained after 2 (P=.02) and, as a trend, after 5 weeks of stroma-dependent long-term culture . We found a median 1.5-fold increase in rhodamine-123 (Rh-123) exclusion in myeloid lineage-committed progeny cells following transduction in the presence of FBMD-1 SCM (P=.0004) . After 2 or 5 weeks of long-term culture, a significantly higher proportion of Rh-123(dull) cells could still be detected in the FBMD-1 SCM transduction group (P=.003 and P=.04, respectively) . L88/5 SCM or HS OS or HS dS was not effective as supplement for improving gene transfer . The FBMD-1 stroma cell line appears to secrete a unique moiety, which can increase retroviral transduction of lineage-committed and primitive progenitor cells . The FBMD-1 stroma activity is not attributable to heparan sulfate.

Drug Resist Updat, 2000 Dec, 3(6), 357 - 363
Does P-glycoprotein play a role in anticancer drug pharmacokinetics?
Sparreboom A, Nooter K.
The multidrug-resistance P-glycoprotein is a drug efflux transport protein abundantly present in various types of human cancer . The protein is encoded by the MDR1 gene and its function is sensitive to modulation by competitive inhibition . Clinical studies have indicated that inhibitors of P-glycoprotein function dramatically decrease the systemic clearance of anticancer agents, necessitating dose reduction . This dose reduction not only complicated the interpretation of toxicity and response data, but also presented a serious obstacle in the development and rational use of P-glycoprotein inhibitors . It is now evident that the pharmacokinetic interference between anticancer drugs and P-glycoprotein inhibitors is due primarily to competition for drug metabolizing enzymes . A wealth of recent experimental data shows that many of the previously tested P-glycoprotein inhibitors, including verapamil, cyclosporin A, and valspodar (SDZ PSC 833), are substrates and/or potent inhibitors of cytochrome P450 3A4 (CYP3A4) . Future development and clinical use of potent P-glycoprotein modulators lacking high affinity for CYP3A4 should decrease the impact of these important drug interactions and will eventually result in improved therapeutic specificity and efficacy .

Drug Resist Updat, 2000 Oct, 3(5), 289 - 302
The (patho)physiological functions of the MRP family; Renes J et al.; The identification of certain members of the large superfamily of ATP binding cassette transport proteins such as MDR1 -P-glycoprotein and the multidrug resistance protein MRP1 as ATP-dependent drug efflux pumps has been a major contribution in our understanding of the multidrug resistance phenotype of cancer cells . Importantly, both transport proteins that exhibit only low structural homology have a very different substrate specificity but confer resistance to a similar spectrum of natural product chemotherapeutic drugs . In contrast to the drug transporter MDR1, MRP1 mainly transports anionic Phase II-conjugates . In addition MRP1-mediated drug resistance is highly dependent on high intracellular glutathione levels which may be linked to the apparent physiological involvement of MRP1 in glutathione-related cellular processes . This review summarizes the current knowledge about functional aspects of MRP1 and its five recently cloned homologues MRP2-MRP6 and discusses their substrate specificities and cellular localization with emphasis on drug resistance .

Drug Resist Updat, 1999 Dec, 2(6), 371 - 381
Photoaffinity analogs for multidrug resistance-related transporters and their use in identifying chemosensitizers; Safa AR; A major obstacle in cancer treatment is the development of resistance to multiple chemotherapeutic agents in tumor cells . The hallmark of this multidrug resistance (MDR) is overexpression of the MDR 1 P-glycoprotein or the multidrug resistance protein MRP1 . It is well documented that these proteins confer MDR in cancer cells . Much evidence indicates that control of intracellular drug levels in MDR cells is determined by P-glycoprotein or MRP, and therefore these proteins are suitable targets for identifying MDR-reversing agents (MDR modulators) . We originally explored the drug-binding ability of P-glycoprotein by synthesizing and using radioactive photoaffinity analogs of vinblastine . Since our initial discovery that P-glycoprotein binds to vinblastine photoaffinity analogs, many P-glycoprotein- and MRP-specific photoaffinity analogs have been developed . In this review, photoaffinity analogs which specifically bind to P-glycoprotein or MRP are discussed . Moreover, utilizing these photoprobes to identify, characterize and localize the drug binding sites of P-glycoprotein and MRP is described . Using P-glycoprotein-specific photoaffinity analogs in combination with site-directed antibodies to several domains of this protein has allowed the localization of the general binding domains of some of the cytotoxic agents an MDR modulators on P-glycoprotein . However, the molecular architecture of the drug binding sites, their exact location on the P-glycoprotein molecule, and the total number of the drug binding sites remain to be determined . This review discusses recent advances in delineating the structure of the drug-binding sites of P-glycoprotein . Moreover, novel MRP1 photoaffinity analogs are reviewed .

Cell Mol Life Sci, 2001 Jun, 58(7), 931 - 59
From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance; Litman T et al.; The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes . The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs . In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated . This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2) . We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR . An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function . Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.

Neuroreport, 2001 Aug 8, 12(11), 2387 - 9
Multidrug resistance-associated protein is involved in the regulation of extracellular levels of phenytoin in the brain; Potschka H et al.; The mechanisms that lead to drug resistance in epilepsy are not known . Recently, overexpression of multidrug transporters, such as multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy . However, it is not known to what extent multidrug transporters such as MRP are involved in transport of antiepileptic drugs . In the present study, we used in vivo microdialysis in rats to study whether the concentration of phenytoin in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of MRP, using the MRP inhibitor probenecid . Local perfusion with probenecid via the microdialysis probe significantly enhanced the extracellular concentration of phenytoin . The data indicate that MRP critically participates in the regulation of extracellular brain concentrations of the major antiepileptic drug phenytoin.

J Biol Chem, 2001 Sep 28, 276(39), 36075 - 8 Epub 2001 Aug 08.
The multidrug resistance P-glycoprotein . Oligomeric state and intramolecular interactions; Taylor JC et al.; The human multidrug resistance P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of transporters, is frequently responsible for the failure of chemotherapy by virtue of its ability to export hydrophobic cytotoxic drugs from cells . Elucidating the inter- and intramolecular interactions of this protein is critical to understanding its cellular function and mechanism of action . Toward this end, we have used both biochemical and genetic techniques to probe potential oligomerization interactions of P-gp . Differentially epitope-tagged P-gp molecules did not co-immunoprecipitate when co-expressed in HEK293 cells or when co-translated in vitro, demonstrating that P-gp is monomeric in both the presence and absence of detergents . The two cytoplasmic domains of P-gp did not interact with each other in vivo when co-expressed as gene fusions in yeast . In contrast, the homologous domains of the transporter associated with antigen processing (TAP), which reside on separate polypeptides and must form a heterodimeric transporter (TAP1/TAP2), did interact in this system, suggesting a role for these domains in TAP dimerization . Implications for understanding the subunit organization of ABC transporters are discussed.

Acta Trop, 2001 Sep 1, 80(1), 39 - 44
Antimalarial activity of azithromycin, artemisinin and dihydroartemisinin in fresh isolates of Plasmodium falciparum in Thailand; Noedl H et al.; Antibiotics with antimalarial activity may offer an interesting alternative for the treatment of multidrug-resistant falciparum malaria . Azithromycin, a relatively recent semisynthetic derivative of erythromycin, was tested for its in vitro activity against fresh isolates of Plasmodium falciparum . As the reportedly slow onset of action of azithromycin suggests its combination with fast-acting substances, such as artemisinin-derivatives, dihydroartemisinin (DHA) was tested parallel as a possible combination partner . The effective concentrations found for azithromycin in this study (EC(50) = 29.3 micromol/l, EC(90) = 77.1 micromol/l blood medium mixture (BMM)) are comparable to those of other antimalarials in the antibiotics class and are considerably higher than those found for mefloquine or quinine . The absence of an activity correlation between azithromycin and chloroquine, quinine and artemisinin emphasises the independence of azithromycin drug response from the sensitivity to these drugs . A weak activity correlation (rho(EC90) = 0.352; p = 0.028), which could point to a potential cross-sensitivity but is probably of little clinical importance, was found with mefloquine above the EC(50) level . Provided that further clinical trials support the combination of these drugs, DHA may offer an interesting combination partner for azithromycin owing to its rapid onset of action and the comparatively low effective concentrations (EC(50) = 1.65 nmol/l, EC(90) = 7.10 nmol/l BMM) . This combination may serve as an interesting alternative for tetracycline and doxycycline, which cannot be used in pregnant women and children, and exhibit phototoxicity . Nevertheless, the relatively high cost of this combination, as well as the controversial reports of the clinical efficacy, may limit the usefulness of azithromycin in malaria therapy and require an adjustment of previously used treatment regimens.

Int J Tuberc Lung Dis, 2001 Aug, 5(8), 741 - 5
Feasibility of home-based and health centre-based DOT: perspectives of TB care providers and clients in an HIV-endemic area of Thailand; Ngamvithayapong J et al.; Focus groups were conducted in a high human immunodeficiency virus (HIV) prevalence area of Thailand to elicit perspectives of health staff and clients regarding the feasibility of directly observed therapy (DOT) for tuberculosis . Most participants perceived health centre-based DOT to be impractical for clients due to severe illness, travel inconvenience, and interference with employment . Most providers perceived home-based DOT to be difficult because of the inconvenience of travel, staff shortages and the high tuberculosis caseload . Most clients except HIV-negative tuberculous females considered home visits to be undesirable due to stigma . The preparedness of providers for home-based DOT might be improved through awareness building among staff about multidrug-resistant tuberculosis.

Blood, 2001 Aug 15, 98(4), 988 - 94
Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin; Linenberger ML et al.; Expression of multidrug resistance (MDR) features by acute myeloid leukemia (AML) cells predicts a poor response to many treatments . The MDR phenotype often correlates with expression of P-glycoprotein (Pgp), and Pgp antagonists such as cyclosporine (CSA) have been used as chemosensitizing agents in AML . Gemtuzumab ozogamicin, an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, is effective monotherapy for CD33(+) relapsed AML . However, the contribution of Pgp to gemtuzumab ozogamicin resistance is poorly defined . In this study, blast cell samples from relapsed AML patients eligible for gemtuzumab ozogamicin clinical trials were assayed for Pgp surface expression and Pgp function using a dye efflux assay . In most cases, surface expression of Pgp correlated with Pgp function, as indicated by elevated dye efflux that was inhibited by CSA . Among samples from patients who either failed to clear marrow blasts or failed to achieve remission, 72% or 52%, respectively, exhibited CSA-sensitive dye efflux compared with 29% (P =.003) or 24% (P <.001) among samples from responders . In vitro gemtuzumab ozogamicin--induced apoptosis was also evaluated using an annexin V--based assay . Low levels of drug-induced apoptosis were associated with CSA-sensitive dye efflux, whereas higher levels correlated strongly with achievement of remission and marrow blast clearance . In vitro drug-induced apoptosis could be increased by CSA in 14 (29%) of 49 samples exhibiting low apoptosis in the absence of CSA . Together, these findings indicate that Pgp plays a role in clinical resistance to gemtuzumab ozogamicin and suggest that treatment trials combining gemtuzumab ozogamicin with MDR reversal agents are warranted . (Blood . 2001;98:988-994)

J Control Release, 2001 Jul 6, 74(1-3), 147 - 58
Water soluble polymers in tumor targeted delivery; Kopecek J et al.; The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability . The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained . Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Eur J Haematol, 2001 Jun, 66(6), 357 - 64
Effects of arsenic trioxide (As2O3) on leukemic cells from patients with non-M3 acute myelogenous leukemia: studies of cytotoxicity, apoptosis and the pattern of resistance; Lehmann S et al.; Arsenic oxide (As2O3) has recently been reported to induce remission in a high percentage of patients with acute promyelocytic leukemia (APL) . The aim of this study was to investigate the effects of As2O3 at therapeutic concentrations on cell viability and apoptosis on leukemic cells from patients with non-M3 acute myelogenous leukemia (AML) and to study the resistance profile compared to conventional AML drugs . Cells from 20 patients were exposed to therapeutic concentrations of As2O3 continuously for 96 h . As2O3 reduced the viability in blast cells from all the 20 tested patients compared to unexposed controls (p-value: 0.02 at 0.05 microM; <0.005 at 1.0 microM and higher concentrations) . An increase in the apoptotic rate was also seen after incubation with As2O3 . Parallel to the incubation with arsenic the in vitro sensitivity to a number of chemotherapeutic agents commonly used in AML was studied . Correlation coefficients for the in vitro sensitivity were highly significant between the conventional AML drugs except for Ara-C . For As2O3, all the correlation coefficients were negative and ranged between -0.05 and -0.51 . Furthermore, increased P-gp expression in a multidrug resistant HL-60 cell line did not decrease the sensitivity to As2O3 as compared to the parental cell line . Neither did a P-gp-transfected variant of the K562 cell line show decreased sensitivity to As2O3 . We conclude that As2O3 at therapeutic concentrations induces apoptosis and cytotoxic effects in blast cells from patients with non-M3 AML, and that As2O3 differs from conventional AML drugs with respect to the mechanisms that confer resistance to the drugs.

Eur J Biochem, 2001 Aug, 268(15), 4151 - 7
Regulation of MDR1 promoter activity in human breast carcinoma cells by protein kinase C isozymes alpha and theta; Gill PK et al.; Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype . Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription . In this study the potential roles of PKC-alpha and PKC-theta in MDR1 gene transcriptional regulation were explored . Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-length PKC-alpha or PKC-theta genes driven by the ecdysone promoter . Stable transfectants were selected by use of the appropriate antibiotics . Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and down-regulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA) . These cells were used to analyse PKC-mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to -8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene . In PKC-alpha expressing cells TPA caused activation of all promoter fragments to -29 bp . This finding suggests that TPA-inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR-1) in this region of the promoter may be due to activation of PKC-alpha . In contrast, PKC-theta activated only two MDR1 fragments, -982 and -612 bp . The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X . These data suggest that MDR1 promoter transcription can be regulated by PKC-alpha and PKC-theta . The results support the search for therapeutic strategies directed specifically against PKC-alpha to ameliorate resistance of tumours against cytotoxic agents.

Cancer Chemother Pharmacol, 2001 Jul, 48(1), 62 - 70
Markedly diminished drug resistance-inducing properties of vinflunine (20',20'-difluoro-3',4'-dihydrovinorelbine) relative to vinorelbine, identified in murine and human tumour cells in vivo and in vitro; Etievant C et al.; PURPOSE: Vinflunine (VFL) is a novel Vinca alkaloid with markedly superior experimental in vivo antitumour activity to its parent molecule, vinorelbine (Navelbine, NVB), against a panel of murine and human tumours . The aim of this study was to establish whether there are differences in the rate and extent of development of resistance, both in vivo and in vitro, to these two newer Vinca alkaloids under identical selection conditions . METHODS: Using P388 leukaemia cells in vivo, it was evident that VFL induced drug resistance far less readily than NVB, as shown by the number of passages required to select for total resistance . Under in vitro conditions, using A549 human lung carcinoma cells, it was also clearly shown by drug sensitivity determinations that VFL was a less-potent inducer of drug resistance than NVB . Resistance resulting from either in vivo or in vitro selection was associated with a classic multidrug resistance profile . Further characterization of the drug-resistance phenotype of the most highly resistant A549 sublines showed that the level of total beta-tubulin expression appeared to be modified exclusively in the NVB-resistant cells . CONCLUSION: The clear demonstration that resistance to VFL developed far less readily than resistance to NVB both in vivo and in vitro may have potential clinical implications.

Eur J Haematol Suppl, 2001 Jul, (64), 41 - 5
DICE (dexamethasone, ifosfamide, cisplatin, etoposide) infusional chemotherapy for refractory or relapsed non-Hodgkin's lymphoma (NHL); Coleman M et al.; Continuous exposure to naturally-derived chemotherapy agents such as etoposide may theoretically override drug resistance due to overexpression of the multidrug resistance gene product, p-glycoprotein . Dexamethasone in high dose may have a similar overriding effect . Data also suggest that ifosfamide both by continuous infusion and when combined with a platinum compound may be more effective . Forty-four chemotherapy-refractory/relapsed lymphoma patients received the DICE infusional regimen . The programme consisted of dexamethasone 40 mg i.v . daily for 4 days, ifosfamide 1500 mg mixed with equal dosing of mesna continuously infused i.v . daily for 4 days, cisplatin 25 mg i.v . each day for 4 days and etoposide 150 mg continuously infused i.v . daily for 4 days, all administered every 3 weeks . Doses of ifosfamide and etoposide were escalated by 250 mg and 25 mg, respectively, based on patient tolerance, usually lack of myelosuppression . Special hydration was not required . G-CSF support was provided to patients as required . All patients had disease that had relapsed from or was resistant to CHOP or a similar anthracycline-containing combination regimen . A majority had previously received at least two regimens and 27% had received three or more . Of 44 patients, 32 (73%) achieved a significant response consisting of 18 complete remissions (CR) (41%) and 14 (32%) partial remissions (PR) . There were 81% objective responses in large cell lymphoma comprised of 50% CR and 31% PR . Previous response to chemotherapy predicted response to DICE: 83% (25/30) of prior responders vs 50% (7/14) of non-responders had a response to the treatment regimen (p = 0.031, Fisher's exact test) . Patients not undergoing transplantation had a median time of 8 months on therapy and a mean of 10 months . Toxicity was haematological (36% developing grade III-IV toxicity) and neurological (9%) . There were only three episodes of clinical cystitis or gross haematuria . Infusional DICE is an easily administered and well tolerated programme with significant activity in refractory or relapsed NHL and may be useful as a tumour-reductive therapy prior to high-dose chemotherapy and autologous stem cell transplantation.

Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 41 - 9
Drug resistant malaria on the Thai-Myanmar and Thai-Cambodian borders; Wongsrichanalai C et al.; We describe the changing epidemiology of drug resistant malaria in Thailand over the past decade . Factors determining the characteristic patterns of the development and spread of resistance to anti-malarial drugs on the Thai-Cambodian border and the Thai-Myanmar border are explored, namely, population dynamics, drug usage and malaria control measures . The introduction of artesunate-mefloquine combination in selected areas along the two borders in 1995 is believed to be one of the multiple factors responsible for stabilizing the multidrug resistance problems in Thailand today . Other control measures and inter-governmental co-operation must continue to be strengthened in order to limit the spread of drug resistance malaria in the Southeast Asian region.

Gene, 2001 Jul 25, 273(1), 89 - 96
Two new genes from the human ATP-binding cassette transporter superfamily, ABCC11 and ABCC12, tandemly duplicated on chromosome 16q12; Tammur J et al.; Several years ago, we initiated a long-term project of cloning new human ATP-binding cassette (ABC) transporters and linking them to various disease phenotypes . As one of the results of this project, we present two new members of the human ABCC subfamily, ABCC11 and ABCC12 . These two new human ABC transporters were fully characterized and mapped to the human chromosome 16q12 . With the addition of these two genes, the complete human ABCC subfamily has 12 identified members (ABCC1-12), nine from the multidrug resistance-like subgroup, two from the sulfonylurea receptor subgroup, and the CFTR gene . Phylogenetic analysis determined that ABCC11 and ABCC12 are derived by duplication, and are most closely related to the ABCC5 gene . Genetic variation in some ABCC subfamily members is associated with human inherited diseases, including cystic fibrosis (CFTR/ABCC7), Dubin-Johnson syndrome (ABCC2), pseudoxanthoma elasticum (ABCC6) and familial persistent hyperinsulinemic hypoglycemia of infancy (ABCC8) . Since ABCC11 and ABCC12 were mapped to a region harboring gene(s) for paroxysmal kinesigenic choreoathetosis, the two genes represent positional candidates for this disorder.

Protein Expr Purif, 2001 Aug, 22(3), 430 - 5
Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis; Oliveira JS et al.; Tuberculosis (TB) resurged in the late 1980s and an estimated 1.87 million people died of TB in 1997 . The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multidrug-resistant strains have created a need to develop new antimycobacterial agents . The existence of a shikimate pathway has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis . The M . tuberculosis aroK-encoded shikimate kinase and aroA-encoded 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase were cloned and the enzymes overexpressed in soluble form . Overexpression was achieved without isopropyl beta-d-thiogalactoside induction, and cells grown to stationary phase yielded approximately 30% of target proteins to total soluble cell proteins . Enzyme activity measurements using coupled assays demonstrated that there was a 328-fold increase in specific activity for shikimate kinase and 101-fold increase for EPSP synthase .

Blood Cells Mol Dis, 2001 May-Jun, 27(3), 637 - 48
5-Azacytidine modulates the response of sensitive and multidrug-resistant K562 leukemic cells to cytostatic drugs; Efferth T et al.; In an endeavor to improve responsiveness of tumor cells to drug combination treatments, we analyzed the effect of 5-azacytidine (5AC) as a model compound for a new class of drugs, DNA-demethylating agents . We used parental K562/WT chronic myelogenous leukemia cells and a multidrug-resistant subline thereof, K562/ADM . Multidrug-resistant cells were more resistant to daunorubicin, but more sensitive to cisplatin than parental K562 cells as measured by growth inhibition and apoptosis assays . Resistance to daunorubicin can be explained by amplification of the MDR1 drug transporter gene . Cisplatin induced more DNA damage in specific genes and in the entire genome of K562/ADM cells compared to K562/WT cells using PCR stop assays and atomic absorption spectroscopy . Pretreatment with 5AC modulated the response of K562/ADM cells toward MDR-type drugs (daunorubicin, vincristine, etoposide) and reduced function and expression of MDR1 as analyzed by flow cytometry and RT-PCR . Analysis of CpG island methylation in the promotor region of the MDR1 gene by bisulfite sequencing and a methylation-sensitive HpaII-digestion/PCR approach revealed that methylation of the MDR1 promotor of K562/ADM cells was greater than in K562/WT cells . 5AC treatment completely abolished MDR1 promotor methylation . The unexpected observation that DNA demethylation by 5AC rather decreases than increases MDR1 expression in K5612/ADM cells points to still unexplored sequences in the MDR1 promotor whose transcriptional activity may be affected by the methylation status . 5AC pretreatment also modulated K562/WT and K562/ADM cells to non-MDR-type drugs such as cisplatin and increased cisplatin-induced DNA damage .

Hepatology, 2001 Aug, 34(2), 351 - 9
Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver; Donner MG et al.; Cholestasis induces down-regulation of multidrug resistance protein 2 (Mrp2, symbol Abcc2), which is localized to the canalicular membrane . Given the overlapping substrate specificities of Mrp2 and multidrug resistance protein 3 (Mrp3, symbol Abcc3), we examined the hypothesis of a different subcellular and lobular localization of these members of the Mrp family in rat liver after bile duct ligation . We raised a polyclonal antibody against rat Mrp3 and detected this protein in the basolateral plasma membrane of hepatocytes surrounding the central veins and of cholangiocytes . The Mrp3 protein level was less than 2% of the expression observed after 72 hours of obstructive cholestasis . After 48 hours of bile duct ligation, the Mrp3 protein was increased and was further enhanced after 72 hours . In 72-hour-cholestatic rat liver Mrp3 was expressed, in addition, in periportal hepatocytes . However, there was a preponderance of Mrp3 in the pericentral area of the liver lobule . In Mrp2-deficient mutant rat liver, the Mrp3 protein expression was most enhanced and its zonation was lost . The Mrp3 immunostaining of cholangiocytes was preserved in cholestatic and in Mrp2-deficient mutant liver . Canalicular Mrp2 decreased and amounted to 34% of normal after bile duct ligation for 72 hours . We conclude that the hepatocellular up-regulation of Mrp3 in cholestasis together with cholangiocellular Mrp3 may compensate for the biliary obstruction and impaired canalicular Mrp2 function by clearing cholephilic anionic substances into the blood.

Hepatology, 2001 Aug, 34(2), 340 - 50
Protein kinase C-dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells; Kubitz R et al.; The subcellular localization of hepatobiliary transport proteins directly affects the rate of bile formation, e.g., the conjugate export pump multidrug resistance protein 2 (MRP2) is regulated on a short-term scale by retrieval from and insertion into the canalicular membrane in the liver . This study reports on the effects of protein kinase C on MRP2 localization and activity in human hepatoblastoma HepG2 cells . MRP2 was detected in HepG2 cells by immunocytochemistry and Western blot analysis . Functional activity was assessed by confocal laser scanning microscopy using fluorescent MRP2 substrates . In untreated HepG2 cells MRP2 was almost exclusively localized at the apical membrane . Treatment of HepG2 cells with phorbol-12-myristate-13-acetate (PMA) resulted in a rapid decrease of apically localized MRP2 and a loss of more than 90% of pseudocanaliculi within 4 hours . This was accompanied by a reduced pseudocanalicular secretion of the MRP2 substrate glutathione-methylfluorescein . Interestingly, PMA treatment (1-100 nmol/L) led to the appearance of immunoreactive MRP2 at the basolateral membrane within 30 minutes . This was shown by its colocalization with MRP1, human dipeptidylpeptidase IV (DPPIV), and transfected rat Ntcp . The effects of PMA on MRP2 localization were sensitive to the protein kinase C (PKC) inhibitor Go6850 but insensitive to inhibition of MEK by PD098059 . Basolateral MRP2-appearance was not inhibited by cycloheximide or by disruption of microtubules or microfilaments . In rat livers cholestasis was induced by PMA (100 nmol) and MRP2 was detected at the basolateral membrane in some areas, colocalizing with Ntcp . The data suggest that retargeting of canalicular MRP2 to the basolateral membrane due to PKC activation may represent a novel mechanism that may contribute to cholestasis.

J Pharm Pharmacol, 2001 Jul, 53(7), 1029 - 39
Apoptosis induced by doxorubicin and cinchonine in P388 multidrug-resistant cells; Furusawa S et al.; Acquired drug resistance is a major factor in the failure of doxorubicin-based cancer chemotherapy . We determined the ability of cinchonine to reverse doxorubicin drug resistance in a doxorubicin-resistant leukaemia cell line (mouse P388/DOX) . A non-cytotoxic concentration of cinchonine (10 microM) increased the sensitivity to doxorubicin of multidrug-resistant P388/DOX cells and significantly enhanced the doxorubicin-induced apoptosis and DNA fragmentation in resistant cells, but had no effect in parent cells . Time-course studies demonstrated that DNA fragmentation was present 24 h after incubation with doxorubicin and cinchonine, indicating that DNA degradation was a preceding event . In cultured cells, cinchonine increased the intracellular accumulation of doxorubicin in the resistant cells in a dose-dependent manner . Using flow cytometry to measure the inhibition of the P-glycoprotein (P-gp) dependent efflux of rhodamine 123, cinchonine was found to be considerably more effective than quinine . The results with cinchonine suggest that there may be quinine derivatives with a similar capacity to inhibit drug transport by P-gp . Additionally, the G2/M phase cell population in resistant cells is increased by doxorubicin/cinchonine treatment . Exposure of resistant cells to 1 microM doxorubicin and 10 microM cinchonine resulted in the expression of Fas (APO-1/CD95) in cells after 6 h . These studies demonstrate that the cell killing effects of doxorubicin and cinchonine in resistant cells

J Pharm Pharmacol, 2001 Jul, 53(7), 1021 - 8
Designing multidrug-resistance modulators circumventing the reverse pH gradient in tumours; Castaing M et al.; Multidrug-resistant tumours often exhibit a reverse pH gradient (acid outside), as they have an acid extracellular pH (pHe) and a neutral alkaline intracellular pH (pHi) . This study was designed to test the hypothesis that the ability of lipophilic drugs to mediate multidrug resistance (MDR) reversal by interacting with the membrane phospholipids may be correlated with pH in resistant tumours . The permeation properties of five MDR modulators were therefore studied at 37 degrees C by quantifying their ability to induce the leakage of Sulfan blue through unilamellar anionic liposomes, over the range pH 6.5-7.7, and in the absence of any membrane potential (pHe = pHi) . The dye leakage induced by two calcium blockers (diltiazem and verapamil) and two antiparasitic agents (thioacridine derivative and mepacrine) was found to significantly increase with the pH of the medium (P < 0.001), whereas that induced by a non-ionic detergent (Triton X-100) showed almost no pH-dependent variations . This process was a cooperative one (0.8 < Hill coefficient < 8.5) and the permeation doses inducing 50% dye leakage (PD50) ranged from 1.6 to 36.0 mM . The permeation ability of the MDR modulators (log(1/PD50)) significantly increased with their octanol-buffer distributions (logD) (slope = 0.35+/-0.06; y intercept = 1.65 +/- 0.14; P < 0.0001) and significantly decreased with their net electric charge (z) (slope = -0.48+/-0.07; y intercept = 2.85+/-0.08; P < 0.0001) . A highly significant multiple correlation was found to exist between the variations of log(1/PD50) with those of logD and z (dlog(1/PD50)/dlogD = 0.21 +/- 0.05; dlog(1/PD50)/dz = -0.34+/-0.07; y intercept = 2.27+/-0.17; P < 0.000001) . The results provide evidence that in resistant tumours (acid pHe and neutral alkaline pHi), the MDR reversal might be enhanced by favourable drug-membrane interactions if the modulators are designed in the form of highly lipophilic (logP approximately equals 4) mono-basic drugs with a near neutral pKa (pKa approximately equals 7-8).

J Biol Chem, 2001 Oct 5, 276(40), 37715 - 21 Epub 2001 Jul 30.
Multiple human vault RNAs . Expression and association with the vault complex; van Zon A et al.; Human vaults are intracellular ribonucleoprotein particles believed to be involved in multidrug resistance . The complex consists of a major vault protein (MVP), two minor vault proteins (VPARP and TEP1), and several small untranslated RNA molecules . Three human vault RNA genes (HVG1-3) have been described, and a fourth was found in a homology search (HVG4) . In the literature only the association of hvg1 with vaults was shown in vivo . However, in a yeast three-hybrid screen the association of hvg1, hvg2, and hvg4 with TEP1 was demonstrated . In this study we investigated the expression and vault association of different vault RNAs in a variety of cell lines, including pairs of drug-sensitive and drug-resistant cells . HVG1-3 are expressed in all cell lines examined, however, none of the cell lines expressed HVG4 . This probably is a consequence of the absence of essential external polymerase III promoter elements . The bulk of the vault RNA associated with vaults was hvg1 . Interestingly, an increased amount of hvg3 was bound to vaults isolated from multidrug-resistant cell lines . Our findings suggest that vaults bind the RNA molecules with different affinities in different situations . The ratio in which the vault RNAs are associated with vaults might be of functional importance.

Int J Cancer, 2001 Aug 15, 93(4), 584 - 9
Association between expression of the MRP3 gene and exposure to platinum drugs in lung cancer; Oguri T et al.; To investigate the roles played by the multidrug resistance-associated protein (MRP1) homologues MRP3 and MRP4 in resistance to platinum drugs, we examined steady-state levels of mRNA for both MRP3 and MRP4 in normal lung and lung cancer specimens as well as peripheral mononuclear cells (PMN) after platinum drug exposure . MRP3 and MRP4 gene expression levels were monitored in the PMN of 10 previously untreated lung cancer patients within 24 hr after carboplatin (CBDCA) administration . Expression levels for both genes were also examined in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients with lung cancer . MRP3 and MRP4 gene expression levels were assessed by quantitative reverse transcription-polymerase chain reaction . MRP3 expression levels in the PMN rose rapidly within 24 hr after administration of CBDCA, whereas MRP4 expression levels remained the same . Furthermore, MRP3 expression levels in normal lung and tumor tissues from autopsy samples that had been exposed to platinum drugs while the patients were alive were significantly higher than those in unexposed tissues, but again MRP4 expression levels remained the same . These results suggest that platinum drugs and/or the physiological stress response to xenobiotics induce expression of the MRP3 gene .

J Biol Chem, 2001 Oct 5, 276(40), 36923 - 30 Epub 2001 Jul 26.
Identification and functional analysis of two novel mutations in the multidrug resistance protein 2 gene in Israeli patients with Dubin-Johnson syndrome; Mor-Cohen R et al.; Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes . The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies . All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing . Two novel mutations were identified in exon 25 . One mutation, 3517A-->T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G-->A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families . Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation . The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry . Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2 . Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells . In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells . These findings provide an explanation for the DJS phenotype in these two patient groups . Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.

Curr Drug Targets, 2000 Jul, 1(1), 85 - 99
P-glycoprotein as a drug target in the treatment of multidrug resistant cancer; Lehne G; Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy . One important mechanism of MDR involves the multidrug transporter, P-glycoprotein (Pgp), which confers upon cancer cells the ability to resist lethal doses of certain cytotoxic drugs by pumping the drugs out of the cells and thus reducing their cytotoxicity . Pgp belongs to the ATP-binding cassette (ABC) family of transporter molecules which require hydrolysis of ATP to run the transport mechanism . The substrates of Pgp may be endogenous (steroid hormones, cytokines) or exogenous (cytostatic drugs) . A number of studies have demonstrated a negative correlation between Pgp expression levels and chemosensitivity or survival in a range of human malignancies . In principle, Pgp mediated drug resistance can be circumvented by treatment regimens that either exclude Pgp substrate drugs or include Pgp inhibitory agents . Experimental studies have demonstrated that certain structural modifications of anthracyclines confer the ability to escape Pgp transport . The therapeutic benefit of Pgp inhibitors as chemosensitizers is currently being explored in phase III clinical trials, and the first promising results have already been reported . Another therapeutic option for Pgp inhibitors has recently evolved as several Pgp inhibitors, many of which are generally low-toxic substances, by themselves constrain proliferation and cause cell death by apoptosis in certain MDR cancer cell lines . The dual effect of Pgp inhibitors, targeting MDR cancer cells selectively, may translate into improved efficacy of cancer chemotherapy and perhaps new and less toxic drug treatment strategies in human MDR cancer.

Ann Hematol, 2001 Jun, 80(6), 349 - 53
Value of Tc-99m sestamibi scintigraphy in the detection of bone lesions in multiple myeloma: comparison with Tc-99m methylene diphosphonate; Alexandrakis MG et al.; Technetium 99m-2-methoxyisobutylisonitrile (Tc-99m MIBI) is a lipophilic agent that accumulates preferentially within living malignant cells due to the higher transmembrane electrical potential as a consequence of the higher metabolic rate than in the surrounding normal cells . It has been effectively used to detect malignant tumors at diagnosis and follow-up and has been reported to be useful in detecting disease lesions in multiple myeloma . We studied 28 consecutive patients with multiple myeloma at diagnosis to determine the value of Tc-99m MIBI in comparison with Tc-99m methylene diphosphonate (MDP), conventional X-rays, computed tomography (CT) scan, and magnetic resonance imaging (MRI) . We found 26 patients with obvious osteolytic lesions in X-rays, 22 patients with positive Tc-99m MIBI scans, and 15 patients with positive Tc-99m MDP scans . There was no coincidence of the positive lesions in the two scans, while in two patients the osteolytic areas were positive in the Tc-99m MDP scans, and in one case the osteolytic area was positive in the Tc-99m MIBI scan . The intensity of Tc-99m MIBI scans correlated with disease activity as determined by lactate dehydrogenase (LDH) (p<0.05), C-reactive protein (CRP) (p<0.01), beta2-microglobulin (p<0.05), and serum ferritin (p<0.01) . We believe that Tc-99m MIBI scintigraphy can detect bone marrow lesions in myeloma patients that cannot be detected by other imaging methods and that it can be useful especially in solitary myeloma to exclude other involved sites . In addition, it could be a prognostic factor related to disease activity and multidrug resistance . We believe that a multicenter study is needed to evaluate the usefulness of this agent.

Mol Med, 2000 Dec, 6(12), 1008 - 15
Potent induction of apoptosis by beta-lapachone in human multiple myeloma cell lines and patient cells; Li Y et al.; BACKGROUND: Human multiple myeloma (MM) remains an incurable hematological malignancy . We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity . MATERIALS AND METHODS: We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay . To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays . The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay . To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis . To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay . RESULTS: We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5) . Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors . In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis . Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM . CONCLUSIONS: Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.

J Clin Microbiol, 2001 Aug, 39(8), 2987 - 90
Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from India; Mani C et al.; Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene were analyzed by DNA sequencing of 50 Mycobacterium tuberculosis clinical isolates (44 resistant and 6 sensitive) from various parts of India . Fifty-three mutations of 18 different kinds, 17 point mutations and one deletion, were observed in 43 of 44 resistant isolates . Three novel mutations and three new alleles within the RRDR, along with two novel mutations outside the RRDR, are reported in this study.

Jpn J Cancer Res, 2001 Jul, 92(7), 785 - 92
A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells; Nokihara H et al.; Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC) . MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR . We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp . In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells . SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression . MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro . Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells . Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells . Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs . These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo . Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.

Jpn J Cancer Res, 2001 Jul, 92(7), 778 - 84
Expression of drug resistance genes in VP-16 and mAMSA-selected human carcinoma cells; Matsumoto Y et al.; The cell lines described in the present study were isolated as part of an effort to understand resistance to topoisomerase (topo) II inhibitors . To that end, 50 sublines were isolated from four human breast cancer cell lines, i.e., MCF-7, T47D, MDA-MB-231, and ZR-75B . As an initial step, a concentration that would be lethal to the majority of cells (IC99) was selected for both VP-16 and mAMSA, for each cell line . The identification of an increasing number of putative drug resistance-related proteins provided the opportunity to examine expression of the corresponding genes in the selected cell lines . Northern blot analysis revealed different responses to the selecting agents in the different cell lines . Previous studies examining expression of multidrug resistance (MDR)-1 in resistant cell lines had found undetectable levels in all cells . In the ZR-75B sublines, increased expression of MDR-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) was observed, and when the relative levels of overexpression were compared, a high correlation was found . In contrast, increased expression of MRP was observed in some of the MDA-MB-231 sublines, without a concomitant increase in cMOAT expression . Finally, in both T47D and MCF-7 sublines, increased expression of cMOAT or MRP was observed infrequently, and where it occurred, was of a much smaller magnitude . In the analysis of expression of MRP, the highest levels were found in the ZR-75B and MDA-MB-231 sublines, with lower levels in the MCF-7 and T47D clones . Similarly, differences in the expression of topo IIalpha were observed among the sublines . Although the differences in expression appear to depend on the parental cell line from which the resistant sublines were derived, a strong correlation was observed between the expression of MRP and the levels of topo IIalpha . Cell lines with low levels of MRP had lower levels of topo IIalpha, while those with high levels of MRP maintained higher levels of topo IIalpha . While a reduced topo IIalpha level was common, there did not appear to be a compensating increase in the expression of topo IIbeta or topo I or casein kinase (CK) IIalpha in any of the cell lines . While the possibility that such compensation could occur has been discussed and even reported in some cell lines, such an adaptation was not observed in the present study, suggesting that it is not common.

Br J Haematol, 2001 Jul, 114(1), 177 - 84
Evidence that natural killer cells express mini P-glycoproteins but not classic 170 kDa P-glycoprotein; Trambas C et al.; Several lines of evidence including reverse transcription polymerase chain reaction, immunoreactivity and their ability to efflux rhodamine 123 have implied the existence of P-glycoprotein in natural killer (NK) cells . It has been a natural tendency to assume that NK-cell P-glycoprotein is identical to the P-glycoprotein of multidrug resistant (MDR) cell lines, however, the present study uncovered major differences . Functionally, NK cells demonstrated a restricted substrate profile, being unable to transport daunorubicin and calcein acetoxymethylester while efficiently transporting other P-glycoprotein substrates . Furthermore, physical differences in NK-cell P-glycoprotein were established by differential reactivity with P-glycoprotein antibodies . NK cells demonstrated strong reactivity with C494 and JSB-1, but did not react appreciably with C219 . In addition, NK cells were unable to bind to the antibody MM4.17 unless they had been fixed and permeabilized, yet this antibody normally recognizes an extracellular epitope of P-glycoprotein . These differences culminated in the demonstration using Western analysis that NK cells did not express detectable levels of 170 kDa P-glycoprotein . Instead, NK cells expressed small-molecular-weight 'mini P-glycoprotein' products, of approximately 70 and 80 kDa . Collectively, these data indicate that the predominant P-glycoprotein species of NK cells are novel mini P-glycoproteins and not the classic P-glycoprotein of MDR models.

Curr Pharm Des, 2001 Sep, 7(13), 1277 - 90
The epothilones, eleutherobins, and related types of molecules; Stachel SJ et al.; Taxol is currently one of the most effective anticancer agents available . However, limitations due to multidrug-resistance (MDR) susceptibility and lack of aqueous solubility render it less than an ideal drug . These limitations, coupled with taxol's unique mechanism of tumor inhibition, involving the stabilization of microtubule assembly, have spurred the search for more effective chemotherapeutic agents . This review will discuss the chemistry and biology of some of the most promising new molecules with "taxol-like" activity . The extended family of microtubule-stabilizing agents now includes the epothilones, eleutherobins, discodermolide, laulimalide and WS9885B . The epothilones have emerged as one of the most exciting new candidates for detailed structure-activity-related studies . A review of our efforts in the synthetic and biological aspects of this research is presented, as are the latest developments reported from other laboratories in academia and the pharmaceutical industry . The synthesis and structure-activity studies of eleutherobins, as well as recent progress with discodermolide, laulimalide and WS9885B are also reviewed . An abundance of exciting advances in chemistry and biology have emerged from these studies, and it is hoped that it will ultimately result in the development of new and more effective chemotherapeutic agents in the fight against cancer.

Curr Pharm Des, 2001 Sep, 7(13), 1259 - 76
Cryptophycins: a novel class of potent antimitotic antitumor depsipeptides; Shih C et al.; The antitumor cryptophycins are synthetic derivatives of the desipeptide cryptophycins isolated from the cyanobacterium Nostoc sp . Cryptophycin 52 that is currently in clinical trial in solid tumors, is prepared by total synthesis of four key fragments that are coupled to form the final product . The mechanism of anticancer activity of the cryptophycins has been associated with their destabilization of microtubules and with their induction of bcl-2 phosphorylation leading to apoptosis . The cryptophycins maintain activity against ovarian and breast carcinoma cells that overexpress the multidrug resistance efflux pump P-glycoprotein . Cryptophycin 52 has demonstrated a broad range of antitumor activity against both murine and human tumors . In the human MX-1 breast carcinoma xenograft cryptophycin 55 produced greater-than- additive tumor response in combination with 5-fluorouracil . In human non-small cell lung carcinoma and human small cell carcinoma xenografts, administration of the cryptophycins along with gemcitabine, cisplatin or carboplatin resulted in antitumor activity greater than either agent alone . The cryptophycins appear to be additive with fractionated radiation therapy in the human H460 non-small cell lung carcinoma . In the human HCT116 colon carcinoma, the cryptophycins resulted in a greater than additive tumor response when administered sequentially with 5-fluorouracil or irinotecan . Treatment of animals bearing intraperitoneal human OVCAR-2 ovarian carcinoma with cryptophycin 52 resulted in survival times that were greater than those achieved with docetaxel or paclitaxel . Cryptophycin 52 is currently in early clinical testing.

J Photochem Photobiol B, 2001 Jul, 60(2-3), 79 - 86
5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells; Li W et al.; To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells . Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells . A P-gp inhibitor could not increase PpIX accumulation . Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production . Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme . Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX . These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.

Gene, 2001 Jul 11, 272(1-2), 141 - 8
Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II alpha; Okada Y et al.; In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha) . We have shown two etoposide resistant hTOP2 alpha mutants, K798L and K798P confer resistance to etoposide . In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature . We purified these mutants to characterize the drug resistant mechanism . Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA . These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast.

J Surg Res, 2001 Aug, 99(2), 179 - 86
Mechanisms of taxotere-related drug resistance in pancreatic carcinoma; Liu B et al.; BACKGROUND: Pancreatic adenocarcinoma (PAC) is generally refractory to most chemotherapeutic agents, including docetaxel (Taxotere; TXT) . Specific mechanisms for TXT-related drug resistance in PAC have not been defined . The hypothesis of this study was that PAC resistance to TXT is primarily related to P-glycoprotein (P-gp), the expression product of multiple drug resistance (MDR)-1, as opposed to lung resistance protein (LRP) or multidrug resistance protein (MRP) . MATERIALS AND METHODS: The sensitivity of the PAC cell line SUIT-2 and its sublines to TXT, doxorubicin (DOX) and 5-fluorouracil (5-FU) was evaluated with a 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay . MDR1 (P-gp), MRP, LRP, and beta-tubulin isotype gene expressions were detected at the messenger RNA level by reverse transcription-polymerase chain reaction (RT-PCR) . Verapamil and indomethacin (IMC) were used to test the functionality of P-gp and MRP, respectively . RESULTS: The SUIT-2 subline S-020 and the TXT-selected SUIT-2 cell line S2/TXT were significantly resistant to TXT . Both showed cross-resistance to DOX but no resistance to 5-FU . RT-PCR demonstrated strong expression of P-gp in S-020 and S2/TXT and weaker or no expression in other cells lines . MRP and LRP expression was found in most of these cell lines but had no relationship to the TXT resistance . TXT resistance in S2-020 and S2/TXT could be reversed by verapamil but not by IMC . Levels of beta-tubulin isotype II and III were increased in S2/TXT compared with S-020 and SUIT-2 . CONCLUSIONS: Intrinsic and acquired TXT resistance is primarily mediated by P-gp, but not by MRP or LRP, and is markedly reversed by the P-gp modulator verapamil . Hence future related studies should focus on the use of agents that block the transporter action of P-gp .

Arch Biochem Biophys, 2001 Aug 1, 392(1), 153 - 61
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation; Cui L et al.; ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes . The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate . Hence mutagenesis of residues in these motifs may interfere with function . This is the case with the MRP1 multidrug transporter . However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein . We have determined now that this latter effect is entirely due to the D792L substitution . This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein . In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome . A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function .

Virchows Arch, 2001 Jun, 438(6), 581 - 90
Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras; Lohr M et al.; Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis . In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene . As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr) . They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel . Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8 . They did not form colonies in soft agar nor did they form tumors in nude mice . Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas . It is concluded that SV 40 immortalized bovine pancreatic

Int J Infect Dis, 2001, 5(2), 93 - 100
Tuberculosis and drug resistance among patients seen at an AIDS Reference Center in São Paulo, Brazil; Pinto WP et al.; OBJECTIVES: To assess the frequency of resistance of Mycobacterium tuberculosis to antituberculosis drugs and the factors associated with it among patients with tuberculosis (TB) and acquired immunodeficiency syndrome (AIDS) . MATERIALS AND METHODS: The medical records of TB and AIDS cases diagnosed from 1992 to 1997 in a public service for AIDS care were reviewed . RESULTS: Resistance was diagnosed in 82 (19%) of 431 cases . The mean and median values between the diagnosis of AIDS and the diagnosis of TB were 214.8 days and 70.5 days, respectively . Multidrug-resistant TB (MDR TB) occurred in 11.3% of cases . Of the 186 patients with no previous treatment, 13 (6.9%) presented primary MDR TB . Of the 90 cases with previous treatment, six (6.7%) presented monoresistance to rifampin and 27 (30%) presented MDR TB . The distribution of cases with sensitive and resistant M . tuberculosis strains was homogeneous in terms of the following variables: gender, age, category of exposure to human immunodeficiency virus (HIV), alcoholism, and homelessness . Multivariate analysis showed an association between resistance and the two following variables: previous treatment and duration of AIDS prior to TB exceeding 71 days . The rates of primary multiresistance and of monoresistance to rifampin were higher than those detected in HIV-negative patients in Brazil . CONCLUSIONS: In this patient series, M . tuberculosis resistance was predominantly of the acquired type, and resistance was independently associated with previous treatment for TB and with duration of AIDS prior to TB exceeding 71 days.

Biochem Biophys Res Commun, 2001 Jul 27, 285(4), 981 - 90
Identification of DNA-protein interactions in the 5' flanking and 5' untranslated regions of the human multidrug resistance protein (MRP1) gene: evaluation of a putative antioxidant response element/AP-1 binding site; Kurz EU et al.; Overexpression of the multidrug resistance protein, MRP1, confers resistance to multiple natural product-type chemotherapeutics . MRP1 amplification is observed in some multidrug-resistant cell lines, while in others, increased transcription occurs in the absence of gene amplification . To investigate mechanisms influencing MRP1 transcription, three small cell lung cancer cell lines were examined: drug sensitive H69 cells with two apparently normal MRP1 alleles, highly resistant H69AR cells in which MRP1 is amplified and low level resistant H69PR cells that contain only one MRP1 allele . Deoxyribonuclease I footprinting and gel mobility shift assays were undertaken using nuclear extracts from the three cell lines and a 1 kb region encompassing the 5' flanking region of MRP1 . Thirteen protein binding sites were identified of which six were sequence specific . Differences in levels of protein binding occurred with a putative antioxidant response element (ARE)/AP-1 binding site at -511 to -477 . Levels of protein binding to this site were 2.5- to 3.0-fold higher in H69AR nuclear extracts versus extracts from H69 or H69PR cells . The AP-1 sequence is required for binding and c-Jun and JunD were identified as components of the protein complex . The ARE/AP-1 element functioned as a transcriptional enhancer but did not mediate induction of a luciferase reporter gene upon beta-naphthoflavone treatment .

Clin Exp Metastasis, 2000, 18(5), 353 - 60
Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966; Hanibuchi M et al.; serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells . Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells . Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes) . Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo . Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype . These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC.

Int J Tuberc Lung Dis, 2001 Jul, 5(7), 648 - 55
Occurrence of serious adverse effects in patients receiving community-based therapy for multidrug-resistant tuberculosis; Furin JJ et al.; SETTING: A community-based treatment program for multidrug-resistant tuberculosis (MDR-TB) in an urban shantytown of Lima, Peru . OBJECTIVES: To ascertain the occurrence of serious adverse effects associated with therapy for MDR-TB in northern Lima, Peru, where therapy was individualized according to drug-susceptibility testing of patients' infecting strains and delivered through a community-based program . DESIGN: A retrospective record review of 60 patients who had received individualized therapy for MDR-TB between September 1996 and October 1998 . RESULTS: Although adverse effects were common, they occurred less frequently than previously reported in the literature and were rarely life-threatening . Effects occurring most frequently in this population included: mild gastritis (100%), dermatological effects (43.3%), peripheral neuropathy (16.7%), depression (18.3%), and anxiety (11.7%) . These effects never resulted in the discontinuation of anti-tuberculosis therapy, and only occasionally resulted in the suspension of an agent (11.7%) . CONCLUSION: In young patients with little comorbid disease, multidrug, long-course regimens rarely caused life-threatening adverse effects . Common side effects may be managed successfully on an out-patient basis through a community-based treatment program in conjunction with MDR-TB experts, even in resource-poor settings . The very low rate of default in this cohort offers hope that strategies to manage the adverse effects may reduce the incidence of abandonment of therapy and increase rates of cure.

Biol Reprod, 2001 Aug, 65(2), 394 - 400
Adenosine triphosphate-binding cassette superfamily transporter gene expression in severe male infertility; Larriba S et al.; Cystic fibrosis transmembrane regulator (CFTR), multidrug-resistant (MDR)1, and multidrug resistance-associated (MRP) proteins belong to the ATP-binding cassette (ABC) transporter superfamily . A compensatory regulation of MDR1 and CFTR gene expression has been observed in CFTR knockout rodent intestine and in an epithelial cell line of human colon, whereas a high homology and similar anion binding site are shared by MRP and CFTR proteins . To provide better insight into the relationship among the expression behavior in vivo of the three genes in human testis, analysis of MDR1 and MRP gene expression in testicular biopsies was performed and related to the presence of CFTR gene mutations in congenital absence of the vas deferens (CAVD: n = 20) and non-CAVD (n = 30) infertile patients with azoospermia or severe oligozoospermia . A CFTR mutation analysis performed in both groups of patients supported the involvement of CFTR gene mutations in CAVD phenotype (85%) and in defective spermatogenesis (19%) . Quantitative reverse transcription-polymerase chain reaction analysis of testicular tissue showed a CFTR-independent MDR1 and MRP gene expression in human testis, suggesting that the mechanisms underlying CFTR gene regulation in testis are different from those in intestine . These findings should contribute to the understanding of patterns of in vivo expression of CFTR, MDR1, and MRP genes in CFTR-related infertility.

Curr Drug Targets, 2001 Mar, 2(1), 57 - 77
Signal transduction pathways and transcriptional mechanisms as targets for prevention of emergence of multidrug resistance in human cancer cells; Shtil AA; Pleiotropic resistance of tumor cells to treatment remains one of the major obstacles for successful cure of cancer patients . Tumor cells may acquire multidrug resistance (MDR) in the course of exposure to various compounds that are used in modern anticancer therapy, including cytotoxic drugs and differentiating agents . Therefore, the recurrence of the disease after the initial treatment may be associated with establishment of secondary MDR in the residual tumor . This phenotype is frequently mediated by P-glycoprotein, an ATP-dependent transmembrane pump capable of effluxing numerous compounds out of the cell . In humans, P-glycoprotein is encoded by the MDR1 gene . Rapid increase of the steady-state level of the MDR1 mRNA in response to stress stimuli is the mechanism of acquisition of P-glycoprotein-mediated MDR in cancer cells . Thus, up-regulation of the MDR1 gene is regarded as part of cellular stress response . This review shows that block of mechanisms that regulate the MDR1 overexpression can prevent the emergence of MDR in tumor cells that expressed null-to-low levels of MDR1 mRNA or P-glycoprotein prior to treatment . In particular, the MDR1 activation can be abrogated by targeting cytoplasmic pathways of signal transduction as well as by interfering with transcriptional up-regulation.

Chemistry, 2001 Jun 18, 7(12), 2663 - 70
Total synthesis of agosterol A: an MDR-modulator from a marine sponge; Murakami N et al.; The first total synthesis of agosterol A, a modulator of multidrug resistance (MDR) mediated by P-gp and MRP1, and isolated from a marine sponge, was achieved from ergosterol by utilizing a regioselective epoxy-cleavage reaction and regioselective dehydroxylation as the key reactions.

Pharm Res, 2001 May, 18(5), 565 - 72
Expression of multidrug resistance-associated protein (MRP) in human retinal pigment epithelial cells and its interaction with BAPSG, a novel aldose reductase inhibitor; Aukunuru JV et al.; PURPOSE: The objective of this study was to determine the expression and activity of multidrug resistance-associated protein (MRP) in the retinal pigment epithelial (RPE) cells and to further assess whether BAPSG, a novel anionic aldose reductase inhibitor, interacts with MRP . METHODS: Functional and biochemical evidence for MRP was obtained in a human retinal pigment epithelial (ARPE-19) cell line and primary cultures of human retinal pigment epithelial (HRPE) cells . Fluorescein accumulation and efflux in the presence and absence of MRP inhibitors was used to obtain functional evidence for MRP . Western blots and RT-PCR were used to obtain biochemical evidence for MRP1 . The influence of MRP inhibitors on BAPSG accumulation and efflux in ARPE-19 cells was determined to understand its interaction with MRP . RESULTS: MRP inhibitors increased fluorescein accumulation and reduced efflux in RPE cells . Both cell types exhibited a 190-kDa western blot band corresponding to MRP1 protein and a 287 bp RT-PCR band corresponding to MRP1 mRNA . MRP inhibitors reduced BAPSG efflux and increased its accumulation in ARPE-19 cells . CONCLUSIONS: MRP is functionally and biochemically active in human RPE cells . Anionic BAPSG is a likely substrate for MRP.

Biochem J, 2001 Aug 1, 357(Pt 3), 859 - 65
Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor; Raggers RJ et al.; The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed . Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell . In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane . We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF) . PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine . {(14)C}PAF synthesized intracellularly from exogenous alkylacetylglycerol and {(14)C}choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport . Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A . Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1) . It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity . While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

Mol Membr Biol, 2001 Apr-Jun, 18(2), 145 - 52
The multi-structural feature of the multidrug resistance gene product P-glycoprotein: implications for its mechanism of action (hypothesis); Zhang JT; P-glycoprotein is a member of the ATP-binding cassette (ABC) transport superfamily . It plays an important role in the development of multidrug resistance in cancers by effluxing a wide variety of anticancer drugs . A large amount of information on the structure and function of P-glycoprotein has been accumulated over recent years from studies using molecular, biochemical, and biophysical approaches . It remains unclear, however, how this protein folds in membranes and how it transports such a wide variety of hydrophobic compounds . This paper highlights the recent progress in the structural and biogenesis aspects of P-glycoprotein . A model mechanism of P-glycoprotein action is proposed as a hypothesis that is based on recent progress in studying the topological folding of P-glycoprotein.

IUBMB Life, 2001 Feb, 51(2), 121 - 6
Intra- and intercellular distribution of mitochondrial probes and changes after treatment with MDR modulators; Diaz G et al.; Fluorescent probes are currently used to evaluate the mitochondrial transmembrane potential in situ . However, in parallel experiments using the probes JC-1 and TMRM in different cell types (human astrocytes, HEp-2, Vero, KB, and HeLa cells), we found that the distribution of JC-1 and TMRM is highly variable not only in different cell types but also in different cells of the same cell type, a condition that has never been documented until our work . This phenomenon depends on a hidden, widespread multidrug resistance (MDR) phenotype that can be recognized only by comparative assays with MDR inhibitors (progesterone, verapamil, and cyclosporin A) and represents a serious risk of error in the evaluation of the mitochondrial potential.

Am J Trop Med Hyg, 2001 May-Jun, 64(5-6), 247 - 56
A clinical and pharmacokinetic trial of six doses of artemether-lumefantrine for multidrug-resistant Plasmodium falciparum malaria in Thailand; Lefevre G et al.; The efficacy-safety and pharmacokinetics of the six-dose regimen of artemether-lumefantrine (Coartem/Riamet; Novartis Pharma AG, Basel, Switzerland) were assessed in a randomized trial in 219 patients (> or = 12 years old) with acute, uncomplicated Plasmodium falciparum malaria in Thailand . One hundred and sixty-four patients received artemether-lumefantrine and 55 received the standard treatment combination of mefloquine-artesunate . Both drugs induced rapid clearance of parasites and malaria symptoms . The 28-day cure rates were 95.5% (90% confidence interval {CI} = 91.7, 97.9%) for artemether-lumefantrine and 100% (90% CI = 94.5, 100%) for mefloquine-artesunate . This high-dose regimen of artemether-lumefantrine was very well tolerated, with very good compliance . The most frequent adverse events were headache, dizziness, nausea, abdominal pain, dyspepsia, vomiting, and skin rash . Overall, only 2% of patients in both groups showed QTc prolongations but without any cardiac complication, and no differences were seen between patients with and without measurable baseline plasma levels of quinine or mefloquine . Plasma levels of artemether, dihydroartemisinin, and lumefantrine were consistent with historical data for the same dose regimen, and were higher, particularly for lumefantrine, than those previously observed with the four-dose regimen, explaining the greater efficacy of the six-dose regimen in a drug-resistant setting . These results confirm the excellent safety and efficacy of the six-dose regimen of artemether-lumefantrine in the treatment of multidrug-resistant P . falciparum malaria.

N Engl J Med, 2001 Jul 19, 345(3), 170 - 4
The treatment of multidrug-resistant tuberculosis in Turkey; Tahaoglu K et al.; BACKGROUND: We evaluated the results of treatment in 158 consecutive patients with multidrug-resistant tuberculosis who were treated at our center in Istanbul . METHODS: A total of 21 female patients and 137 male patients (age range, 15 to 68 years) received treatment for multidrug-resistant tuberculosis between March 1992 and October 1999 . The patients had previously received a mean of 5.7 antituberculosis drugs and were infected with organisms that were resistant to a mean of 4.4 drugs . All patients were infected with organisms that were resistant to both isoniazid and rifampicin . The regimens we used were selected on the basis of previous treatment protocols and the results of susceptibility tests . All patients received at least three drugs thought to be active; the treatment was continued for at least 18 months after the conversion to a negative culture and for at least 24 months in the absence of first-line drugs . RESULTS: The mean number of drugs given during the study was 5.5 (range, 3 to 9) . Surgical resection was performed in 36 patients . Adverse effects led to discontinuation of one or more drugs in 62 patients (39 percent) . Cultures became negative in 150 patients (95 percent) after a mean of 1.9 months (range, 1 to 9) . The overall success rate of treatment was 77 percent, with cures in 78 patients (49 percent) and probable cures in 43 (27 percent) . Treatment failed in 13 patients (8 percent) . Seven patients died (4 percent) . Seventeen patients (11 percent) did not complete the treatment regimen . The patients with unsuccessful outcomes were older than those with successful outcomes (mean age, 42 years vs . 36 years; P=0.008), had received a larger number of drugs previously (median, six vs . five; P=0.048), were more likely to have been treated previously with ofloxacin (57 percent vs . 30 percent, P=0.004), and were less likely to have received ofloxacin as part of the study protocol (65 percent vs . 84 percent, P=0.018) . Thirty-eight percent of the patients with unsuccessful outcomes were infected with organisms that were resistant to more than five drugs . In a step-down logistic-regression analysis, a successful outcome was independently associated with a younger age (P=0.013) and the absence of previous treatment with ofloxacin (P=0.005) . CONCLUSIONS: Most patients with multidrug-resistant tuberculosis can be cured with the use of appropriate, intensive treatment regimens.

Eur J Vasc Endovasc Surg, 2001 Jul, 22(1), 31 - 6
Lipid metabolism and molecular changes in normal and atherosclerotic vessels; Petruzzo P et al.; OBJECTIVES: a positive correlation between cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT), multidrug resistance (MDR1) gene expression and atherosclerotic lesions has been shown in human arteries . The objective of this study was to map the expression of MDR1, ACAT genes and the cholesteryl ester content in normal, atherosclerotic and varicose human vessels . MATERIALS: vascular segments were obtained from seven cadaveric donors, 27 patients undergoing vascular surgery for severe atherosclerotic disease and 11 patients with saphenous vein varicosities . METHODS: lipid analysis and RT-PCR of MDR1 and ACAT mRNAs were performed . RESULTS: an increase in cholesteryl ester content and in ACAT and MDR1 expression was demonstrated in relation to the age in the arteries prone to atherosclerosis; this expression was maximal in arteries from symptomatic patients . In resistant arteries and in veins cholesteryl ester accumulation was rare and light, while ACAT and MDR1 expression was not related to the age of the subjects . CONCLUSIONS: the results showed that an increase in MDR1 and ACAT expression may be responsible for the accumulation of cholesteryl esters as well as for cell growth rate acceleration in vessel sites prone to atherosclerosis .

Mar Environ Res, 2000 Jul-Dec, 50(1-5), 319 - 23
Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis; McFadzen I et al.; Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux . This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay . The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages . The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater . Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil . Larval growth was also significantly impaired in the presence of verapamil . Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive . This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.

Anticancer Drugs, 2001 Jul, 12(6), 549 - 54
Relationship between cytocidal activity and glutathione-S-transferase inhibition using doxorubicin coupled to stereoisomers of glutathione with different substrate specificity; Hashizume Y et al.; To determine the cytotoxic mode of action of a glutathione (GSH)--doxorubicin (DXR) conjugate, which exhibited potent cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH--DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated . The following four GSH molecules with stereoisomeric forms were prepared: L-Glu--L-Cys--Gly (LL-GSH), D-Glu--L-Cys--Gly (DL-GSH), L-Glu--D-Cys--Gly (LD-GSH) and D-Glu--D-Cys--Gly (DD-GSH) . The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST . Addition of DXR conjugated with each isomer (10 microM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH--XR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control . Moreover, IC50 values for these conjugates were 30, 20 and 250 nM, respectively . The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer . These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH--XR, DL-GSH-DXR and LD-GSH--XR, respectively . These findings suggested that GSH--DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity.

Bioconjug Chem, 2001 Jul-Aug, 12(4), 523 - 8
New TFO conjugates containing a carminomycinone-derived chromophore; Capobianco ML et al.; Conjugates obtained by linking the anthracycline intercalating chromophore to triple helix forming oligonucleotides (TFOs) have been used in a physicochemical study of the stability of triple helices with DNA sequences of pharmacological relevance . The intercalating moiety is represented by carminomycinone derivatives obtained upon O-demethylation and hydrolysis of the glycosidic linkage of daunomycin followed by the introduction of an alkylating residue at two different positions . Results of experiments with a polypurinic region present in the multidrug resistance (MDR) gene indicate that the stability of the triple helix is significantly enhanced by replacement of C's with (5-Me)C's in the TFO sequences tested . The stability is not changed when a 3'-TpT is present in place of a 3'-CpG at the presumed intercalation site of the anthraquinone chromophore . The same carminomycinone derivatives were used for the preparation of conjugates able to form triple helices with the polypurine tract (PPT) present in the human integrated genome of HIV-1 infected cells . Three different TFOs (T(4)(Me)CT(4)(Me)CC, C2; T(4)(Me)CT(4)(Me)CC(Me)CC(Me)CCT, C6; and T(4)(Me)CT(4)G(6), G6) were designed and linked to the anthraquinone moiety . These conjugates showed a significantly enhanced ability to bind the PPT region of HIV with respect to the nonconjugated TFOs.

Eur J Pharm Sci, 2001 Aug, 14(1), 29 - 36
SB203580, a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance; Barancik M et al.; P-glycoprotein (P-gp) is the plasma membrane transport pump responsible for efflux of chemotherapeutic agents from cells and is one of the systems that secures multidrug resistance (MDR) of neoplastic cells . In the present study, drug sensitive L1210 and multidrug resistant L1210/VCR (characterized by overexpression of P-gp) mouse leukemic cell lines were used as an experimental model . We have found that SB203580, a specific inhibitor of p38-MAPK pathway, significantly reduced the degree of the vincristine resistance in L1210/VCR cells . This phenomenon was accompanied by a decrease in the LC(50) value of vincristine from 3.203+/-0.521 to 0.557+/-0.082 microM . The LC(50) value of sensitive cells for vincristine was about 0.011 microM . The effect of SB203580 on L1210/VCR cells was associated with significantly increased intracellular accumulation of {3H}-vincristine in the concentration dependent manner . Prolonged exposure of resistant cells to 30 microM SB203580 did neither significantly influence the gene expression of P-gp, nor change the protein levels of p38-MAPK . Western blot analysis revealed that the MDR phenotype in L1210/VCR cells was associated with increased level and activity of cytosolic p38-MAPK . In resistant cells, the enhanced phosphorylation of both, p38-MAPK and ATF-2 (endogenous substrate for p38-MAPK) was found as well . In conclusion we could remark that SB203580, an inhibitor of p38 kinase pathway, reversed the MDR resistance of L1210/VCR cells . MDR phenotype of these cells is connected with increased levels and activities of p38-MAPK . These findings point to the possible involvement of the p38-MAPK pathway in the modulation of P-gp mediated multidrug resistance in the L1210/VCR mouse leukemic cell line . However, the mechanisms of SB203580 action should be further investigated.

Mol Pharmacol, 2001 Aug, 60(2), 302 - 9
Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic; Liu J et al.; Recent work shows that long-term exposure to low levels of arsenite induces malignant transformation in a rat liver epithelial cell line . Importantly, these chronic arsenic-exposed (CAsE) cells also develop self-tolerance to acute arsenic exposure . Tolerance is accompanied by reduced cellular arsenic accumulation, suggesting a mechanistic basis for reduced arsenic sensitivity . The present study examined the role of xenobiotic export pumps in acquired arsenic tolerance . Microarray analysis of CAsE cells showed increased expression of the genes encoding for glutathione S-transferase Pi (GST-Pi), multidrug resistance-associated protein genes (MRP1/MRP2, which encode for the efflux transporter Mrp1/Mrp2) and the multidrug resistance gene (MDR1, which encodes for the efflux transporter P-glycoprotein) . These findings were confirmed at the transcription level by reverse transcription-polymerase chain reaction and at the translation level by Western-blot analysis . Acquired arsenic tolerance was abolished when cells were exposed to ethacrynic acid (an inhibitor of GST-Pi), buthionine sulfoximine (a glutathione synthesis inhibitor), MK571 (a specific inhibitor for Mrps), and PSC833 (a specific inhibitor for P-glycoprotein) in dose-dependent fashions . MK571, PSC833, and buthionine sulfoximine markedly increased cellular arsenic accumulation . Consistent with a role for multidrug resistance efflux pumps in arsenic resistance, CAsE cells were found to be cross-resistant to cytotoxicity of several anticancer drugs, such as vinblastine, doxorubicin, actinomycin-D, and cisplatin, that are also substrates for Mrps and P-glycoprotein . Thus, acquired tolerance to arsenic is associated with increased expression GST-Pi, Mrp1/Mrp2 and P-glycoprotein, which function together to reduce cellular arsenic accumulation.

Mol Pharmacol, 2001 Aug, 60(2), 254 - 61
Transmembrane domain (TM) 9 represents a novel site in P-glycoprotein that affects drug resistance and cooperates with TM6 to mediate {125I}iodoarylazidoprazosin labeling; Song J et al.; The multidrug resistant cell line DC-3F/ADII was obtained by stepwise selection for growth in actinomycin D (ActD) . Compared with parental cells, it displays high resistance to ActD and vincristine and low resistance to colchicine and daunorubicin . These cells overexpress a form of P-glycoprotein (Pgp1) containing a double mutation, I837L and N839I, in transmembrane domain (TM) 9; when transfected into DC-3F, this mutation confers the DC-3F/ADII phenotype . We have shown previously that another cell line, DC-3F/ADX, also displays this phenotype and overexpresses a mutant form of Pgp1 containing a double mutation in TM6 (G338A, A339P) . Hence, mutations in TM9 and TM6 are independently capable of conferring the same cross-resistance phenotype . The TM6 mutations inhibit the ability of cyclosporin A to reverse cross-resistance and to block labeling of the protein by {125I}iodoarylazidoprazosin (IAAP), whereas the TM9 mutations do not show similar effects . A chimeric protein containing both pairs of mutations confers twice the level of resistance to ActD than expected from the sum of the individual mutations, but it cannot be labeled to detectable levels with {125I}IAAP . Thus, TM9 represents a novel site that cooperates with TM6 to mediate drug resistance and {125I}IAAP labeling.

J Biol Chem, 2001 Sep 14, 276(37), 35194 - 200 Epub 2001 Jul 13.
Novel low molecular weight spirodiketopiperazine derivatives potently inhibit R5 HIV-1 infection through their antagonistic effects on CCR5; Maeda K et al.; Novel low molecular weight spirodiketopiperazine derivatives which potently inhibit R5 human immunodeficiency virus type 1 (HIV-1) infection through their antagonistic effects on CCR5 were identified . One such compound E913 (M(r) 484) specifically blocked the binding of macrophage inflammatory protein-1alpha (MIP-1alpha) to CCR5 (IC(50) 0.002 microm) and MIP-1alpha-elicited cellular Ca(2+) mobilization (IC(50) approximately 0.02 microm) . E913 potently inhibited the replication of laboratory and primary R5 HIV-1 strains as well as various multidrug-resistant monocyte/macrophage tropic (R5) HIV-1 at IC(50) values of 0.03 to 0.06 microm . E913 was inactive against T cell tropic (X4) HIV-1; however, when combined with a CXCR4 antagonist AMD-3100, E913 potently and synergistically inhibited the replication of dualtropic HIV-1 and a 50:50 mixture of R5 and X4 HIV-1 . Antagonism in anti-HIV-1 activity was not seen when E913 was combined with the reverse transcriptase inhibitor zidovudine or protease inhibitors . E913 proved to compete with the binding of antibodies to CCR5 which recognize the C-terminal half of the second extracellular loop (ECL2B) of CCR5 . E913 and its analogs are acid-resistant and orally bioavailable in rodents . These data warrant that spirodiketopiperazine derivatives be further developed as potential therapeutics for HIV-1 infection.

Drug Metab Dispos, 2001 Aug, 29(8), 1080 - 3
Evaluation of the interaction of loratadine and desloratadine with P-glycoprotein; Wang EJ et al.; The absorption of many drugs is affected by their interaction with ATP-binding cassette (ABC) transporters . The most extensively studied of these ABC transporters is the proein product of MDR1 (multidrug resistance) that encodes a 170-kDa integral plasma membrane phosphorylated glycoprotein known as P-glycoprotein (P-gp) . The purpose of this study was to determine, using two different methods, whether the nonsedating antihistamine loratadine (L) and its active metabolite desloratadine (DL) interact with P-gp . MDR cells presenting human P-gp were incubated with the fluorescent P-gp substrate daunorubicin with or without L, DL, and several positive controls . The IC(50) of loratadine (approximately 11 microM) was approximately 160 times the maximum observed plasma concentration (C(max)) following a dose of 10 mg . The IC(50) of desloratadine (approximately 43 microM) was approximately 880 times the C(max) following a dose of 5 mg . The positive control, cyclosporin A, had an IC(50) of approximately 1 microM . ATP hydrolysis activity was measured in the membrane fraction prepared from MDR cells presenting P-gp, which were exposed to various concentrations of test compounds . Known substrates of P-gp demonstrated clear, repeatable, concentration-dependent increases in ATP hydrolysis activity . L caused an increase in ATPase activity above basal levels . L had a V(max) about 200% basal activity and K(m) of approximately 3 microM for P-gp . In contrast, DL had no significant effect on baseline ATP hydrolysis . L inhibited human P-gp much less than verapamil or cyclosporin A . DL inhibited human P-gp significantly less than L (4 times) . DL therefore is not a significant inhibitor of P-gp and should not cause clinical drug interactions with agents that are P-gp substrates.

Cancer Res, 2001 Jul 15, 61(14), 5461 - 7
Resistance to mitoxantrone in multidrug-resistant MCF7 breast cancer cells: evaluation of mitoxantrone transport and the role of multidrug resistance protein family proteins; Diah SK et al.; We examined the role of multidrug resistance protein (MRP) 1 (ABCC1) in the emergence of mitoxantrone (MX) cross-resistance in a MCF7 breast cancer cell line selected for resistance to etoposide . The resistant cell line, MCF7/VP, expresses high levels of MRP1, whereas the parental cell line, MCF7/WT, does not . MCF7/VP cells are 6-10-fold cross-resistant to MX when compared with MCF7/WT cells . Drug transport studies in intact MCF7/VP cells revealed that MX resistance is associated with reduced MX accumulation due to enhanced MX efflux . MX efflux is ATP dependent and inhibited by sulfinpyrazone and cyclosporin A . Inhibition of MX efflux with these agents sensitizes cells to MX cytotoxicity and partially reverses MX resistance in MCF7/VP cells . Whereas resistance is partially attributable to increased MX efflux in MRP1-expressing MCF7/VP cells, we found no evidence for glutathione or other conjugates of MX in these cells . Moreover, glutathione depletion with buthionine sulfoximine had no effect on MX transport or sensitivity in MCF7/VP cells . MRP1 substrates are generally amphiphilic anions such as glutathione conjugates or require the presence of physiological levels of glutathione for MRP1-mediated transport . Therefore we conclude that MRP1 overexpression is unlikely to be responsible for increased MX efflux and resistance in MCF7/VP cells . In considering the potential involvement of other MRP family isoforms, a 3-fold increase in the expression of MRP5 was observed in MCF7/VP cells . However, stable expression of a transduced MRP5 expression vector in MCF7/WT cells failed to confer MX resistance . Because other transporters known to be associated with MX resistance, including P-glycoprotein and BCRP/MXR (ABCG2), are not expressed in MCF7/VP cells, we conclude that increased MX efflux and resistance in MCF7/VP cells is attributable to a novel transport mechanism or that MX represents a novel class of cationic, glutathione-independent MRP1 substrates.

Liver, 2001 Aug, 21(4), 247 - 53
Expression of hepatic transporters OATP-C and MRP2 in primary sclerosing cholangitis; Oswald M et al.; BACKGROUND/AIMS: In chronic cholestatic liver diseases, biliary excretion of organic anions from blood into bile is impaired . The aim of this study was to identify the underlying mechanism . METHODS: Expression of the basolateral organic anion transporting polypeptide OATP-C (SLC21A6) and the canalicular multidrug resistance protein 2 (MRP2) was studied in patients with primary sclerosing cholangitis (PSC) (n=4), a chronic cholestatic liver disease, and in non-cholestatic controls (n=4) (two with chronic hepatitis C, one with idiopathic liver cirrhosis and one with fatty liver) . Total RNA was isolated from liver tissue, reverse transcribed and subjected to polymerase chain reaction (PCR) amplification using primers specific for OATP-C, MRP2 and beta-actin . PCR products were quantified densitometrically . RESULTS: When normalized for beta-actin expression, the level of OATP-C mRNA in liver tissue of patients with PSC was 49% of controls (OATP-C/beta-actin 1.60+/-0.25 vs . 3.24+/-0.69; p<0.05) and the level of MRP2 mRNA was 27% of controls (MRP2/beta-actin 0.70+/-0.36 vs . 2.54+/-0.56; p<0.01) . CONCLUSIONS: Both OATP-C and MRP2 are decreased as measured by mRNA level in PSC . Downregulation of OATP-C might be the consequence of impaired canalicular secretion of organic anions and could serve to reduce the organic anion load of cholestatic hepatocytes.

Pediatr Hematol Oncol, 2001 Jul-Aug, 18(5), 325 - 34
Effect of P-glycoprotein expression on outcome in the Ewing family of tumors; Perri T et al.; This study was designed to determine the prognostic significance of multidrug resistance, mediated by P-glycoprotein (Pgp) expression, in Ewing sarcoma . The clinical and laboratory features, treatment protocol, and outcome of 75 patients with Ewing sarcoma or peripheral neuroectodermal tumor treated between 1972 and 1997 were reviewed . Pgp expression was tested with the monoclonal antibody JSB-1 . Thirty-four (64%) of the 53 tissue samples from untreated patients stained positive for Pgp . Progression-free and overall survival were 44 and 59%, respectively, in patients with negative findings, and 28 and 41% in those with positive findings; neither difference was significant . Of the 12 relapsed patients, 6 (50%) expressed more Pgp after chemotherapy than at diagnosis and 4 (33%) expressed less . Within these subgroups, 5 out of 6 and 3 out of 4 died from the disease . No correlation was found between Pgp and known prognostic factors of Ewing tumors . Pgp expression is probably an intrinsic factor of Ewing tumors but has no correlation to prognosis.

Aquat Toxicol, 2001 Sep, 54(1-2), 29 - 38
7-Ethoxyresorufin O-deethylase induction in rainbow trout gill epithelium cultured on permeable supports: asymmetrical distribution of substrate metabolites; Carlsson C et al.; The induction of 7-ethoxyresorufin O-deethylase (EROD) has been measured in cultured epithelia from rainbow trout gills . Epithelia incubated with water on the apical side and culture media at the basolateral side were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (betaNF), benzo{k}fluoranthene (B(k)F), and 3,3',4,4',5-pentachlorobiphenyl (PCB#126) from the water . EROD activity was measured as the formation of resorufin from 7-ethoxyresorufin over time in intact epithelia . The EC(50) values obtained after 24 h of exposure (mean+/-S.D.) were for TCDD (n=9) 4.1+/-3.2x10(-11) M, for betaNF (n=6) 1.6+/-3.8x10(-9) M, for B(k)F (n=4) 5.4+/-3.0x10(-9) M and for PCB#126 (n=4) 6.15+/-10.1x10(-9) M . When assaying for EROD activity, it was found that the resorufin concentrations differed between the apical and the basolateral compartments, indicating an asymmetrical distribution of the enzymatically formed resorufin molecules . Generally, the resorufin concentration was highest in the basolateral compartment, but there were differences between epithelia obtained from different fish individuals . Of a total of 13 preparations 10 had the highest resorufin concentration in the basolateral compartment, while in three preparations, the resorufin was uniformly distributed or slightly higher in the apical compartment . The reasons for this asymmetrical distribution of substrate metabolites are not known, and the addition of multidrug resistance inhibitors (verapamil and cyclosporin A) did not alter the asymmetrical pattern . The transepithelial electrical resistance (TER) was also measured to diagnose the tightness of the epithelia . The change from culture media to experimental water (containing TCDD, betaNF, or DMSO as control) in the apical compartment resulted in a large increase in TER, followed by a decline, measured after 24 h . The cytochrome P450 1A (CYP1A) inducers had no effect on the TER and were judged, therefore, not to affect the tightness of the epithelia.

J Hepatol, 2001 Jun, 34(6), 881 - 7
Effect of phenobarbital on the expression of bile salt and organic anion transporters of rat liver; Hagenbuch N et al.; BACKGROUND/AIMS: The hepatic clearance of drugs and cholephilic organic anions is stimulated by phenobarbital (PB) . Our aim was to analyze the effects of PB on the expression of hepatocellular bile salt and organic anion transporters . METHODS: Male Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg/d) or saline for 5 days . Transporter expression was quantified by northern and western blot analysis and initial uptake rates of bromosulphophthalein (BSP) and digoxin were measured in isolated hepatocytes . RESULTS: Compared to control rats, PB treatment increased expression of the organic anion transporting polypeptide 2 (Oatp2; Slc21aS) more than 2-fold on the RNA (P < 0.05) and protein (P < 0.001) levels . Expression of Oatpl (Slc21al), Oatp4 (Slc21a6) and the Na+-taurocholate cotransporting polypeptide (Ntcp; Slc10a1) was unaltered . At the canalicular pole, expression of the bile salt export pump (Bsep; ABCB11) and of the multidrug resistance proteins 2 (Mrp2; ABCC2) and 6 (Mrp6; ABCC6) was not significantly changed . Whereas hepatocellular BSP uptake was unaffected by PB, digoxin uptake was stimulated 4-fold . CONCLUSIONS: The induction of digoxin uptake by PB correlates with Oatp2 expression . In contrast, the lack of increase of Oatpl and Oatp4 expression is in accordance with unchanged BSP uptake . These data challenge the previously held view that PB induces hepatocellular BSP uptake systems.

Pharm Res, 2001 Apr, 18(4), 432 - 8
Inhibition of P-glycoprotein transport function by grapefruit juice psoralen; Wang EJ et al.; PURPOSE: The grapefruit juice component bergamottin is known to inactivate cytochrome P450 3A4, with grapefruit juice consumption causing increased absorption and enhanced oral bioavailability of many cytochrome P450 3A4 substrates . Many of these substrates are also recognized by the efflux transporter P-glycoprotein . The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics . METHODS: Using a whole ceil assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of grapefruit juice components to inhibit the function of this transporter . RESULTS: In a cell line presenting an overexpressed amount of the human transporter, the enzyme exhibited a 40 microM IC50 for inhibition by bergamottin . Additionally, using the ATP-hydrolysis assay, we showed that bergamottin increases P-gp-mediated ATP hydrolysis by approximately 2.3 fold with a Km of 8 microM . The concentration for this interaction is similar to that for CYP3A4 inactivation . CONCLUSIONS: These results suggest that observed grapefruit juice drug pharmacokinetic clinical interactions may be due to P-gp inhibition rather than or in addition to CYP3A4 inhibition . Inhibition of P-gp by citrus psoralens could present ways both to enhance bioavailability of therapies without increasing the dose and to diminish drug resistance in refractory cells.

Scand J Infect Dis, 2001, 33(6), 466 - 9
Early bactericidal activity of amoxicillin in combination with clavulanic acid in patients with sputum smear-positive pulmonary tuberculosis; Donald PR et al.; The early bactericidal activity (EBA) of an antituberculosis agent is the rate of decrease in viable colony-forming units (CFU) per milliliter of sputum during the first 2 d of treatment of patients with previously untreated smear-positive pulmonary tuberculosis . The objective of this open randomized study was to evaluate the EBA of the combination of amoxicillin 3 g and clavulanic acid 750 mg . Ten patients with a mean age of 34 y and a mean weight of 56 kg received amoxicillin/clavulanic acid and 5 patients with a mean age of 34 y and a mean weight of 57 kg received no drug . In the patients receiving 1 dose of amoxicillin/clavulanic acid daily for 2 d the mean log10CFU/ml of sputum before treatment was 6.7402 (SD 0.539) and after 2 d of treatment 6.7046 (SD 0.609); the corresponding values in patients receiving no drug were 6.7823 (SD 0.563) and 6.7502 (SD 0.673), respectively . The EBA of 0.018 (SD 0.130) in patients receiving amoxicillin/clavulanic acid did not differ significantly from that of 0.016 (SD 0.069) in patients receiving no drug . It is unlikely that the combination of amoxicillin/clavulanic acid has an important place in the treatment of tuberculosis with the exception of those patients with multidrug-resistant tuberculosis who are otherwise therapeutically destitute.

Biochem Pharmacol, 2001 Aug 15, 62(4), 417 - 24
Verapamil-stimulated glutathione transport by the multidrug resistance-associated protein (MRP1) in leukaemia cells; Cullen KV et al.; Multidrug resistance mediated by the multidrug resistance-associated protein MRP1 is associated with decreased drug accumulation, which is in turn dependent on cellular glutathione . We have reported that verapamil, an inhibitor of drug transport, caused a decrease in cellular glutathione in CCRF-CEM/E1000 MRP1-overexpressing leukaemia cells (Biochem Pharmacol 55;1283--9, 1998) . We now demonstrate that other inhibitors of MRP1-mediated drug transport (e.g . MK571, indomethacin, genistein, and nifedipine) deplete cellular glutathione in these leukaemia cells (>30% decrease; P < 0.01) while having no effect on the parental CCRF-CEM cells . However, treatment with etoposide or vincristine (at similar molar concentrations) caused a 20% decrease in glutathione . Verapamil-stimulated glutathione transport correlated with MRP1 expression in a series of drug-resistant cells, and glutathione was quantitatively recovered in the extracellular media . Further, verapamil-stimulated glutathione transport was rapid (50% decrease in 10 min), dose-dependent, and inhibited by vanadate, an inhibitor of ATPase activity, but not by sulphobromophthalein (BSP) or methionine, inhibitors of hepatic glutathione transporters . Incubation of CCRF-CEM/E1000 cells in 25 mM glutathione not only showed that verapamil-mediated efflux occurred against the concentration gradient, but also demonstrated the MRP1-mediated uptake of glutathione (P < 0.01 compared to the parental CCRF-CEM cells), which was not inhibited by vanadate . These results demonstrate that while MRP1 transports glutathione in the presence of inhibitors of drug transport, there is no convincing evidence for co-transport of glutathione with drug . They further demonstrate that MRP1 mediates the facilitated transport of glutathione into the MRP1-overexpressing CEM/E1000 cells, suggesting that MRP1 may play a major role in cellular glutathione homeostasis.

J Biol Chem, 2001 Sep 7, 276(36), 33747 - 54 Epub 2001 Jul 10.
Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4 . Resistance to 6-mercaptopurine and 6-thioguanine; Chen ZS et al.; Human multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate . However, neither its substrate selectivity nor physiological functions have been determined . Here we report the results of investigations of the in vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus . It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP, and estradiol 17-beta-D-glucuronide (E(2)17 beta G) . cGMP, cAMP, and E(2)17 beta G are transported with K(m) and V(max) values of 9.7 +/- 2.3 microm and 2.0 +/- 0.3 pmol/mg/min, 44.5 +/- 5.8 microm and 4.1 +/- 0.4 pmol/mg/min, and 30.3 +/- 6.2 microm and 102 +/- 16 pmol/mg/min, respectively . Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs . On the basis of its capacity to transport cyclic nucleotides and E(2)17 beta G, it is concluded that MRP4 may influence diverse cellular processes regulated by cAMP and cGMP and that its substrate range is distinct from that of any other characterized MRP family member.

Int J Oncol, 2001 Aug, 19(2), 367 - 71
Differential expression of the multidrug resistance-related protein MRP1 in the histological compartments of nephroblastomas; Efferth T et al.; Nephroblastomas (Wilms' tumors) are curable with survival rates above 80% . Some tumors, however, fail to respond to therapy and those patients have a poor prognosis . In a search for prognostic markers, we investigated the expression of the multidrug resistance-related protein 1 (MRP1) in 32 nephroblastomas by means of immunohistochemistry . The immunohistochemical results were validated with a real-time RT-PCR technique . MRP1 expression was heterogeneous and predominantly found in the blastemal and epithelial compartments compared to the stromal elements of nephroblastomas . We found significant relationships of MRP1 expression to survival of patients and to expression of p53, HSP70, and LRP/MVP . The relationship between MRP1 and p53 expression is a clue that the transcriptional control of MRP1 by p53 reported for other tumor types may also take place in nephroblastomas . The correlation of MRP1 to other drug resistance genes, e.g . HSP70 and LRP/MVP in nephroblastomas indicates that the co-expression of different drug resistance genes may be under a common regulation of still unknown transcription factors.

Pharmacotherapy, 2001 Jul, 21(7), 778 - 96
Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation; Matheny CJ et al.; P-glycoprotein (P-gp) is a cell membrane-associated protein that transports a variety of drug substrates . Although P-gp has been studied extensively as a mediator of multidrug resistance in cancer, only recently has the role of P-gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined . P-glycoprotein is present in organ systems that influence drug absorption (intestine), distribution to site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues . Many marketed drugs inhibit P-gp function, and several compounds are under development as P-gp inhibitors . Similarly, numerous drugs can induce P-gp expression . While P-gp induction does not have a therapeutic role, P-gp inhibition is an attractive therapeutic approach to reverse multidrug resistance . Clinicians should recognize that P-gp induction or inhibition may have a substantial effect on the pharmacokinetics and pharmacodynamics of concomitantly administered drugs that are substrates for this transporter.

Am J Physiol Cell Physiol, 2001 Aug, 281(2), C369 - 85
Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance; Lange K et al.; The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling . According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV) . The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm . The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds . Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism . Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV . The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells . The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

J Vet Pharmacol Ther, 2001 Jun, 24(3), 171 - 7
Influence of verapamil on the efflux and metabolism of 14C moxidectin in cultured rat hepatocytes; Dupuy J et al.; Moxidectin (MOX) is an antiparasitic drug widely used in cattle, sheep and companion animals . As a result of the implication of cytochrome P450 3 A in the metabolism of MOX and the role of competitor substrates of P-glycoprotein (Pgp) in modification of the bioavailability of endectocides, we studied the influence of verapamil (a multidrug-resistance reversing agent) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h . The metabolism of MOX remained low: 10.79 +/- 1.99% of the total 14C moxidectin for the main detected metabolite in verapamil-treated cells and 7.17 +/- 0.74% for the control cells after 24 h . The main detected metabolite in rat hepatocytes was the same as that detected in rat hepatic microsomes (the C29 monohydroxymethyl metabolite) . Verapamil increased the quantity of MOX in the cells after 24, 48 and 72 h . Examination of the Area Under the concentration time Curve (AUC) of the main detected metabolite revealed a significant increase in the exposure of cells to MOX after verapamil treatment throughout the experiment . It is hypothesized that verapamil interfered with MOX as a substrate for Pgp during the initial incubation period . After this initial interaction, verapamil metabolites were able to interfere with Pgp . This experiment demonstrated the implication of Pgp in the transport of MOX and allowed prediction of the drug-drug interactions which might modify the bioavailability of endectocides.

Microb Drug Resist, 2001 Summer, 7(2), 191 - 6
Self-transmissible multidrug resistance plasmids in Escherichia coli of the normal intestinal flora of healthy swine; Sunde M et al.; The resistance genes and their surroundings on three self-transmissible plasmids found in Escherichia coli of the enteric normal flora of healthy pigs have been characterized . The resistance elements found are similar to those commonly found in clinical isolates, like the transposon Tn1721 including the Tet A tetracycline resistance determinant, Tn10 with the Tet B determinant, Tn21 including a class 1 integron with the aadA1a cassette inserted, sulII encoding sulfonamide resistance, and the strA-strB genes responsible for streptomycin resistance . The plasmids were able to mobilize into various recipients, including swine pathogens, zoonotic bacteria, and commensals when conjugation experiments were carried out . Transfer of plasmids did not require optimal conditions concerning nutrition and temperature as plasmids were transferred in 0.9% saline at room temperature, suggesting that in vivo transfer might be possible . This study shows that transferable resistance elements appearing in normal flora bacteria from animals are similar to those commonly found in clinical isolates of human origin . The results indicate a probable communication between pathogens and the normal flora with respect to exchange of resistance factors.

Scand J Infect Dis, 2001, 33(5), 329 - 32
The global control of tuberculosis: what are the prospects?
Nunn P.
Tuberculosis (TB) still imposes a huge burden of ill health, premature death and emotional suffering on the developing world . Over the past 30 y it has been greatly neglected by those concerned about international public health and there are now nearly 8 million new cases annually and 1.86 million deaths . An epidemic of HIV-associated TB is now affecting Africa and threatening parts of Asia . Multidrug-resistant TB has emerged as a huge threat in Russia and its former satellites . However, with the advent of the directly observed treatment, short-course (DOTS) strategy in 1995, high-burden countries have started to seriously address the problem . Recent political commitment on the part of the rich nations, together with significant increases in funding from private foundations and great scientific advances in our understanding of Mycobacterium tuberculosis, give rise to cautious optimism that TB will be controlled during this century.

Biochem Biophys Res Commun, 2001 Jul 6, 285(1), 111 - 7
Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells; Ozvegy C et al.; ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer . Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations . ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein . ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A . Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity . The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer . We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents .

Arch Biochem Biophys, 2001 Jul 15, 391(2), 171 - 9
RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes; Sharma R et al.; We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al . Biochemistry 39, 9327-9334) . Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes . Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes . In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes . Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function . Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used . In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70% . On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed . These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes .

Blood, 2001 Jul 15, 98(2), 405 - 13
Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild-type TP53, and vicinal thiol/disulfide status; Pallis M et al.; Nonresponse to remission-induction chemotherapy, which remains a major problem in acute myeloblastic leukemia (AML), has been linked to cellular resistance to apoptosis . Because the apoptosis induced by chemotherapeutic drugs is mediated by loss of mitochondrial transmembrane potential (MTP), it was postulated that sensitivity to mitochondrial membrane depolarization might be heterogeneous in AML . Using the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (mClCCP), the mitochondrial membrane sensitivity to depolarization (mClCCP concentrations that inhibit 50% of the transmembrane potential {IC(50)}) in AML blasts was measured and demonstrated marked interclonal heterogeneity, with the existence of comparatively sensitive (median mClCCP IC(50), 4 microM) and resistant (median mClCCP IC(50), 10 microM) clones . Furthermore, the mClCCP IC(50) was inversely associated with spontaneous in vitro apoptosis (P =.001) . It was high in cases with mutant TP53 and correlated with the total cellular level of the multidrug resistance-associated protein (P =.019) but not of bcl-2, bax, or bcl-x . It was also found that the dithiol oxidant diamide, in contrast to the monovalent thiol oxidant diethyl maleate, increased the sensitivity of mitochondrial membranes to mClCCP . To confirm that TP53 directly affects MTP in leukemic cells and to establish the role of vicinal thiol oxidation in the TP53-dependent pathway, CEM 4G5 leukemia cells with forced, temperature-dependent expression of TP53 were studied . Monobromobimane, which inhibits mitochondrial membrane depolarization by preventing dithiol cross-linking, inhibited depolarization and apoptosis in 4G5 cells . It was concluded that in leukemia, TP53 and vicinal thiol/disulfide status are determinants of mitochondrial membrane sensitivity to depolarization, which is in turn associated with spontaneous apoptosis.

Breast Cancer Res, 2001, 3(4), 253 - 63 Epub 2001 Apr 02.
Reversal effects of nomegestrol acetate on multidrug resistance in adriamycin-resistant MCF7 breast cancer cell line; Li J et al.; BACKGROUND: Chemotherapy is important in the systematic treatment of breast cancer . To enhance the response of tumours to chemotherapy, attention has been focused on agents to reverse multidrug resistance (MDR) and on the sensitivity of tumour cells to chemical drugs . Hundreds of reversal drugs have been found in vitro, but their clinical application has been limited because of their toxicity . The reversal activity of progestogen compounds has been demonstrated . However, classical agents such as progesterone and megestrol (MG) also have high toxicity . Nomegestrol (NOM) belongs to a new derivation of progestogens and shows very low toxicity . We studied the reversal activity of NOM and compared it with that of verapamil (VRP), droloxifene (DRO), tamoxifen (TAM) and MG, and investigated the reversal mechanism, i.e . effects on the expression of the MDR1, glutathione S-transferase Pi (GSTpi), MDR-related protein (MRP) and topoisomerase IIalpha (TopoIIalpha) genes, as well as the intracellular drug concentration and the cell cycle . The aim of the study was to examine the reversal effects of NOM on MDR in MCF7/ADR, an MCF7 breast cancer cell line resistant to adriamycin (ADR), and its mechanism of action . METHODS: MCF7/ADR cells and MCF7/WT, an MCF7 breast cancer cell line sensitive to ADR, were treated with NOM as the acetate ester . With an assay based on a tetrazolium dye {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MTT}, the effects of various concentrations of NOM on MDR in MCF7/ADR cells were studied . Before and after the treatment with 5 microM NOM, the expression of the MDR-related genes MDR1, GSTpi, TopoIIalpha and MRP were assayed with a reverse transcriptase polymerase chain reaction (RT-PCR) immunocytochemistry assay . By using flow cytometry (FCM), we observed the intracellular ADR concentration and the effects of combined treatment with NOM and ADR on the cell cycle . Results collected were analysed with Student's t test . RESULTS: NOM significantly reversed MDR in MCF7/ADR cells . After treatment NOM at 20, 10 and 5 microM, chemosensitivity to ADR increased 21-fold, 12-fold and 8-fold, respectively . The reversal activity of NOM was stronger than that of the precursor compound MG, and comparable to that of VRP . After treatment with 5 microM NOM, the expression of both the MDR1 and the GSTpi mRNA genes began to decline on the second day (P <0.05 and P <0.01, respectively), and reached the lowest level on the third day (both P <0.01); however, on the fifth day the expression levels began to increase again (both P <0.05) . The expression of MRP and TopoIIalpha had no significant changes . Changes in the expression of P-glycoprotein (P-gp) and GSTpi were similar to those of their mRNA expressions, showing early declines and late increases . Two hours after treatment with 20, 10 and 5 microM NOM, the intracellular ADR concentration increased 2.7-fold, 2.3-fold and 1.5-fold respectively . However, NOM did not increase ADR accumulation in MCF7/WT cells . FCM data showed that after 48 h of combined administration of NOM (20 microM) and ADR (from low to high concentration), MCF7/ADR cells showed a gradual arrest at the G2M phase with increasing ADR dose . The arrest effect with combined drug treatment was stronger than that with the single ADR treatment . CONCLUSION: MDR is the major mechanism of drug resistance in malignant tumour cells . To overcome MDR and to increase chemosensitivity, many reversal agents have been found . Most progestogen compounds have been demonstrated to have reversal effects, but we found no data on NOM, a new progestogen compound . Our results show that NOM has strong reversal activity . The reversal effects were stronger than those of the precursor compound, MG, and were comparable to that of VRP . Because NOM has low toxicity, it might have good prospects in clinical application . Using RT-PCR and immunocytochemistry assays, we studied the effects of NOM on MDR-related genes . The results were that NOM could markedly downregulate the mRNA and protein expression levels of MDR1 and GSTpi . TopoIIalpha and MRP gene expression showed no significant changes . It is known that P-gp induces MDR in tumour cells mainly by decreasing the intracellular drug concentration . After treatment with NOM, the intracellular drug concentration in MCF7/ADR cells increased significantly . Combined treatment with NOM and ADR induced arrest at the G2M phase . It is worth noting that NOM caused an early decrease and a late increase in the expression of some MDR-related genes in a time-dependent manner . The phenomena raise a question for the continued administration of reversal agents in clinics that merits further study . We demonstrate that NOM has strong reversal effects on MDR in MCF7/ADR cells . (ABSTRACT TRUNCATED)

Breast Cancer Res, 2001, 3(4), 224 - 9 Epub 2001 Jun 14.
Tumour-stromal interactions . Integrins and cell adhesions as modulators of mammary cell survival and transformation; Chrenek MA et al.; Stromal-epithelial interactions modulate mammary epithelial cell (MEC) growth and apoptosis by influencing cell adhesion and tissue organization . Perturbations in the mammary stroma and cell adhesion characterize breast tumors and underlie the altered tissue organization, disrupted tissue homeostasis and enhanced survival phenotype of the disease . Apoptosis resistance likely arises during malignant transformation via genetic and epigenetic modification of cell adhesion pathways induced by a changing tissue microenvironment . Acquisition of adhesion-linked survival networks that enhance MEC viability in the absence of basement membrane interactions probably promote malignant transformation, and may render breast tumors sufficiently resistant to exogenous apoptotic stimuli to generate multidrug resistance.

Hepatology, 2001 Jul, 34(1), 146 - 57
A cellular gene up-regulated by hepatitis B virus-encoded X antigen promotes hepatocellular growth and survival; Lian Z et al.; Polymerase chain reaction (PCR) select complementary DNA (cDNA) subtraction of hepatitis B x antigen (HBxAg)-positive compared with -negative HepG2 cells resulted in the up-regulated expression of a cellular gene that encodes a transcript of 745 bases and a polypeptide 99 amino acids long . GenBank analysis revealed extensive homology with the amino terminal domain of cellular multidrug resistant proteins (MRP), although overexpression of this gene did not confer an MRP phenotype . In situ hybridization and immunostaining showed colocalized expression with HBxAg in the liver of hepatitis B carriers . Overexpression of this protein stimulated the growth of HepG2 cells in serum-free medium, and partially protected cells from anti-Fas-mediated killing, but did not promote growth in soft agar or tumor formation in nude mice . Introduction of the dominant negative inhibitor of nuclear factor kappaB (IkappaBalpha) into HBxAg-positive HepG2 cells decreased the levels of messenger RNA (mRNA) and protein, suggesting that its up-regulation is nuclear factor kappaB (NF-kappaB) dependent . Hence, HBxAg activation of NF-kappaB may result in the up-regulation of a cellular protein that promotes growth factor-independent survival and protects against Fas-mediated killing . This factor may contribute to the persistence of infected hepatocytes during chronic infection, which is important for the later development of hepatocellular carcinoma (HCC).

Cancer Lett, 2001 Aug 28, 169(2), 181 - 8
To predict chemotherapy response using technetium-99m tetrofosmin and compare with p-glycoprotein and multidrug resistance related protein-1 expression in patients with untreated small cell lung cancer; Shiau YC et al.; The aim of this study was to investigate the relationships among technetium-99m tetrofosmin (Tc-TF) accumulation in untreated small cell lung cancer (SCLC), the expression of P-glycoprotein (Pgp) and multidrug resistance related protein-1 (MRP1), and the response to chemotherapy in patients with untreated SCLC . Thirty patients with SCLC were studied with chest scintigraphy 15 to 30 min after intravenous injection of Tc-TF before chemotherapeutic induction . Tc-TF chest scans were interpreted both visually and quantitatively . The response to chemotherapy was evaluated upon completion of chemotherapy . Immunohistochemical analyses were performed on multiple non-consecutive sections of biopsy specimens to detect Pgp and MRP1 expression . Fifteen patients with good response to chemotherapy had a significantly higher incidence (100.0%) of positive Tc-TF chest single photon emission computed tomography (SPECT) findings and negative Pgp or MPR expression than 15 patients with poor response (20%) (P<0.05) . The tumor/background (T/B) ratios were 1.8+/-0.3 and 1.2+/-0.3 for patients with good response and poor response, respectively (P<0.05) . However, other prognostic factors (performance status, tumor size and stage) were not significantly related to Tc-TF chest scan findings and response to chemotherapy . Tc-TF chest scintigraphy correlated well with Pgp or MRP1 expression and accurately predicted the response to chemotherapy in patients with SCLC.

J Biol Chem, 2001 Sep 14, 276(37), 34966 - 74 Epub 2001 Jun 27.
Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17; Zhang DW et al.; Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly . By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance . However, Glu(1089) mutations had no effect on E(2)17betaG transport . We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen . Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold . Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance . However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16 . To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys . These mutations decreased E(2)17betaG transport 2-fold . Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold . The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested . As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG . These results demonstrate the important role of Thr(1242) for E(2)17betaG transport . They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.

J Biol Chem, 2001 Aug 24, 276(34), 31800 - 5 Epub 2001 Jun 27.
Cross-linking of human multidrug resistance P-glycoprotein by the substrate, tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis . Evidence for rotation of a transmembrane helix; Loo TW et al.; We identified a thiol-reactive substrate, Tris-(2-maleimidoethyl)amine (TMEA), to explore the contribution of the TM segments 6 and 12 of the human multidrug resistance P-glycoprotein (P-gp) during transport . TMEA is a trifunctional maleimide and stimulated the ATPase activity of Cys-less P-gp about 7-fold . Cysteine-scanning mutagenesis of TM12 showed that the activity of mutant V982C was inhibited by TMEA . P-gp mutants containing V982C (TM12) and another cysteine in TM6 were constructed and tested for cross-linking with TMEA . A cross-linked product was observed in SDS-polyacrylamide gel electrophoresis for mutant L339C(TM6)/V982C(TM12) . Cross-linking by TMEA also inhibited the ATPase activity of the mutant protein . Substrates such as cyclosporin A, vinblastine, colchicine, or verapamil inhibited cross-linking by TMEA . In the presence of ATP at 37 degrees C, cross-linking of mutant L339C/V982C was decreased . In contrast, there was enhanced cross-linking of mutant F343C(TM6)/V982C(TM12) in the presence of ATP . These results show that cross-linking must be within the drug-binding domain, that residues L339C(TM6)/V982C(TM12) must be at least 10 A apart, and that ATP hydrolysis promotes rotation of one or both TM helices.

AIDS Res Hum Retroviruses, 2001 Jun 10, 17(9), 807 - 18
Selection by AZT and rapid replacement in the absence of drugs of HIV type 1 resistant to multiple nucleoside analogs; Lukashov VV et al.; We studied the intrahost evolution and dynamics of a multidrug-resistant HIV-1, which contains an insertion of two amino acids (aa) and several aa changes within the reverse transcriptase (RT) gene . From an individual receiving intermittent therapy, sequences of 231 full-length molecular clones of HIV-1 RT were obtained from serum-derived viruses at 12 consecutive time points over a period of 6 years, 17 to 20 clones per time point . In the 3.5-year period prior to the first course of therapy, only wild-type (wt) viruses were found . As soon as 6 months after the start of zidovudine (AZT) monotherapy, all viruses contained an insertion of two aa between positions 68 and 69 of the RT and aa changes at positions 67 and 215, a combination conferring resistance to multiple nucleoside analogs . After termination of therapy, the insertion mutants were rapidly and completely replaced by the wt viruses . In turn, the insertion mutants replaced the wt viruses after initiation of therapy with 3TC, d4T, and saquinavir . After termination of triple therapy, the wt viruses completely replaced the mutants within 1 month, which is markedly faster than has been observed earlier for the replacement of AZT-resistant viruses . Fast replacements of the mutant virus populations after termination of therapy indicate gross competitive disadvantage of the insertion mutant in the absence of therapy, which we estimated by using several models . The insertion mutants attained high virus loads, demonstrating that virus load cannot be used as a direct measure of virus fitness.

J Med Chem, 2001 Jul 5, 44(14), 2308 - 18
Heterocycle-containing retinoids . Discovery of a novel isoxazole arotinoid possessing potent apoptotic activity in multidrug and drug-induced apoptosis-resistant cells; Simoni D et al.; In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared . Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4 seems to have particular importance for their apoptotic activity, novel retinoid analogues with a restricted or, vice versa, a larger flexibility in this region were designed and prepared . The new compounds were evaluated in vitro for their ability to activate natural retinoid receptors and for their differentiation-inducing activity . Cytotoxic and apoptotic activities were, in addition, evaluated . In general, these analogues showed low cytotoxicity, with the restricted structures being slightly more active than the more flexible ones . As an exception, however, the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations <5 microM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA, and 9-cis-RA and the previously described synthetic retinoid 4 . 15b also exhibited a good affinity for the retinoid receptors . Interestingly, another important property of 15b was its ability to induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration as is effective in HL60 . Therefore, 15b represents a new retinoid possessing high apoptotic activity in an MDR cell line . The ability of 15b to act on K562 and HL60R cells suggests that this compound may have important implications in the treatment of different leukemias, and its structure could offer an interesting model for the design of new compounds endowed with apoptotic activity on MDR- and retinoid-resistant malignancies.

Semin Cell Dev Biol, 2001 Jun, 12(3), 247 - 56
Molecular genetic analysis and biochemical characterization of mammalian P-glycoproteins involved in multidrug resistance; Hrycyna CA; A variety of human cancers become resistant or are intrinsically resistant to treatment with conventional drug therapies . This phenomenon is due in large part to the overexpression of a 170 kDa plasma membrane ATP-dependent pump known as the multidrug resistance transporter or P-glycoprotein . P-glycoprotein is a member of the large ATP binding cassette (ABC) superfamily of membrane transporters . This review focuses on the use of structure-function analyses to elucidate further the mechanism of action of mammalian P-glycoproteins . Ultimately, a complete understanding of the mechanism is important for the development of novel strategies for the treatment of many human cancers .

Semin Cell Dev Biol, 2001 Jun, 12(3), 225 - 37
Transcriptional regulation of multidrug efflux pumps in bacteria; Grkovic S et al.; As integral membrane proteins demonstrating an extraordinarily wide substrate range, some degree of regulatory control over the expression of bacterial multidrug-resistance (MDR) transporters is to be expected . Excessive expression could be deleterious, due to direct, physical disruption of membrane integrity, or the unwanted export of essential metabolites, a potential side-effect of their broad substrate specificity . There are limited clues as to the physiological functions of most MDR transporters, but their expression is likely to be up-regulated in response to the presence of natural substrates of these pumps . Thus, it is no surprise that MDR genes are subject to regulation at the local level, consisting of examples of both transcriptional repression and activation by proteins encoded adjacent to that for the transporter . Furthermore, an increasing number of MDR genes have also been found to be controlled by global transcriptional activator proteins .

J Pharm Pharmacol, 2001 Jun, 53(6), 779 - 87
Triton-X-100-modified polymer and microspheres for reversal of multidrug resistance; Liu Z et al.; Triton X-100 is a non-ionic detergent capable of reversing multidrug resistance (MDR) due to its interaction with cell membranes . However, it interacts with cells in a non-specific way, causing cytotoxicity . This work aimed to develop polymeric chemosensitizers that possess the ability to reverse MDR and lower toxic side effects . When being delivered to tumours, the polymeric chemosensitizers may also have longer retention times in tumours than the free detergent . Triton-X-100-immobilized dextran microspheres (T-MS) and inulin (T-IN) were prepared and characterized . Their cytotoxicity against multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was compared with that of free Triton X-100 solutions . The in-vitro effect of the products on 3H-vinblastine accumulation by CH(R)C5 cells was determined . Both T-MS and T-IN showed a marked decrease in the cytotoxicity, as compared with free Triton solutions at equivalent concentrations . Drug accumulation by CH(R)C5 cells was increased over two fold in the presence of T-MS or T-IN . These results suggest that polymeric drug carriers with MDR-reversing capability and lower cytotoxicity may be prepared by immobilization of chemosensitizers.

Int J Pharm, 2001 Jul 17, 222(2), 169 - 82
Mechanisms of transport across cell membranes of complexes contained in antitumour drugs; Szachowicz-Petelska B et al.; Various mechanism of antitumour drug transport across cell membranes has been described . Particular attention has been paid to a passive transport, active transport and multidrug resistance of complexes contained in antitumour drugs . A drug supply to the target site depends on the blood circulation within the tumour, on characteristic drug diffusion in the tissue, and also on binding protein . The physiologic transfer of hydrophilic compounds across the membrane is usually intermediated by means of a specific receptor or a carrier in that membrane, which facilitates the transport of compounds to and from the cell . Some drugs, e.g . doxorubicin and annamycin, can pass across the membrane by intermediacy of liposomes which exhibit a great activity in penetrating into tumour cells . The efficiency of antitumour drugs is limited by the appearance of resistance, i.e . by the lack of sensitivity of the cell to the administered drug . The presence in the membrane of specific proteins belonging to the ABC carriers group is postulated in a resistance theory; they would be responsible for 'pumping out' lipophilic drug molecules from the cell . Participation of high-energy ATP molecule is required by P-glycoprotein (Pgp) and by MRP protein described in this paper for their action . The mechanisms that are responsible for the cell resistance to drugs have been presented by analysing the resistance to antimetabolites, particularly to folate and fluoropyrimidine analogues, to alkylating agents, e.g . cisplatinum, and to heterocyclic compounds being responsible for so-called multidrug resistance.

Leuk Lymphoma, 2000 Dec, 40(1-2), 191 - 5
Differential effects of the MDR1 (multidrug resistance) gene-activating agents on protein kinase C: evidence for redundancy of mechanisms of acquired MDR in leukemia cells; Shtil AA et al.; Human leukemia cells may acquire MDR1/P-glycoprotein-mediated multidrug resistance (MDR) in the course of short-term (within hours) exposure to many stress stimuli . This effect is thought to be associated with the activity of protein kinase C (PKC) (Chaudhary, Roninson, 1992 . 1993) . However, we show here that cytosine beta-D-arabinofuranoside (Ara C) and 12-O-tetradecanoylphorbol 13-acetate (TPA), agents that activated the MDR1 gene in the H9 T-cell leukemia line, caused different effects on PKC . Namely, TPA activated PKC whereas Ara C was without the effect . Furthermore, cell permeable ceramide, a lipid messenger known to mediate cellular effects of chemotherapeutic drugs and TPA, activated the MDR1 gene and down-regulated PKC . These results suggest that the MDR1 gene can be activated via the pathway(s) that requires PKC activity as well as via bypass of PKC . The redundancy of signaling pathways that regulate the acquisition of MDR should be taken into consideration for prevention of secondary drug resistance in hematological malignancies.

Lancet, 2001 Jun 16, 357(9272), 1948 - 50
Fake artesunate in southeast Asia; Newton P et al.; Artesunate is a key antimalarial drug in the treatment of multidrug-resistant Plasmodium falciparum malaria in southeast Asia . We investigated the distribution of counterfeit artesunate tablets by use of the validated, simple, and inexpensive Fast Red TR dye technique . We also aimed to identify distinguishing characteristics of the fake drugs . Of 104 shop-bought "artesunate" samples from Cambodia, Laos, Myanmar (Burma), Thailand, and Vietnam, 38% did not contain artesunate . Characteristics such as cost and physical appearance of the tablets and packaging reliably predicted authenticity . The illicit trade in counterfeit antimalarials is a great threat to the lives of patients with malaria . The dye test will assist national malaria control authorities in urgently needed campaigns to stop this murderous trade.

Kidney Int, 2001 Jul, 60(1), 156 - 66
Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients; Koziolek MJ et al.; BACKGROUND: The multidrug resistance (MDR) gene product P-glycoprotein (P-gp) is a transmembrane efflux pump for hydrophobic, potentially toxic compounds, including the immunosuppressant cyclosporine A (CsA) . We have previously shown that CsA increases P-gp expression in proximal tubule and endothelial cells in vitro . The aim of the present study was to investigate the in vivo relevance of these observations in renal allograft biopsies from CsA-treated patients . METHODS: P-gp expression was determined by immunohistochemistry of paraffin sections using two different monoclonal antibodies (UIC2 and MRK16) . Biopsies were taken from CsA-treated renal transplant patients with different histopathological diagnoses (N = 79) and were compared with biopsies from normal human kidneys (N = 13) or with allograft biopsies from patients under a CsA-free immunosuppression (N = 15) . Moreover, biopsies from 10 donor kidneys before implantation and during rejection episodes ("zero biopsies") were investigated . RESULTS: P-gp expression in biopsies with acute tubular necrosis (ATN; N = 10) after CsA treatment was significantly higher in arterial endothelia, proximal tubules, and epithelial cells of Bowman's capsule (BC), whereas P-gp was sparsely induced in CsA nephrotoxicity (N = 19) compared with controls . Acute cellular (N = 30) and vascular rejection (N = 10) or chronic allograft nephropathy (N = 10) after CsA was associated with strong P-gp expression in infiltrating leukocytes and increased P-gp expression in arterial endothelia, proximal tubules, and BC . In contrast, biopsies of patients treated with a CsA-free immunosuppression regimen did not show increases in P-gp expression compared with controls . Zero biopsies showed a weak, homogeneous, nonpolarized expression of P-gp in tubules and an increased expression of P-gp after CsA therapy in the brush border, arterial endothelia, and BC . CONCLUSIONS: CsA treatment was associated with increased P-gp expression in parenchymal cells of kidney transplants with ATN, acute or chronic transplant rejection, but P-gp was not increased in patients with CsA nephrotoxicity . This indicates that CsA induces its own detoxification by P-gp and that inadequate up-regulation of P-gp in renal parenchymal cells contributes to CsA nephrotoxicity . Increased expression of P-gp in infiltrating leukocytes correlated with the severity of allograft rejection, suggesting that P-gp may decrease the immunosuppressive efficacy of CsA . Thus, individual differences in the P-gp induction response of CsA-exposed renal parenchymal cells and/or infiltrating leukocytes may predispose to either CsA nephrotoxicity or rejection, respectively.

Res Microbiol, 2001 Apr-May, 152(3-4), 365 - 74
Multidrug transport by ATP binding cassette transporters: a proposed two-cylinder engine mechanism; van Veen HW et al.; The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms . Multidrug transporters interact directly with the drug substrates . This review summarizes current insights into the mechanism(s) by which ATP hydrolysis is coupled to drug transport in bacterial LmrA and its human homolog P-glycoprotein . In addition, the relevance of these insights for other ABC transporters will be discussed.

J Membr Biol, 2001 Jun 1, 181(3), 153 - 62
The involvement of sphingolipids in multidrug resistance; Sietsma H et al.; Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety in structure and molecular targets . Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently been identified as an important mediator of this process . Indeed, one of the events elicited by application of many cytotoxic drugs is an accumulation of this lipid . Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance, in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents . Different cellular mechanisms underlying this phenomenon have been described . Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated proteins are the most extensively studied examples . Recently, an increased cellular capacity for ceramide glycosylation has been recognized as a novel multidrug resistance mechanism . Indeed, virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically, increased levels of glucosylceramide . On the other hand, several direct molecular interactions between sphingolipids and drug efflux proteins have been described . Therefore, in addition to a role in the multidrug resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated . In this review, we present an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug resistance.

Lancet, 2001 Apr 21, 357(9264), 1267 - 8
Airborne outbreak of nosocomial Scedosporium prolificans infection; Guerrero A et al.; We describe six inpatients with acute non-lymphocytic leukaemia who developed invasive infection with Scedosporium prolificans resistant to amphotericin B, flucytosine, ketoconazole, fluconazole, and itraconazole . All six patients died . Phenotypic and genotypic assessment of samples from clinical material and ambient air from the isolation rooms where the patients were being treated showed that the epidemic was caused by a single strain . After implementation of aerial control measures, there were no further infections with this organism . We conclude that fatal multidrug-resistant S prolificans epidemics can be aerially transmitted and can be prevented with implementation of appropriate infection-control measures.

Oncol Res, 2000, 12(5), 219 - 29
Indoloquinoxaline compounds that selectively antagonize P-glycoprotein; Smith CD et al.; Tumor cells often develop drug resistance through overexpression of membrane transport proteins that effectively efflux anticancer agents . The pharmacologies of the two best-studied transporters, P-glycoprotein (Pgp) and MRP1, are partially overlapping but distinct . To improve the therapeutic potential of drug resistance reversing agents, we have developed a program to identify compounds with selectivity for Pgp or MRP1 . Screening of a commercial library of compounds identified indoloquinoxaline compounds with transporter selectivity, and certain examples were synthesized and further evaluated . 1,4-Dibutoxy-6H-indolo{2,3-b}quinoxaline and 4,7-dibutoxy-2,3-dihydrobenzimidazole-2-spiro-3-indolin-2-one were synthesized by condensation of 3,6-dibutoxy-1,4-diaminobenzene and isatin . Neither compound was cytotoxic to MCF-7 cells, nor did either one affect the sensitivity of MCF-7/VP or HL-60/ADR cells at doses up to at least 20 microM, indicating that they do not antagonize MRP1 . In contrast, each compound, at doses as low as 0.25 microM, sensitized NCI/ADR cells to vinblastine, actinomycin D, Taxol, and doxorubicin, indicating that they effectively reverse Pgp-mediated multidrug resistance (MDR) . Furthermore, the compounds sensitized two additional cell lines that overexpress Pgp to this panel of anticancer drugs . However, these compounds did not affect the sensitivities of MCF-7 or T24 cells to these cytotoxic drugs, and did not alter the sensitivities of any of the tested cell lines to cisplatin or 5-fluorouracil . Both compounds enhanced the intracellular accumulation of {3H}vinblastine by NCI/ADR cells, but did not inhibit photoaffinity labeling of Pgp by {3H}azidopine at concentrations up to at least 100 microM . Therefore, these novel nontoxic indoloquinoxalines selectively sensitize Pgp-overexpressing cells to drugs that are subject to transport by this protein, without modulating the sensitivities of MRP1-overexpressing or non-Pgp cells to cytotoxic drugs . Because of this transporter selectivity, we predict that these compounds will be effective MDR modulators in vivo.

Leukemia, 2001 Jun, 15(6), 936 - 41
Treatment with inhibitors of caspases, that are substrates of drug transporters, selectively permits chemotherapy-induced apoptosis in multidrug-resistant cells but protects normal cells; Blagosklonny MV; Many chemotherapeutic agents induce apoptosis in tumor cells, but killing of normal cells remains a major obstacle . Development of multidrug resistance further limits chemotherapy in cancer . Here, I show that multidrug resistance can be exploited for selective killing of multidrug-resistant cells by a combination of an apoptosis-inducing agent that is not a substrate of either Pgp or MRP (e.g . flavopiridol) with a caspase inhibitor that is a substrate (e.g . Z-DEVD-fmk) . In normal cells, treatment with caspase inhibitors prevented PARP cleavage, nuclear fragmentation, and cell death caused by flavopiridol or epothilone B . In contrast, Pgp- and MRP-expressing cells were not rescued by caspase inhibitors . Furthermore, reversal of drug resistance renders Pgp cells sensitive to caspase inhibitors abolishing therapeutic advantage . Thus, caspase inhibitors, that are inactive in multidrug-resistant cells, protect normal but not multidrug-resistant cells against chemotherapy, permitting selective eradication of multidrug-resistant cells . Clinical application of this approach may diminish the toxic side-effects of chemotherapy in patients with multidrug-resistant tumors.

Dig Dis Sci, 2001 Jun, 46(6), 1290 - 8
Modifying hepatic phospholipid synthesis associates with biliary phospholipid secretion rate in a transporter-independent manner in rats: relation to canalicular membrane fluidity; Yasumiba S et al.; Biliary phospholipid secretion is mediated by a multidrug resistance gene product, and its molecular subselection occurs at the site of secretion to modulates bile metastability . The aim of this study was to determine the effect of modifying hepatic phospholipid synthesis on canalicular phospholipid transporter expression and membrane fluidity . Bile-duct cannulation was performed in male Sprague-Dawley rats pretreated with or without intravenous infusion of dimethylethanolamine, an intermediate phospholipid metabolite along the pathway of phosphatidylcholine synthesis of phosphatidylethanolamine N-methylation (0.01 mg/min/100 g body wt) for 15 hr, followed by sodium taurocholate infusion (50 nmol/min/100 g body wt) with or without sulfobromophthalein (50 nmol/min/100 g body wt) . Dimethylethanolamine enhanced biliary phospholipid secretion in association with a decrease in biliary phospholipid hydrophobicity . Dimethylethanolamine also increased canalicular membrane fluidity defined by 1,6-diphenyl-1,3,5-hexatriene fluorescence depolarization, whereas the expression of multidrug resistance gene product and multidrug resistance associated protein was unchanged . In contrast, a disproportionate reduction of biliary phospholipid secretion caused by sulfobromophthalein (uncoupling) was enhanced by under the treatment with dimethylethanolamine . In conclusion, the increase in biliary phospholipid secretion and canalicular membrane fluidity without a drastic change of its canalicular transporter by dimethylethanolamine suggests that such a canalicular membrane fluidity facilitates the transporter activity and/or phospholipid molecular movement from the canalicular outer membrane into the bile . A more drastic reduction in phospholipid secretion under sulfobromophthalein-caused uncoupling indicates the possibility of a preferential distribution of relatively hydrophilic phosphatidylcholine molecules to bile salt micelles since sulfobromophthalein is known to reduce the micellar capacity to extract membrane lipids for biliary secretion.

J Chem Inf Comput Sci, 2001 May-Jun, 41(3), 505 - 11
Comparison of a neural net-based QSAR algorithm (PCANN) with Hologram- and multiple linear regression-based QSAR approaches: application to 1,4-dihydropyridine-based calcium channel antagonists; Viswanadhan VN et al.; A QSAR algorithm (PCANN) has been developed and applied to a set of calcium channel blockers which are of special interest because of their role in cardiac disease and also because many of them interact with P-glycoprotein, a membrane protein associated with multidrug resistance to anticancer agents . A database of 46 1,4-dihydropyridines with known Ca2+ channel binding affinities was employed for the present analysis . The QSAR algorithm can be summarized as follows: (1) a set of 90 graph theoretic and information theoretic descriptors representing various structural and topological characteristics was calculated for each of the 1,4-dihydropyridines and (2) principal component analysis (PCA) was used to compress these 90 into the eight best orthogonal composite descriptors for the database . These eight sufficed to explain 96% of the variance in the original descriptor set . (3) Two important empirical descriptors, the Leo-Hansch lipophilic constant and the Hammet electronic parameter, were added to the list of eight . (4) The 10 resulting descriptors were used as inputs to a back-propagation neural network whose output was the predicted binding affinity . (5) The predictive ability of the network was assessed by cross-validation . A comparison of the present approach with two other QSAR approaches (multiple linear regression using the same variables and a Hologram QSAR model) is made and shows that the PCANN approach can yield better predictions, once the right network configuration is identified . The present approach (PCANN) may prove useful for rapid assessment of the potential for biological activity when dealing with large chemical libraries.

Biochem Biophys Res Commun, 2001 Jun 22, 284(4), 863 - 9
Transport of fluorescein in MDCKII-MRP1 transfected cells and mrp1-knockout mice; Sun H et al.; The multidrug resistant-associated protein 1 (MRP1) is a membrane-bound transport protein that is involved in the efflux of organic anions and has been implicated in multidrug resistance in cancer . MRP1 has also been reported to be ubiquitously expressed in normal tissues, including the brain . The presence of functional organic anion transporters in the blood-brain and blood-CSF barriers that influence the distribution of various compounds to the brain has long been known . The purpose of this study was to examine the role of MRP1 in the brain distribution of a model organic anion, fluorescein . The substrate specificity of MRP1 for fluorescein was initially determined by examining the accumulation of fluorescein in MDCKII MRP1-transfected cells . The distribution of fluorescein in the brain was then examined in wild-type and mrp1 gene knockout mice . The results show that in MDCKII MRP1-transfected cells, the accumulation of fluorescein was significantly lower (about 40% lower) than that in wild-type MDCKII cells . MRP1 inhibitors such as probenecid, MK-571, and LY402913 enhanced fluorescein accumulation in MDCKII MRP1-transfected cells to a greater extent than in wild-type MDCKII cells . In an in vivo study, after intravenous injection of fluorescein, the fluorescein brain-to-plasma concentration ratio in mrp1 knockout mice was not significantly different than that in wild-type mice . However, when probenecid was co-administered with fluorescein in wild-type mice, the fluorescein brain-to-plasma ratio was significantly increased (1.5-fold) . These findings suggest that fluorescein is a substrate for MRP1 . Furthermore, the in vivo study also suggests that MRP1 has a limited role in the transport and distribution of fluorescein in the brain . Therefore, other organic anion transport proteins, including the various isoforms of the MRP family, may be responsible for the accumulation and transport of organic anions in the brain .

Int J Tuberc Lung Dis, 2001 Jun, 5(6), 575 - 8
A case series: initial outcome of persons with multidrug-resistant tuberculosis after treatment with the WHO standard retreatment regimen in Ho Chi Minh City, Vietnam; Lan NTN et al.; Few data address the outcomes of patients who have multidrug-resistant tuberculosis (MDR-TB), defined as resistance to at least isoniazid and rifampin, and who receive a standard World Health Organization (WHO) recommended retreatment regimen after relapse or failure with initial treatment . In this case series, we examined treatment outcomes of a convenience sample of 42 relapse or failure patients who had documented MDR-TB and who had received a standard WHO retreatment regimen (2SHRZE/1HRZE/5H3R3E3) . One patient died of tuberculosis in the last month of treatment; the remaining 41 patients completed retreatment . Of the 42, 14 (33%) were sputum smear-negative on completion of therapy . The proportion of patients cured of MDR-TB with the WHO retreatment regimen was similar to historic outcomes when no chemotherapy for TB was given.

Int J Tuberc Lung Dis, 2001 Jun, 5(6), 551 - 8
Familial outbreak of disseminated multidrug-resistant tuberculosis and meningitis; Sofia M et al.; Rapidly progressive multidrug-resistant tuberculosis (MDR-TB) is well documented in human immunodeficiency virus (HIV) positive subjects, but it is not fully recognised in HIV-negative subjects in the familial environment . We report three cases of MDR-TB in three young HIV-negative subjects from the same family . All the patients showed signs of meningitis during the course of their disease, and in two cases a resistant strain of Mycobacterium tuberculosis was isolated in cerebrospinal fluid . Two of the three subjects died from neurological complications; the other was successful treated utilising both systemic and intrathecal therapy for tuberculous meningitis . By a retrospective analysis of DNA obtained from Lowenstein-Jensen cultures, the strains were confirmed as M . tuberculosis resistant to rifampicin and isoniazid, and were closely related in the two cases where specimens were available for analysis . The resistance was acquired in two patients initially infected with a susceptible strain; in the other patient, the resistance was present on the first sensitivity test for which results were available . This report demonstrates the high risk of fatality from MDR-TB for HIV-negative subjects in the absence of reliable early diagnostic and preventive tools . It also reinforces the concept that genetic susceptibility to M . tuberculosis may be an important factor in the clinical presentation and outcome of MDR-TB.

Int J Tuberc Lung Dis, 2001 Jun, 5(6), 546 - 50
Increased resistance to ciprofloxacin and ofloxacin in multidrug-resistant mycobacterium tuberculosis isolates from patients seen at a tertiary hospital in the Philippines; Grimaldo ER et al.; SETTING: A hospital-based study at the Makati Medical Center, Makati City, Philippines, a hyperendemic area for tuberculosis (TB) . OBJECTIVE: To determine the susceptibility of Mycobacterium tuberculosis to ciprofloxacin and ofloxacin . DESIGN: Retrospective analysis of drug susceptibility tests (DST) of M . tuberculosis isolated from 1995-2000 . RESULTS: Resistance to ciprofloxacin was 26.8%, ofloxacin 35.3%, and multidrug resistance (MDR) was 17.2% . Of the MDR strains, 51.4% were resistant to ciprofloxacin and ofloxacin . Acquired resistance was significantly higher for all first-line drugs and for ciprofloxacin, but not for ofloxacin . A significant increase in resistance to ciprofloxacin and ofloxacin was noted compared to 1989-1994, while resistance to the firstline drugs was not significantly different . CONCLUSION: Ciprofloxacin and ofloxacin are now a significantly less effective alternative therapy in tuberculosis, particularly MDR-TB, due to a selection pressure from their widespread use in the treatment of TB and possibly other infections in the community, which is hyperendemic for tuberculosis.

J Exp Bot, 2001 Apr, 52(357), 877 - 9
A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome; Yazaki K et al.; A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica . The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids . Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.

Biol Pharm Bull, 2001 Jun, 24(6), 612 - 7
Reversal of vinblastine resistance in human leukemic cells by haloperidol and dihydrohaloperidol; Kataoka Y et al.; Haloperidol, an antipsychotic, was investigated in cells overexpressing P-glycoprotein to detemine whether it was a clinically effective drug to reverse for reversing multidrug resistance (MDR) mediated by P-glycoprotein . A nontoxic concentration of haloperidol (1-30 microM) enhanced the cytotoxic effects of vinblastine (VBL) concentration-dependently in VBL-resistant human leukemia (K562/VBL) cells, but had no effect in the parent cells . Haloperidol also enhanced the cytotoxicities of epirubicin, doxorubicin and actinomycin D in the K562/VBL cells, but not those of idarubicin or cisplatin; this enhancement was less than that of the VBL toxicity in the VBL-resistant tumor line . Haloperidol increased the intracellular accumulation of VBL in the K562/VBL cells, and the binding of {3H}-azidopine to the cell-surface protein, P-glycoprotein, was inhibited by haloperidol in a concentration-dependent manner . Haloperidol was less potent than verapamil . Thus, haloperidol appeared to potentiate anticancer agents through the reversal of MDR by competitively inhibiting drug-binding to P-glycoprotein . In contrast, the main metabolite of haloperidol, dihydrohaloperidol, without antipsychotic activity, had less of an effect . Therefore, haloperidol might be useful in reversing drug-resistance.

Oncol Rep, 2001 Jul-Aug, 8(4), 815 - 9
Effects of MDR1/P-glycoprotein expression on prognosis in advanced colorectal cancer after surgery; Tokunaga Y et al.; Resistance to chemotherapeutic agents is a major problem for successful cancer treatment . P-glycoprotein (Pgp), a product of the multidrug resistance (MDR)1 gene expressed in cancer cells, is one of the mechanism of MDR . However, there are few reports regarding the effects of Pgp on prognosis of colorectal cancer (CRC) after surgery . We examined a total of 80 patients (45 males and 35 females with an average age of 69 years) whose CRCs were classified into stage 2-4 and completely resected surgically in our institute between January 1990 and September 1999 . To evaluate Pgp expression in CRC, immunohistochemical stain was performed with a monoclonal antibody . Relationships between Pgp expression and clinicopathological variables which may have affected prognosis were evaluated . Survival curves were calculated using the Kaplan-Meier method, and differences were evaluated with the log-rank test . The Cox's proportional hazards model was used in the univariate and multivariate survival analysis . Pgp expression showed a significant correlation with histological differentiation (p=0.023) . However, no correlation was observed with gender, tumor location, lymph node metastasis, lymphatic invasion, venous invasion, and cancer stages . Survival rates after surgery tended (p=0.093) to be higher in Pgp (+) than Pgp (-) patients . Pgp was not a significant prognostic factor by univariate analysis and multivariate analysis adjusted for other clinicopathologic variables . Survival rates after surgery tended to be higher in Pgp (+) than Pgp (-) patients and Pgp expression was correlated with histological differentiation of CRC . Thus, a relative resistance of CRC to conventional chemotherapy may be partly caused by Pgp expressed in well or moderately differentiated CRC . However, Pgp expression was not a significant independent prognostic factor in advanced CRC after surgery.

Clin Cancer Res, 2001 Jun, 7(6), 1798 - 804
Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels; Young LC et al.; Previously (L . C . Young et al., Clin . Cancer Res., 5: 673-680, 1999), we found, in a panel of 23 lung cancer cell lines that had not been selected for in vitro drug resistance, that the mRNA levels of MRP3 and MRP1, two members of the ATP-binding cassette superfamily of transport proteins, correlated with resistance to doxorubicin, vincristine, VP-16, and cis-diamminedicholoroplatinum(II) . To extend these studies, we measured multidrug resistance protein (MRP)1, MRP2, and MRP3 protein levels in a panel of 30 lung cancer cell lines that included the original 23 cell lines as well as an additional 7 unselected lung cancer cell lines . In the case of MRP3, a polyclonal antibody was developed that was found to be a sensitive reagent for the detection of MRP3 by Western blot analysis . We found good agreement in the original 23 cell lines between the cognate mRNA and protein levels for MRP1, MRP2, and, especially, MRP3 (r, 0.852), supporting the use of semiquantitative PCR to predict MRP1, MRP2, and MRP3 protein levels in patient samples . There were also strong correlations between the mRNA and protein levels of MRP3 and MRP1, which suggested that these genes might be expressed in a coordinate manner . MRP3, MRP1, and MRP2 protein levels were higher in the non-small cell lung cancer (NSCLC) than in the SCLC cell lines and, in addition, MRP3 and MRP2 were detected almost exclusively in the NSCLC cell lines . Finally, we found that both MRP3 and MRP1, but not MRP2, protein levels correlated with decreased sensitivity of these lung cancer cell lines to doxorubicin, VCR, VP-16, and cis-diamminedicholoroplatinum(II) . These findings are consistent with our hypothesis that both MRP3 and MRP1 are components of the multifactorial multidrug resistance phenotype of lung cancer and that MRP3 contributes to the intrinsic resistance of NSCLC cells.

Haematologica, 2001 May, 86(5), 470 - 7
The prognostic value of Bcl-XL gene expression for remission induction is influenced by cytogenetics in adult acute myeloid leukemia; Schaich M et al.; BACKGROUND AND OBJECTIVES . There is growing evidence that altered expression of genes belonging to the BcL-2 family of apoptosis regulators might influence chemotherapy-induced apoptosis in malignant cells and therefore could confer multidrug resistance . So far expression studies of apoptosis-regulating genes on acute myeloid leukemia (AML) have mainly focused on Bcl-2 itself and most of them have not included other factors involved in drug resistance or apoptosis as parameters determining response to chemotherapy, disease progression and survival . DESIGN AND METHODS . We therefore examined Bcl-2, Bcl-XL and Bax gene expression in 235 adult patients with de novo or secondary myeloid leukemia . The expression levels were correlated with established prognostic factors such as age, cytogentic aberrations, mdr1 gene expression and clinical outcome in a multivariate analysis . RESULTS . Bcl-2 and Bcl-XL positive patients had a much lower white blood cell count than negative patients (p<0.001 and p=0.003, respectively) . Bcl-2 expression correlated with FAB subtype M0 (p=0.03), Bax with M5b (p=0.02) and Bcl-XL with M6 (p=0.005) . Mdr1 expression was more frequently seen in Bcl-2 and Bcl-XL positive patients (p=0.03 and p=0.02, respectively) . Remarkably Bax was significantly less frequently expressed in de novo AML patients with high risk cytogenetics (p=0.007) . No difference in expression was recognized for Bcl-2 or Bcl-XL when statistical analyses were done for cytogenetic risk groups . However, in the multivariate analysis regarding the group of de novo AML patients < or =60 years with intermediate risk cytogenetics, Bcl-XL expression was found to be an independent negative prognostic factor for response to induction therapy (p=0.04) . In contrast, no prognostic impact of Bcl-XL expression on treatment response was seen within the group of patients with high risk cytogenetic findings . Neither Bcl-2 nor Bax nor Bcl-XL expression had a significant influence on overall or disease-free survival . INTERPRETATION AND CONCLUSIONS . These data indicate that the prognostic value of Bcl-XL gene expression for treatment response in AML patients < or =60 years is dependent on cytogenetics.

Biochim Biophys Acta, 2001 Jun 15, 1526(3), 293 - 300
Reduced glutathione protect cells from ouabain toxicity; Capella LS et al.; It is widely accepted that a prolonged ouabain blockade of the Na(+),K(+)-ATPase makes cells detach from each other and from the substrate, leading to their death and that cellular resistance to ouabain is due to the presence of isoforms of Na(+),K(+)-ATPase with low affinity to this glycoside . In the present work the effect of reduced glutathione in the response of two types of renal cells to ouabain: MDCK, a ouabain-sensitive cell line and Ma104, a ouabain-resistant one, was studied . Glutathione protected MDCK cells from ouabain toxicity and inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine sensitized Ma104 cells to ouabain . As glutathione is involved with multidrug resistance (MDR) in cells expressing the multidrug resistance-related protein MRP1 and as Ma104 cells have a MDR phenotype, it was investigated whether Ma104 cells express this protein . The expression of the MRP1-mRNA in Ma104 cells was detected by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay, and the protein was detected by Western blotting and immunofluorescence . Treatment of Ma104 cells with ouabain increased MRP1-mRNA expression and altered the localization of MRP1 in these cells . Our results suggest that some cells may have mechanisms to protect themselves from ouabain toxicity and that MRP1 may have a role in controlling the toxic effects of ouabain.

Pharm Res, 2001 Feb, 18(2), 183 - 90
Screening of multidrug-resistance sensitive drugs by in situ brain perfusion in P-glycoprotein-deficient mice; Cisternino S et al.; PURPOSE: This study was conducted to assess the influence of P-glycoprotein (P-gp) on brain uptake of multidrug resistance sensitive drugs using an in situ brain perfusion technique in P-gp-deficient (mdr1a{-/-}) and wild-type mice . METHODS: The blood-brain transport of radiolabeled vinblastine, vincristine, doxorubicin, colchicine, and morphine was evaluated in mdr1a(-/-) and wild-type CF-1 mice with the in situ brain perfusion technique . Brain uptake of drugs after intravenous pretreatment with P-gp reversal agents, (PSC 833, GF 120918, or (+/-)-verapamil), or vehicle also was studied in wild-type mice . In all experiments, cerebral vascular volume was determined by co-perfusion of sucrose . RESULTS: Cerebral vascular volume was preserved during perfusion, indicating maintenance of blood-brain barrier integrity in both types of mice within the concentration range of substrates in the perfusate . The apparent brain transport of colchicine . vinblastine, doxorubicin, and morphine was increased 3.0, 2.7, 1.5, and 1.4-fold, respectively, in mdr1a(-/-) mice compared with the wild-type: the brain uptake of vincristine was not affected by P-gp . Preadministration of PSC 833 or GF 120918 in wild-type mice led to a -3-fold increase in the brain transport of colchicine and vinblastine, but no effect was observed for the other compounds . Intravenous verapamil enhanced colchicine brain transport (1.8-fold), but failed to increase the brain uptake of vinblastine and morphine . CONCLUSION: The in situ brain perfusion technique appears to be a sensitive and powerful tool for medium throughput screening of the brain uptake of multidrug resistance sensitive drugs . The effect of P-gp is characterized more efficiently with mdr1a(-/-) mice than by using modulators of P-gp in wild-type mice.

Pharm Res, 2001 Feb, 18(2), 171 - 6
A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells; Gao J et al.; PURPOSE: To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers . METHODS: A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of {3H}-taxol, a known substrate of P-gp . The apparent affinities (K(I) values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of {3H}-taxol . Intact Caco-2 cell monolayers were utilized for transport inhibition studies . Samples were analyzed by liquid scintillation counting . RESULTS: {3H}-Taxol (0.04 microM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction . This difference in {3H}-taxol flux could be totally abolished by inclusion of (+/-)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium . However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of {3H}-taxol under the same conditions . These results suggest that P-gp, not MRP, was involved in taxol transport . Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of {3H}-taxol with resulting K(I) values of 1.5+/-0.8, 2.5+/-1.0, 3.0+/-0.3, 7.3+/-0.7, 8.5+/-2.8, 36.5+/-1.5, 276+/-69, and 313+/-112 microM, respectively . With the exception of that of quinidine, these K(I) values were comparable with literature values . CONCLUSIONS: This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp, however, this assay does not permit the differentiation of substrates and inhibitors . The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

Biophys Chem, 2001 Jun 15, 91(1), 21 - 35
Cooperativity of phospholipid reorganization upon interaction of dipyridamole with surface monolayers on water; Caetano W et al.; Results from various surface sensitive characterization techniques suggest a model for the interaction of the piperidinopyrimidine dipyridamole (DIP)--known as a vasodilator and inhibitor of P-glycoprotein associated multidrug resistance of tumor cells--with phospholipid monolayers in which the drug is peripherally associated with the membrane, binding (up to) five phospholipids at a time . These multiple interactions are responsible for a very strong association of the drug with the lipid monolayer even at exceedingly low concentrations (approximately 0.2 mol%) . Electrostatic interactions and hydrogen bonding are likely involved in the binding of DIP to DPPC . Cooperative effects among the lipids are invoked to explain the macroscopically measurable changes of lipid monolayer properties even when only one out of 100 DPPC molecules is directly associated with a DIP molecule . A reversal of the observed changes upon drug association with the membrane as the DIP concentration surpasses a threshold concentration (c(crit)approximately 0.5 mol%) may be explained by cooperativity in a different context, the self-aggregation of drug molecules . With its implications for the interaction of DIP with phospholipid films, this work provides a first approach to the explanation of the high sensitivity of cell membranes to piperidinopyrimidine drugs on a molecular level.

Int J Oncol, 2001 Jul, 19(1), 163 - 8
Differential expression of the lung resistance-related protein/major vault protein in the histological compartments of nephroblastomas; Efferth T et al.; Nephroblastomas (Wilms' tumors) are curable with survival rates above 80% . Some tumors, however, fail to respond to therapy and those patients have a poor prognosis . In a search for molecular markers of drug resistance, we investigated the expression of lung resistance protein (LRP) in tissue samples from 32 children with nephroblastoma by means of immunohistochemistry . LRP is a human major vault protein (MVP) and is associated with multidrug resistance of tumors . LRP/MVP expression was found in the blastemal and epithelial compartments but to a significantly lesser extent in the stromal compartment of Wilms' tumors . Expression was generally heterogeneous with respect to staining intensity and percentage of positive cells . We found significant relationships between LRP/MVP expression and chemotherapeutic pre-treatment of tumors and tumor stage . The immunohistochemical results were validated with a real-time RT-PCR technique and a significant association between protein and mRNA expression was observed.

J Pharmacol Exp Ther, 2001 Jul, 298(1), 234 - 9
Arsenic induces expression of the multidrug resistance-associated protein 2 (MRP2) gene in primary rat and human hepatocytes; Vernhet L et al.; Metals, such as arsenic or cadmium, have recently been demonstrated to interact with metabolic pathways, including phase I and phase II enzymes and the phase III efflux pump P-glycoprotein . In the present study, we investigated the effects of heavy metals and metalloids on the expression of the multidrug resistance-associated protein 2 (MRP2), a major hepatic transporter . Treatment of primary rat hepatocytes by sodium arsenite {As(III)}, sodium arsenate and potassium antimony tartrate, but not cadmium chloride, was shown to markedly increase MRP2 mRNA and protein levels; As(III)-mediated induction was dose- and time-dependent and paralleled a strong increase in MRP2 amounts as assessed by Western blotting . As(III) was also demonstrated to markedly up-regulate MRP2 gene expression in primary human hepatocytes . MRP2 mRNA induction occurring in As(III)-treated rat hepatocytes was fully blocked by actinomycin D, indicating that it required active gene transcription . It was associated with an activation of the c-Jun N-terminal kinase pathway and with a reduction of cellular glutathione levels . Quercetin, a flavonoid compound known to block As(III)-related induction of P-glycoprotein, was also found to prevent up-regulation of MRP2 gene expression in rat hepatocytes exposed to As(III) . Such an effect was unlikely to be due to alteration of JNK pathway since quercetin failed to abolish As(III)-induced JNK phosphorylation . It may rather be linked to the increase of cellular glutathione levels by quercetin, thus limiting the depleting effects of As(III) on glutathione amounts . Finally, these results confirm that some metals strongly regulate expression of detoxifying proteins, including biliary drug transporters.

J Clin Oncol, 2001 Jun 15, 19(12), 3130 - 41
Phase I and pharmacokinetic study of the novel MDR1 and MRP1 inhibitor biricodar administered alone and in combination with doxorubicin; Peck RA et al.; PURPOSE: To evaluate the safety, tolerability, and pharmacokinetics of biricodar (VX-710), an inhibitor of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1), alone and with doxorubicin in patients with advanced malignancies . The effect of VX-710 on the tissue distribution of (99m)Tc-sestamibi, a P-gp and MRP1 substrate, was also evaluated . PATIENTS AND METHODS: Patients with solid malignancies refractory to standard therapy first received a 96-hour infusion of VX-710 alone at 20 to 160 mg/m(2)/h . After a 3-day washout, a second infusion of VX-710 was begun, on the second day of which doxorubicin 45 mg/m(2) was administered . Cycles were repeated every 21 to 28 days . (99m)Tc-sestamibi scans were performed before and during administration of VX-710 alone . RESULTS: Of the 28 patients who enrolled, 25 patients were eligible for analysis . No dose-limiting toxicity (DLT) was observed in the nine assessable patients who received 120 mg/m(2)/h or less . Among seven patients receiving VX-710 160 mg/m(2)/h, two DLTs were seen: reversible CNS toxicity and febrile neutropenia . All other adverse events were mild to moderate and reversible . Plasma concentrations of VX-710 in patients who received at 120 and 160 mg/m(2)/h were two- to fourfold higher than concentrations required to fully reverse drug resistance in vitro . VX-710 exhibited linear pharmacokinetics with a harmonic mean half-life of 1.1 hours . VX-710 enhanced hepatic uptake and retention of (99m)Tc-sestamibi in all patients . CONCLUSION: A 96-hour infusion of VX-710 at 120 mg/m(2)/h plus doxorubicin 45 mg/m(2) has acceptable toxicity in patients with refractory malignancies . The safety and pharmacokinetics of VX-710 plus doxorubicin warrant efficacy trials in malignancies expressing P-gp and/or MRP1.

J Clin Oncol, 2001 Jun 15, 19(12), 2983 - 93
Phase I/II trial of the multidrug-resistance modulator valspodar combined with cisplatin and doxorubicin in refractory ovarian cancer; Baekelandt M et al.; PURPOSE: To determine the maximum-tolerated dose (MTD) of doxorubicin when given in combination with cisplatin and the multidrug-resistance (MDR) modulator valspodar and the remission rate induced by this combination in patients with platinum- and anthracycline-resistant ovarian cancer . PATIENTS AND METHODS: Fifty-nine patients who had failed prior platinum- and anthracycline-based chemotherapy were enrolled . During the dose-finding phase, patients received a loading dose of valspodar (1.5 or 2 mg/kg) via 2-hour intravenous (IV) infusion on day 1 and continuous IV infusion (CIVI) of valspodar (2, 4, or 10 mg/kg/d) over 3 days . Doxorubicin (starting from 20 up to 50 mg/m(2)) and cisplatin (50 mg/m(2)) were administered via 15- to 20-minute IV infusions on day 3 . During the efficacy phase, patients received at least two treatment cycles unless toxicity was unacceptable, and responding patients and those with stable disease received four to six cycles . RESULTS: All patients completed at least one cycle of combined treatment . The MTD of doxorubicin was determined to be 35 mg/m(2) when administered with valspodar at 2 mg/kg loading dose and 10 mg/kg/d CIVI plus 50 mg/m(2) cisplatin . At these doses, valspodar blood concentrations known to reverse MDR in vitro were reached in all patients . Valspodar was well tolerated at all dose levels . Dose-limiting toxicities of the combination were primarily hematologic and included febrile neutropenia and prolonged leucopenia . The addition of valspodar to the treatment did not worsen cisplatin-related toxicity . Among 33 patients treated at the MTD for doxorubicin, one (3%) had a complete response, and four (12%) had a partial response . An additional seven patients experienced a stabilization of their previously progressive disease . The survival rates at 6 and 12 months were 59% and 19%, respectively . CONCLUSION: Valspodar can be safely coadministered with doxorubicin and cisplatin . Although the regimen used in this trial produced renewed responses in patients with heavily pretreated, refractory ovarian cancer, the value of valspodar in reversing resistance mediated by P-glycoprotein remains to be determined.

J Clin Oncol, 2001 Jun 15, 19(12), 2975 - 82
Phase II study of paclitaxel and valspodar (PSC 833) in refractory ovarian carcinoma: a gynecologic oncology group study; Fracasso PM et al.; PURPOSE: A phase II study was conducted to determine the efficacy of paclitaxel and valspodar (PSC 833) in patients with advanced epithelial ovarian cancer . Valspodar, a nonimmunosuppressive cyclosporine D analogue that reverses P-glycoprotein-mediated multidrug resistance, in combination with paclitaxel might be active in paclitaxel-resistant and refractory ovarian cancer . PATIENTS AND METHODS: Patients received valspodar 5 mg/kg orally qid x 12 doses . Paclitaxel (70 mg/m(2) intravenously for 3 hours) was administered on day 2, 2 hours after the fifth or sixth dose of valspodar . This treatment was repeated every 21 days . One blood sample was collected before the sixth dose of valspodar for the first three cycles to evaluate valspodar trough concentration . Tumor tissue was obtained from patients for immunohistochemical staining of P-glycoprotein . RESULTS: Of 60 patients entered, 58 were assessable for response . There were five partial responses (8.6%; 90% confidence interval {CI}, 3.8 to 20.0; median duration of response, 5.0 months {range, 1.9 to 10.5 months}) . Median progression-free survival was 1.5 months (90% CI, 1.4 to 2.4) . Grade 3 or 4 toxicities observed were neutropenia, anemia, nausea and vomiting, peripheral neuropathy, and cerebellar ataxia . The trough concentrations of valspodar were > or = 1,000 ng/mL in all but two of 40 patients in the first cycle . Immunohistochemical staining for P-glycoprotein was positive for one of two responding patients . CONCLUSION: Valspodar in combination with paclitaxel has limited activity in patients with paclitaxel-resistant ovarian carcinoma . An international randomized clinical trial of paclitaxel and carboplatin with or without valspodar as first-line therapy in advanced ovarian cancer is underway.

Aquat Toxicol, 2001 Aug, 53(3-4), 215 - 28
Prochloraz and nonylphenol diethoxylate inhibit an mdr1-like activity in vitro, but do not alter hepatic levels of P-glycoprotein in trout exposed in vivo; Sturm A et al.; P-glycoproteins (P-gps) encoded by multidrug resistance 1 (mdr1) genes are ATP-dependent transporters located in the cytoplasmic membrane which mediate the efflux of a broad spectrum of hydrophobic compounds from the cell . The tissue distribution of P-gps suggests their role in the organismal defense against xenobiotics by effecting xenobiotic excretion and reducing xenobiotic uptake . In the present work, the interaction of P-gp(s) in the liver and in primary cultured hepatocytes of rainbow trout with two model pollutants was studied - the imidazole fungicide prochloraz and the alkylphenolic surfactant nonylphenol diethoxylate (NP2EO) . Using a monoclonal antibody (mAB C219) directed against a conserved P-gp epitope, an immunoreactive protein of 160 kDa was detected in immunoblots of liver extracts from control trout . In sections of control trout livers, immunohistochemistry with the mAB C219 resulted in specific staining of bile canaliculi . In juvenile trout exposed for 7 days to sublethal concentrations of prochloraz (0.027 microM; 0.27 microM) or NP2EO (0.32 microM; 1.30 microM), no changes in levels of hepatic P-gp(s) were found in immunoblot and immunochemical investigations . The efflux of the fluorescent mdr 1 substrate rhodamine 123 (Rh123) from cultured isolated trout hepatocytes was partly inhibited by verapamil and vinblastine, compounds known to interfere with mdr 1-dependent transport . This demonstrates the presence of a mdr1-like mechanism in trout liver which is probably involved in the biliary excretion of hydrophobic xenobiotics . Non-cytotoxic concentrations of prochloraz and NP2EO were tested for effects on the efflux of Rh123 from trout hepatocytes . Prochloraz was a potent inhibitor of the mdr1-like mechanism, being effective at 0.3 microM and above . NP2EO inhibited Rh123 efflux only at the highest concentration tested (31.6 microM) . The accumulation and elimination of 14C-prochloraz by cultured trout hepatocytes was not affected by mdr 1-type substrates (Rh123, vinblastine) and a mdr 1 inhibitor (verapamil) . This shows that prochloraz is, despite its inhibitory potency, not a substrate of the mdr1-like mechanism in trout liver . The inhibition by prochloraz and NP2EO of the md r1-like mechanism in trout hepatocytes suggests that water pollutants can interfere with P-gp-function in fish and thus may impair the organismal defense against xenobiotics.

Eur J Pharmacol, 2001 Jun 1, 421(1), 1 - 9
Kinetics of glutathione and daunorubicin efflux from multidrug resistance protein overexpressing small-cell lung cancer cells; Salerno M et al.; The present study examined how the multidrug resistance protein (MRP1), which is an ATP-dependent anionic conjugate transporter, also mediates the transport of reduced glutathione (GSH) and the co-transport of the cationic drug, daunorubicin, with GSH in living GLC4/Adr cells . To obtain information on the affinity of GSH for the multidrug resistance protein in GLC4/Adr cells, we investigated the GSH concentration dependence of the ATP-dependent GSH efflux . The intracellular GSH concentration was modulated by preincubation of the cells with 25 microM buthionine sulfoximine, an inhibitor of GSH synthetase, for 0-24 h . The transport of GSH was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued . Fitting of the obtained data according to the Michaelis-Menten equation revealed a Km of 3.4+/-1.4 mM and a Vmax of 1.5+/-0.2x10(-18) mol/cell/s . The ATP-dependent transport of GSH was inhibited by 3-({{3-(2-{7-chloro-2-quinolinyl}ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl}thio)propanoic acid (MK571), with 50% inhibition being obtained with 1.4 microM MK571 . We investigated the GSH concentration dependence of the MRP1-mediated ATP-dependent transport of daunorubicin under conditions where the transport of daunorubicin became saturated . The daunorubicin transport was related to the intracellular GSH concentration up to approximately 5 mM and then plateaued . We were therefore in the situation where GSH acted as an activator: its presence was necessary for the binding and transport of daunorubicin by MRP1 . However, GSH was also transported by the multidrug resistance protein . The concentration of GSH that gave half the maximal rate of daunorubicin efflux was 2.1+/-0.8 mM, very similar to the Km value obtained for GSH . In conclusion, the rate of daunorubicin efflux, under conditions where the transport of daunorubicin became saturated, and the rate of GSH efflux determined at any intracellular concentration of GSH were very similar, yielding a 1:1 stoichiometry with respect to GSH and daunorubicin transport . These results support a model in which daunorubicin is co-transported with GSH.

Hematology, 2001, 5(5), 359 - 367
P-Glycoprotein Expression in Acute Myeloid Leukaemia Cells at Diagnosis: Its relationship to Daunorubicin or Idarubicin Induction Therapy and Survival; Malignancy; Pogliani EM et al.; We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo diagnosed acute myeloid leukaemia (AML) and the relationship between presence of P-gp in leukaemic cells and efficacy, as remission induction and survival rate, of two different anthracyclines, daunorubicin (DNR) and idarubicin (IDR) . We found that 30 out of 50 patients (60%) were negative (Group 1) and 20 (40%) were positive (Group 2) for P-gp expression evaluated by mean of MRK16 MoAb using a cut-off of 10% positive cells . Thirty-five out of 50 patients (70%) obtained complete remission (CR); depending on P-gp expression, the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005) . The median duration of overall survival was 20 months for patients in Group 1 as compared with 10 months for patients of Group 2 (p < 0.005) . Regarding the anthracycline used, no significant difference in CR was observed in patients of Group 1 (75% of CR with DNR vs . 90% with IDR); Group 2 obtained 40% of CR with DNR vs . 70% with IDR (p < 0.005) . The median duration of overall survival (OS) with the two regimens was comparable in Group 1, while it was significantly longer in patients of Group 2 treated with IDR compared with DNR regimen (p < 0.005) . These results confirm the prognostic value of P-gp expression in AML at first appearance and we suggest that idarubicin could be a valid anthracycline drug in the treatment of AML to be evaluated as potential drug of choice in patients with primary or drug-induced multidrug resistance.

Hematology, 2001, 5(5), 343 - 358
Application of Resistance Reversal Agents in Hematologic Malignancies; Malignancy; Current Clinical Practice; Fishman MN et al.; The clinical application of resistance reversal drugs for patients with hematologic malignancies is reviewed . The phenomenon of multidrug resistance versus other mechanisms are discussed . The pump-like mechanisms of P-glycoprotein, multidrug resistance associated protein, lung resistance protein and of other ATP binding cassette transporter proteins are reviewed briefly, as well as the important substrate drugs and pump-blocking compounds . The problems associated with resistance protein assays in clinical samples and the concept of prognostic versus therapeutic clinical relevance are described, within the context of selected hematologic malignancies . Toxicities and treatment outcomes of phase II and III trials of reversal agents in lymphoma, multiple myeloma, myelodysplastic syndromes, acute myeloid leukemia and blast phase of chronic myeloid leukemia are reviewed . Finally, current options for on-study management of relapsed or refractory hematologic malignancy patients are discussed.

Nucl Med Commun, 2001 Jun, 22(6), 645 - 50
Verapamil decreases accumulation of 99Tcm-MIBI and 99Tcm-tetrofosmin in human breast cancer and soft tissue sarcoma cell lines; Rodrigues M et al.; 99Tcm-methoxyisobutylisonitrile (99Tcm-MIBI) and 99Tcm-tetrofosmin are cationic tracers recognized by the efflux pump P-glycoprotein (Pgp) . Verapamil has been shown to be a competitive inhibitor of Pgp, and was one of the first multidrug-resistant reversing agents identified . The aim of this preclinical in vitro study was to evaluate the effects of verapamil on the accumulation of 99Tcm-MIBI and 99Tcm-tetrofosmin in the human breast cancer cell lines MCF-7 and SK-BR-3 and in the human soft tissue sarcoma cell lines SW 982 and SW 1353, in comparison with respective control cells, i.e . without preincubation with verapamil . After preincubation with 10 or 100 microM of verapamil for 15 or 30 min, the 99Tcm-MIBI and 99Tcm-tetrofosmin accumulation in cells was assessed at 10, 30 and 60 min after incubation with these tracers . Addition of verapamil caused a decline in the accumulation of the two tracers at all incubation times, as compared with control cells . These effects of verapamil were neither dose- nor preincubation time-dependent in most cells . Our data indicate that verapamil is not a promising agent for increasing the sensitivity of scintigraphy with 99Tcm-MIBI or 99Tcm-tetrofosmin, or for evaluating Pgp tumour status in these types of tumours.

Br J Haematol, 2001 May, 113(2), 369 - 74
Technetium-99m-sestamethoxyisobutylisonitrile scan as a predictor of chemotherapy response in malignant lymphomas compared with P-glycoprotein expression, multidrug resistance-related protein expression and other prognosis factors; Kao CH et al.; The purpose of the present study was to predict the response of malignant lymphomas (MLs) to chemotherapy using technetium-99m methoxyisobutylisonitrile (Tc-MIBI) scan and to compare it with the predictive ability of P-glycoprotein (P-gp) expression, multidrug resistance-related protein (MRP) expression and other prognosis factors . Twenty-five ML patients were enrolled in this study prior to initiation of chemotherapy . Images were obtained 10 min after intravenous injection of Tc-MIBI, interpreted visually and the tumour-to-background (T/B) ratios calculated . Immunohistochemical analyses were performed on sections of the biopsy specimens to determine P-gp and MRP expression . Chemotherapy response was evaluated in the first 1-2 years after completion of chemotherapy . The mean T/B ratio of the 15 patients with a good response (3.3 +/- 0.6) was significantly higher than that of the 10 patients with a poor response (1.2 +/- 0.1) . All 15 patients with a good chemotherapy response had positive Tc-MIBI scan results and negative P-gp and MRP expression . All 10 patients with a poor response had negative Tc-MIBI scan results and either positive P-gp or MRP expression . Other prognosis factors showed no significant difference in the incidence of good and poor responses . Tc-MIBI scan results represent P-gp or MRP expression more accurately than other prognosis factors and predict the chemotherapy response in ML patients.

Br J Haematol, 2001 May, 113(2), 339 - 46
Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide; Lotfi K et al.; Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy . In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP . The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively . Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIalpha (P < 0.001) and beta protein (P < 0.026), formed substantially lower amounts of the topo II-DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression . Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line . No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein . The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells . However, there was an increase in the activity of the cytosolic 5'-nucleotidases (5'-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0.001) for the high Km and 134% (P < 0.331) for the low Km 5'-NT in resistant cells . The high Km 5'-NT is probably responsible for the decreased amount of the active metabolite CdA 5'-triphosphate {40% decreased (P < 0.045)}, as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells . In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0.001) was observed in the resistant cells . Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5'-NT and higher dCTP levels . Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.

Anticancer Res, 2001 Mar-Apr, 21(2A), 879 - 85
Modulation of multidrug resistance in a cancer cell line by anti-multidrug resistance-associated protein (MRP) ribozyme; Hatanaka H et al.; Multidrug resistance-associated protein (MRP) is the major candidate molecule responsible for non-P-glycoprotein (PGp)-mediated multidrug resistance . We used a hammerhead anti-MRP ribozyme (alpha MRP-Rz) to inactivate MRP function in a multidrug resistant cancer cell line, KB8-5 . The beta-actin promoter-driven alpha MRP-Rz sequence (pH beta/alpha MRP-Rz) was introduced into KB8-5 cells (KB8-5/alpha MRP-Rz) and we evaluated growth of the cell line . The gene expression of multidrug resistance-related molecules was estimated . Drug sensitivity was estimated by MTT assay in vitro . MRP mRNA expression was decreased in KB8-5/alpha MRP-Rz cells . The MTT assay showed increased IC50 values or resistance to doxorubicin (DOX), etoposide (VP-16) and cisplatin (CDDP) in KB8-5/alpha MRP-Rz cells . No significant differences were observed in expression of multidrug resistance gene (MDR1), thymidylate synthase, glutathione S-transferase pi or topoisomerase II alpha . The hammerhead ribozyme-mediated simple suppression of MRP mRNA expression was not sufficient to reverse multidrug resistance in the cancer cell line KB8-5.

Anticancer Res, 2001 Mar-Apr, 21(2A), 847 - 56
Influence of beta-adrenergic antagonists, H1-receptor blockers, analgesics, diuretics, and quinolone antibiotics on the cellular accumulation of the anticancer drug, daunorubicin: P-glycoprotein modulation; Ibrahim S et al.; BACKGROUND: Treatment of patients with several drugs simultaneously may result in modulation of the naturally expressed P-glycoprotein (Pgp) at different tissues . With this possibility in mind, we have assessed the ability of different classes of drugs to modulate Pgp function in vitro . Modulation of the Pgp function was studied at in vitro drug concentrations comparable to therapeutic blood levels of the drugs . MATERIALS AND METHODS: Human blood brain barrier endothelial cells and human colon adenocarcinoma cells were transduced or transfected with the multidrug resistance gene (MDR1) to express Pgp . The uptake of fluorescent substrates of Pgp, Rhodamine 123 and daunorubicin, into these cells and NIH3T3/MDR1 and MDCK/MDR1 cells was measured by flow cytometry and in monolayers in the presence and absence of the different drugs . RESULTS: From the tested six H1-receptor blockers, seven beta-adrenergic antagonists, four analgesics, ten diuretics and five quinolons, five drugs inhibited Pgp at therapeutic blood levels and two at somewhat higher concentrations . Significant synergism for blocking Pgp could be demonstrated for several drugs . CONCLUSION: We conclude that administration of several drugs which modulate the function of Pgp to patients may adversely affect the natural function of this efflux pump and may cause drug-drug interactions induced side effects.

Anticancer Res, 2001 Mar-Apr, 21(2A), 1189 - 94
Inhibitory effect of steroidal alkaloids on drug transport and multidrug resistance in human cancer cells; Lavie Y et al.; Intrinsic or acquired resistance of tumor cells to multiple cytotoxic drugs (multidrug resistance MDR) is a major cause of failure of cancer chemotherapy . MDR is often caused by elevated expression of drug transporters such as P-glycoprotein (P-gp) or multidrug resistance protein (MRP) . A number of compounds, termed chemosensitizers, have little or no cytotoxic action of their own, but inhibit (P-gp) or MRP-mediated drug export and are capable of sensitizing MDR cells to the cytotoxic effects of chemotherapeutic drugs . Here we examined the ability of steroidal alkaloids of plant origin, namely the Veratrum sp . alkaloid cyclopamine and the Lycopersicon sp . alkaloid tomatidine, to act as potent and effective chemosensitizers in multidrug resistant tumor cells . Drug uptake was determined by measuring accumulation of tetramethylrosamine in multidrug resistant NCI AdrR human adenocarcinoma cells . Resistance to adriamycin and vinblastine was determined by utilizing the MTT cell survival assay . Cyclopamine and tomatidine elevate tetramethylrosamine uptake by NCI AdrR cells and sensitize the cells to the cytotoxic action of adriamycin and vinblastine . In both cases these agents are comparable in patency and efficacy to verapamil, a reversal agent commonly used in MDR research . It is concluded that steroidal alkaloids of plant origin act as inhibitors of P-gp-mediated drug transport and multidrug resistance and therefore may serve as chemosensitizers in combination chemotherapy with conventional cytotoxic drugs for treating multidrug resistant cancer.

Anticancer Res, 2001 Mar-Apr, 21(2A), 1023 - 7
Modulation of cancer cell multidrug resistance by an extract of Ficus citrifolia; Simon PN et al.; Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer . Previous studies have shown that natural compounds such as prenyl flavonoids are able to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells . A fraction from the dichloromethane extract of a common Guadalupe Ficus, Ficus citrifolia was studied for its direct interaction with the purified C-terminal cytosolic domain of P-glycoprotein, and for its induced accumulation and cytotoxicity of vinblastine and daunomycin in two model cell lines overexpressing P-glycoprotein, namely K562/R7 and MESSA/Dx5 . The fraction bound with high affinity to P-glycoprotein C-terminal cytosolic domain and was as efficient as cyclosporin A to increase intracellular accumulation of daunomycin in K562/R7 leukemic cells . Moreover, the fraction markedly enhanced the cytotoxic effect of vinblastine on the growth of MESSA/Dx5 cells . These results suggest that Ficus citrifolia possesses important therapeutic potential for improving the efficacy of cancer chemotherapy.

Bratisl Lek Listy, 2001, 102(2), 66 - 72
Immunohistochemical detection of LRP protein in the normal human lung; Rybarova S et al.; In this study, we have determined the LRP (lung resistance-related protein) by immunohistochemical method . LRP belongs to proteins which cause the multidrug resistance (MDR) . It has been found in various normal human tissues, where it plays a protective role against toxic compounds . Multidrug-resistant cells distribute the cytotoxic drug into the perinuclear region and then redistribute it back into the cytoplasm . It is just a hypothesis today that LRP can mediate drug resistance by regulating both the cytoplasmic redistribution and the nucleocytoplasmic transport of drugs . In order to detect LRP we have used the paraffin-embedded sections of the normal human lung tissue . LRP was predominantly located in two regions: 1) in bronchial epithelial cells and 2) in alveolar macrophages . Positive cells were coloured brown and showed strong reactivity . Negative control included the omitting of primary antibody and replacing it by buffer solution . Bronchial epithelial cells and alveolar macrophages stayed uncoloured, i.e . unreactive . (Fig . 5, Ref . 17.).

Anticancer Drugs, 2001 Jun, 12(5), 475 - 83
Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells; Johansson S et al.; The saponin digitonin, the aglycone digitoxigenin and five cardiac glycosides were evaluated for cytotoxicity using primary cultures of tumor cells from patients and a human cell line panel (representing different cytotoxic drug-resistance patterns) . Of these seven compounds, proscillaridin A was the most potent (IC(50): 6.4--76 nM), followed by digitoxin, and then ouabain, digoxin, lanatoside C, digitoxigenin and digitonin . Correlation analysis of the log IC(50) values for the cell lines in the panel showed that compound cytotoxicity was only slightly influenced by resistance mechanisms that involved P-glycoprotein, topoisomerase II, multidrug resistance-associated protein and glutathione-mediated drug resistance . Digitoxin and digoxin expressed selective toxicity against solid tumor cells from patients, while proscillaridin A expressed no selective toxicity against either solid or hematological tumor cells . The results revealed marked differences in cytotoxicity between the cardiac glycosides, both in potency and selectivity, and modes of action for cytotoxicity that differ from that of commonly used anticancer drugs.

Anticancer Drugs, 2001 Jun, 12(5), 441 - 51
Altered DNA-cleavage activity of topoisomerase II from WEHI-3B leukemia cells with specific resistance to ciprofloxacin; Pessina A et al.; In order to investigate the mechanisms of drug resistance arising in tumor cells, we investigated the capacity of fluoroquinolones to inhibit the in vitro growth of WEHI-3B monomyelocytic leukemia cells and then we established a variant of this line (currently maintained in the absence of drug) . The line, named WEHI-3B/CPX, expresses a specific resistance to ciprofloxacin (CPX; resistance index=17.3+/-2.2), and does not show cross-resistance with other fluoroquinolones, camptothecin and topoisomerase II inhibitors such as doxorubicin, etoposide and teniposide . Although a little decrease in intracellular accumulation of CPX is observed in WEHI-3B/CPX cells, these cells do not express MDR or LRP markers, and the resistance is not circumvented by verapamil . Purified nuclear extracts from WEHI-3B and WEHI-3B/CPX cells were tested for topoisomerase I catalytic activity and checking in vitro topoisomerase I sensitivity to CPX and camptothecin inhibition, but no difference was observed . As the treatment with CPX showed that the resistant cell line suffers a significantly lower number of breaks in the DNA molecule we also addressed our investigations to the topoisomerase II-dependent DNA cleavage that, in the resistant clone, was found dramatically less susceptible to be enhanced by CPX both in pre-strand and post-strand DNA passage conditions . WEHI-3B/CPX cells do not express any character of multidrug resistance and represent a rare case of specific drug resistance to CPX . The specific resistance to CPX observed in these cells is related to a functional decrease of topoisomerase II cleavage activity . It could be consequent to a decreased binding affinity of CPX for the topoisomerase II--DNA complex or to a decreased affinity or specificity of topoisomerase II for its DNA cleavage sites.

Anticancer Drugs, 2001 Jun, 12(5), 419 - 32
Active transepithelial transport of irinotecan (CPT-11) and its metabolites by human intestinal Caco-2 cells; Yamamoto W et al.; Irinotecan (CPT-11) is a camptothecin analog with low (about 10--20%) and variable oral bioavailability in animal models . Here, Caco-2 cells were used to evaluate the transepithelial transport of CPT-11 and its metabolites . Caco-2 cells demonstrated significant expression of P-glycoprotein (P-gp), multidrug resistance-associated protein and canalicular multispecific organic anion transporter . Both the lactone and carboxylate forms of CPT-11 and SN-38 were actively transported across the cell monolayers, mainly by the apical-localized P-gp pump . Cellular permeability of CPT-11 at a concentration of 17 microM converted from active to passive-diffusional transport between the 2 and 6 h exposure time points . Antiproliferative effects of CPT-11 were related to permeability of the lactone form, whereas for SN-38 efficacy was dependent on lactone accumulation . Exposure of CPT-11 with cyclosporin A significantly enhanced its efficacy, whereas this was not observed with verapamil and R101933 . In contrast, SN-38 efficacy decreased in the presence of P-gp inhibitors due to active transport toward the basolateral side, thereby reducing drug accumulation . Hence, multiple-active transport systems could be demonstrated to be responsible for not only accumulation profiles but also cytotoxic efficacy of CPT-11 and SN-38 in the intestinal Caco-2 cells . It is suggested that CPT-11 might act in a time-dependent manner and that SN-38-mediated cytotoxicity relates to (dose-dependent) lactone kinetics . The results detailed in this report could contribute toward the development of a clinically useful oral formulation of CPT-11 with improved absorption characteristics and suggest that cyclosporin A is a suitable agent for further research of this concept.

Anticancer Drugs, 2001 Jun, 12(5), 401 - 17
Quinone isomers of the WS-5995 antibiotics: synthetic antitumor agents that inhibit macromolecule synthesis, block nucleoside transport, induce DNA fragmentation, and decrease the growth and viability of L1210 leukemic cells more effectively than ellagic acid and genistein in vitro; Perchellet EM et al.; Antibiotic WS-5995A (code name J4) and two of its synthetic analogs, o-quinone J1 and model p-quinone J7, which show some structural similarity with both ellagic acid (EA) and genistein (GEN), were compared for their antileukemic activity in L1210 cells in vitro . Overall, J4 is more cytostatic and cytotoxic than J1 and J7, suggesting that methyl and methoxy substitutions, a p-quinone moiety, and a hydrogen bonding phenolic group may enhance the antitumor potential of these naphthoquinone lactones, which are all more potent than EA and GEN . For instance, the lead compound J4 inhibits tumor cell proliferation and viability at day 4 (IC(50): 0.24--0.65 microM) more effectively than EA (IC(50): 5--6 microM) and GEN (IC(50): 7 microM) . Since J4 does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression like EA and GEN . A 1.5- to 3-h pretreatment with J4 is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC(50): 2.0--2.5 microM) determined over 30- to 60-min periods of pulse-labeling in L1210 cells in vitro, whereas EA (IC(50): 20-130 microM) and GEN (IC(50): 40--115 microM) are less effective against macromolecule synthesis . In contrast to 156 microM EA, which is inactive, a 15-min pretreatment with 10--25 microM J4 has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides over a 30 s period in vitro, an effect which can be mimicked by 156 microM GEN . Hence, the WS-5995 analogs and GEN may prevent the incorporation of {(3)H}adenosine and {(3)H}thymidine into DNA because they rapidly block the uptake of these nucleosides by the tumor cells . After 24 h, the concentration-dependent induction of DNA cleavage by J4 peaks at 10 microM and declines at 25 microM, whereas EA and GEN are ineffective at 10 microM but maximally stimulate DNA cleavage at 62.5 microM . Like EA and GEN, the mechanism by which J4 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO(4), suggesting that J4 triggers apoptosis by caspase and endonuclease activation . Because they are more potent than EA and GEN, and affect both nucleoside transport and DNA cleavage, the WS-5995 antitumor antibiotics might be valuable in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Int J Cancer, 2001 Jul 1, 93(1), 114 - 22
Determining MDR1/P-glycoprotein expression in breast cancer; Faneyte IF et al.; The mechanism of chemotherapy resistance in breast cancer is unresolved . MDR1/P-glycoprotein (P-Gp) over-expression confers multidrug resistance in vitro and might play a role in clinical breast cancer . Studies using clinical samples have yielded conflicting results . MDR1/P-Gp mRNA expression was determined relative to the expression in normal human liver using TaqMan real-time RT-PCR (corrected for expression of the housekeeping gene PBGD) . Immunohistochemistry (IHC) was performed with monoclonal antibodies against P-Gp (JSB1, C219) . The positive control was SW1573/2R160, the intermediate control SW1573 and the negative control GLC4/ADR . We assayed 9 breast-cancer cell lines by RT-PCR and IHC, 52 carcinoma samples by RT-PCR and 168 samples by IHC . SW1573/2R160 contained high levels of MDR1/P-Gp mRNA (1.0, equal to liver) and showed strong membranous staining . Expression of MDR1/P-Gp mRNA in SW1573 (0.05) and GLC4/ADR (3.2 x 10(-5)) was not detectable by IHC . Very low levels of MDR1/P-Gp mRNA were measured in breast-cancer cell lines (mean 3.1 x 10(-4), range 1 to 12 x 10(-4)), but P-Gp was not detected by IHC . In 25 specimens from chemotherapy-naive patients, MDR1/P-Gp mRNA levels varied from 1 to 11 x 10(-2) (mean 3.9 x 10(-2)) . In sections of 80 chemotherapy-naive tumors, no membrane-bound staining was observed in the tumor cells . Tumors of 27 anthracycline-treated patients had comparable MDR1/P-Gp mRNA expression levels (mean 5.4 x 10(-2)) . P-Gp was undetectable in 88 tumor samples of patients who had received anthracycline-based chemotherapy . In breast cancer, MDR1/P-Gp mRNA is low or absent and P-Gp levels in cancer cells are too low to detect by IHC . Chemotherapy exposure does not result in detectable MDR1/P-Gp over-expression .

Int J Cancer, 2001 Jul 1, 93(1), 107 - 13
Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol A on MRP1 function; Chen ZS et al.; We previously isolated agosterol A (AG-A) from a marine Spongia sp . and found that it completely reversed colchicine resistance in P-glycoprotein (Pgp)-over-expressing KB-C2 cells and vincristine resistance in multidrug-resistance protein (MRP)1-over-expressing CV60 cells . However, a tri-deacetylated derivative of AG-A (IAG-A) showed almost no activity in reversing Pgp- or MRP1-mediated drug resistance . In this study, we examined the mechanisms by which AG-A reverses MRP1-mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1-over-expressing human KB carcinoma (KB/MRP) cells . {3H}-Leukotriene C4 (LTC4), {3H}-2,4-dinitrophenyl-S-glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular {3H}-vincristine accumulation and efflux in KB/MRP cells were measured with or without AG-A and/or inactive IAG-A . AG-A reduced MRP1-mediated {3H}-LTC4 transport in a dose-dependent manner, but IAG-A did not . Inhibition by AG-A was competitive, with a K(i) value of 31 microM . AG-A at 10 microM enhanced the accumulation of {3H}-vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent . Likewise, ATP-dependent efflux of {3H}-vincristine from KB/MRP cells was enhanced compared with KB-3-1 cells and inhibited by AG-A . In addition, AG-A reduced intracellular levels of glutathione, a compound required for MRP1-mediated transport of some anti-cancer drugs . These findings suggest that AG-A reverses MRP1-mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs . In addition, the capacity of AG-A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1 .

Int J Cancer, 2001 Jul 1, 93(1), 62 - 6
Expression of multidrug-resistance P-glycoprotein (MDR1) in human brain tumors; Demeule M et al.; Multidrug resistance (MDR) is associated with the expression of P-glycoprotein (P-gp), an ATP-dependent transporter which expels anti-cancer drugs from cells . In the present study, MDR1 P-gp was immunodetected by Western blot analysis in 60 human brain tumors, including meningiomas, schwannomas, low-grade gliomas (astrocytomas, pilocytic astrocytomas) and high-grade gliomas (anaplastic astrocytomas, glioblastomas and anaplastic oligodendrogliomas) . Most samples from primary tumors expressed P-gp at the same levels as normal brain tissue except for schwannomas, in which levels were reduced by 65%, and meningiomas, in which levels were more than 10-fold higher in 7 of 10 samples . P-gp levels were 70% and 95% lower in brain metastases from melanomas and lung adenocarcinomas, respectively, than in normal brain tissue . These results indicate that the majority of primary brain tumors express MDR1 P-gp and that its high expression levels in meningiomas may be a marker for this type of brain tumor .

Hepatology, 2001 Jun, 33(6), 1425 - 31
Induction of Mdr1b expression by tumor necrosis factor-alpha in rat liver cells is independent of p53 but requires NF-kappaB signaling; Ros JE et al.; The multidrug resistance protein Mdr1b in rats is up-regulated during liver regeneration after partial hepatectomy or after endotoxin treatment . We hypothesize that up-regulation of Mdr1b in these models is TNF-alpha-dependent . The mechanism of Mdr1b activation by TNF-alpha is unknown as TNF-alpha can signal through various pathways, including NF-kappaB and p53, transcription factors for which binding sites in the Mdr1b promoter have been identified . We aimed to elucidate the mechanism of up-regulation of Mdr1b by TNF-alpha . We selectively used constructs expressing dominant negative Fas-associated death domain protein (FADD), TNF receptor associated factor-2 (TRAF2) or IkappaB to inhibit pathways downstream of the TNF receptor . Further, the proteasome inhibitor MG-132 was used, which prevents the breakdown of IkappaB . We show a critical role for NF-kappaB in activation of Mdr1b gene expression both in primary rat hepatocytes and in rat hepatoma H-4-II-E cells . Because p53 is up-regulated by TNF-alpha in an NF-kappaB-dependent manner and the Mdr1b promoter contains a p53 binding site, we used liver cells expressing a dominant negative p53 to show that TNF-alpha up-regulation of Mdr1b is independent of functional p53 . Using transient transfection assays, we show that Mdr1b up-regulation correlates with activation of the promoter . Mutation of the NF-kappaB site in the Mdr1b promoter prevents its induction by TNF-alpha . In conclusion our results show that activation of the rat Mdr1b gene by TNF-alpha is a result of NF-kappaB signaling and independent of p53.

J Acquir Immune Defic Syndr, 2001 Apr 15, 26(5), 423 - 34
A pilot study of the use of mycophenolate mofetil as a component of therapy for multidrug-resistant HIV-1 infection; Coull JJ et al.; Mycophenolic acid (MPA) increases the activity of both abacavir (ABC) and didanosine (ddI) in vitro against wild-type and multinucleoside-resistant HIV . We treated 7 patients with diagnosed AIDS who did not respond to eight or more antiretroviral therapies in an open label pilot study with mycophenolate mofetil (MMF), ABC, ddI, amprenavir (APV), and ritonavir (RTV), with or without efavirenz (EFV).Therapy was well tolerated despite the patients' advanced disease states . No significant decline in lymphocyte or other blood counts was observed . Median HIV RNA was 5.26 log10 copies/ml at entry, 4.53 log10 copies/ml at 4 weeks, and 5.13 log10 copies/ml at 16 weeks . Median CD4+ count was 34 cells/microl at entry and 39 cells/microl at 16 weeks of therapy . CD4+ counts increased further in five study subjects on extended therapy to 25 weeks (median 27 cells/microl at entry, 66 cells/microl at close), despite loss of virologic suppression in 4 of 5 cases . MPA can induce apoptosis in lymphocytes in vitro . However despite viral rebound, cell surface markers of apoptosis and activation declined in total CD3+ cells and CD3+/CD4+ cells twofold to fourfold in 4 of 5 adherent study subjects at 16 weeks, reaching levels comparable with those found in seronegative donors.Although low-dose MMF appears safe in late-stage HIV disease, this study did not demonstrate virologic efficacy . Higher doses of MMF may be more effective . With careful monitoring of toxicities and pharmacokinetics, MMF deserves further testing in HIV therapy.

Biochem Pharmacol, 2001 Jul 15, 62(2), 199 - 206
Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1; Bagrij T et al.; The ATP-dependent transport of natural product drugs, e.g . vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not . The present results suggest, however, that GSH can modulate transport of anionic species . Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells . As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells . Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF . Transport of the anionic conjugate dinitrophenyl-glutathione ({(3)H}DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells . At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 microM vincristine inhibited uptake but only in the presence of 1--3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport . Agents shown to reduce efflux of calcein from resistant cells, i.e . indomethacin, MK-571, and probenecid, also inhibited {(3)H}DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein . At concentrations achievable within cells, GSSG (70 microM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake . We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.

Clin Infect Dis, 2001 Jul 1, 33(1), 48 - 53 Epub 2001 May 23.
Bacteremia due to quinolone-resistant Escherichia coli in a teaching hospital in South Korea; Cheong HJ et al.; Quinolone-resistant Escherichia coli (QREC) strains are being isolated with increasing frequency . From 1993 to 1998, 40 cases of QREC bacteremia were observed in a teaching hospital; 25 episodes (63.5%) were community-acquired . The incidence of QREC bacteremia increased steadily, from 6.7% to 24.6% during 5 years, and correlated with the significantly increased use of fluoroquinolones (P = .003, r = 0.98) . When the 40 QREC bacteremic patients were compared with 80 patients with bacteremia due to quinolone-susceptible E . coli, prior fluoroquinolone use was the only independent risk factor for QREC bacteremia (P = .001) . A high APACHE II score was the only independent risk factor for death . The rate of multidrug resistance of QREC was much higher (60%) than that of quinolone-susceptible isolates (13.8%) . Pulsed-field gel electrophoresis patterns of these isolates were diverse . Therefore, the isolates revealed little evidence of clonal spread and may have emerged in direct response to the selective pressure exerted by prior fluoroquinolone use.

Blood, 2001 Jun 15, 97(12), 3882 - 9
Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor cells from patients with acute myeloid leukemia; Feuring-Buske M et al.; Efflux of Hoechst 33342 from normal hematopoietic cells identifies a "side population" (SP(+)) of negatively staining cells that, in the mouse, are largely CD34(-) and are enriched for primitive progenitors . To further characterize human SP(+) cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression . The mean percentages of SP(+) and MDR(+) cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values . The percentages of SP(+) cells that were CD34(+)CD38(-), CD34(+)CD38(+), or CD34(-) were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%) . Cytogenetically abnormal cells were always detected in the SP(-)CD34(+)CD38(-) and SP(+)CD34(-) fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia-IC were detected in the former fraction . No progenitors were detected among SP(+)CD34(-) cells in any of these assays from 9 of 10 samples . In contrast, exclusively normal cells were detected in the SP(+)CD34(+)CD38(-) fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively . In contrast to murine studies, primitive progenitors are enriched among SP(+)CD34(+)CD38(-) cells from patients with AML . The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression . (Blood . 2001;97:3882-3889)

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Dec, 30(4), 360 - 2
{Study on the reversal of cancer multidrug resistance by Chinese medicine Fw13-te41 in nude mice}; Zhang X et al.; We have reported that three reversal agents were sifted out from 32 Chinese galenicals through a series of cell culture tests . Among them, Fw13-te41 has the best effect of reversal cancer multidrug resistance (MDR) in vitro . In this study, the reversal action of Fw13-te41 in vivo was studied on the animal model of nude mice with human leukemia k562/ADR . Twenty SPF BALB/c-nu/nu nude mice with xenograft tumor were randomly divided into the control group (n = 6), VCR group {intraperitoneal (i.p.) VCR 250 micrograms/week, n = 5}, VCR + Fw13-te41 group (i.p VCR 250 micrograms/week + Fw13-te41 0.2 ml/day, equivalent to crude drug 10 g/kg, n = 5), and Fw13-te41 group (i.p Fw13-te41 0.2 ml/day, equivalent to crude drug 10 g/kg, n = 4) . After 18 days, the rate of tumor inhibition (RTI) of VCR group was 19.79%, but the RTI of VCR + Fw13-te41 group was as high as 86.95% (P < 0.05) . There results demonstrate that the Chinese medicine Fw13-te41 has an evident reversal action of malignancy MDR in vitro and in vivo.

Eur J Obstet Gynecol Reprod Biol, 2001 Jun, 96(2), 202 - 8
Expression of glutathione S-transferase pi (GST-pi) in human malignant ovarian tumors; Satoh T et al.; OBJECTIVES: In recent years, glutathione S-transferase pi (GST-pi) has attracted much attention and has been studied as a mechanism of multidrug resistance of tumors to anticancer drugs . In the present study, we immunohistologically measured the expression of GST-pi in tumor tissues using surgical specimens obtained from patients with malignant ovarian tumors . METHODS: Of 137 patients with malignant ovarian tumors treated and managed during a period of 20 years since the establishment of Tsukuba University Hospital, 117 patients were selected as subjects because of the presence of complete data on their clinical courses as well as paraffin blocks preserved in a good condition . GST-pi in these specimens was immunohistochemically stained to determine the correlation between GST-pi stainability and clinical outcomes . Stainability was graded as 0 when GST-pi was completely absent, 1 when less than 20% of tumor cells were stained, 2 when 20--60% were stained, and 3 when more than 60% were stained . RESULTS: When the correlation between stainability and clinical outcomes was analyzed with Kaplan--Meier method, excluding stage Ia cases that did not receive adjuvant chemotherapy at our hospital, significantly better clinical outcomes were observed in the low stainable group, compared with the high stainable group (P<0.01--0.05, Cox--Mantel test, Wilcoxon's test) . CONCLUSION: Since the stainability for GST-pi was high in tumors of histological types with strong resistance to anticancer drugs, and better clinical outcomes were observed in cases having a lower stainability score, the expression of GST-pi was thought to play some role in the resistance of malignant ovarian tumors to anticancer drugs.

Curr Infect Dis Rep, 2001 Jun, 3(3), 302 - 308
Immune Reconstitution Strategies in HIV; Leibowitz MR et al.; Although potent antiretroviral therapy can dramatically decrease HIV replication and improve some aspects of host immunity, incomplete immune reconstitution persists even after several years of fully suppressive therapy . In addition, long-term toxicities of antiretroviral medications and the probability of developing multidrug-resistant virus with long-term use indicate that alternate means of controlling viral replication are needed for more durable suppression of HIV . Immune-based therapies may help potentiate the host's own defenses against HIV and other pathogens, and may ultimately result in more durable viral suppression and lower incidence of antiretroviral therapy-related side effects and toxicities.

Br J Haematol, 2001 Jun, 113(3), 713 - 26
Comparison of 'sequential' versus 'standard' chemotherapy as re-induction treatment, with or without cyclosporine, in refractory/relapsed acute myeloid leukaemia (AML): results of the UK Medical Research Council AML-R trial; Liu Yin JA et al.; This aim of the acute myeloid leukaemia (AML)-R trial was to compare sequential (Seq) ADE (cytarabine, daunorubicin, etoposide) with standard (Std) ADE as remission re-induction treatment and to assess any benefit of cyclosporine (CSA) as a multidrug resistance modulator in refractory/relapsed AML patients . Seq ADE, based on the concept of Timed Sequential Therapy, comprised the same drugs as Std ADE but given at higher doses and in a different sequence . Between 1992 and 1997, 235 patients with relapsed (175) and refractory (60) AML were entered: 170 were randomized between Std versus Seq ADE and 213 between CSA versus no CSA . CSA was initially given at a dose of 5 mg/kg/d and increased to 10 mg/kg/d in the latter part of the trial . Overall, the complete remission (CR) rate was 43%, with Std ADE being significantly better than Seq ADE (54% versus 34%, P = 0.01) . CR rates did not differ between the CSA and no CSA arms (41% versus 45%, P = 0.6) . Overall, 3 year disease-free survival (DFS) of remitters was 16%, with a relapse risk of 70% . DFS was not significantly different between the chemotherapy or the CSA arms . Overall, 3 year survival was 8% . Survival with Std ADE was significantly better than with Seq ADE (12% versus 6%, P = 0.03) . CSA did not affect overall survival, except in patients > or = 60 years, who fared worse on CSA (P = 0.0003) . No difference in haematological toxicity between the chemotherapy or CSA arms was seen . Survival was better with longer first CR duration (P < 0.0001) . We conclude that Std ADE was superior to Seq ADE for CR achievement and survival, with no benefit with CSA, at the doses used in this studyPublication Types:

	Clinical Trial
	Randomized Controlled Trial

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