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