J Clin Invest, 2002 Dec, 110(11), 1651 - 8 Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion; Ma T et al.; Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults . The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis . We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR . Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified . The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM . CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models . At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters . A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours . Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas. J Chemother, 2002 Oct, 14(5), 518 - 25 Multidrug resistance in ovarian cancer: comparing an immunocytochemical study and ATP-tumor chemosensitivity assay; Raspollini MR et al.; The aim of our study was to evaluate the possible prognostic and predictive significance of the expression of P-glycoprotein, a transmembrane transport protein related to multidrug resistance, in previously untreated patients with FIGO stage III ovarian cancer; to compare the results of immunocytochemical analysis of tissue sections of tumors to the in vitro chemosensitivity to cytotoxic drug of fresh samples of the same tumors; and to evaluate survival in women who underwent the same surgical treatment and the same adjuvant chemotherapy. Cancer Res, 2002 Dec 1, 62(23), 6938 - 43 Protracted low-dose effects on human endothelial cell proliferation and survival in vitro reveal a selective antiangiogenic window for various chemotherapeutic drugs; Bocci G et al.; Recent preclinical studies have shown that frequent administration in vivo of low doses of chemotherapeutic drugs ("metronomic" dosing) can affect tumor endothelium and inhibit tumor angiogenesis, reducing significant side effects (e.g., myelosuppression) involving other tissues, even after chronic treatment . This suggests that activated endothelial cells may be more sensitive, or even selectively sensitive, to protracted ("high-time") low-dose chemotherapy compared with other types of normal cells, thus creating a potential therapeutic window . To examine this hypothesis, we assessed the effects of several different chemotherapeutic drugs--namely paclitaxel, 4-hydroperoxycyclophosphamide, BMS-275183 (an oral taxane), doxorubicin, epothilone B (EpoB) and its analogue 5-methylpyridine EpoB--on human microvascular or macrovascular endothelial cells, fibroblasts, and drug-sensitive or multidrug-resistant breast cancer cell lines in cell culture, using both short-term (24 h) versus long-term (144 h), continuous exposures, where drug-containing medium was replaced every 24 h . Whereas little differential and only weak effects were observed using the short-term exposure, a striking trend of comparative vascular endothelial cell hypersensitivity was induced using the continuous long-term exposure protocol . Potent differential growth inhibition effects as well as induction of apoptosis were observed with IC(50) values in the range of 25-143 pM for paclitaxel, BMS-275183, EpoB, and 5-methylpyridine-EpoB . In contrast, the IC(50) values for tumor cells and fibroblasts tested were in the range of 500 pM to >1 nM for these drugs . Similar differential IC(50) values were noted using 4-hydroperoxycyclophosphamide . The results are consistent with the possibility that continuous low-dose therapy with various chemotherapeutic drugs may have a highly selective effect against cycling vascular endothelial cells, and may be relevant to the use of continuous or frequent administration of low doses of certain types of drugs as an optimal way of delivering antiangiogenic therapy. Eur J Cancer, 2002 Dec, 38(18), 2455 - 62 Bcl-2 has differing effects on the sensitivity of breast cancer cells depending on the antineoplastic drug used; Del Bufalo D et al.; The aim of this paper was to evaluate the role of bcl-2 in the susceptibility of the MCF7 ADR human breast carcinoma line overexpressing the P-170 glycoprotein (P-170) to various drugs . The sensitivity to four multidrug resistance (MDR)-related drugs (doxorubicin (ADR), vincristine (VCR), vinblastine (VBL), actinomycin D (ACTD)) and three MDR-non-related drugs (cisplatin (DDP), bischloroethylnitrosourea (BCNU), 5-fluorouracil (5-FU)) was evaluated by the 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) assay in three bcl-2-overexpressing clones obtained from the MCF7 ADR line . We found that the bcl-2-overexpressing clones show increased resistance to DDP and BCNU, while no difference to 5-FU were observed between the control cells and bcl-2 transfectants . Surprisingly, bcl-2-overexpressing clones displayed an increased sensitivity compared with the control cells to the MDR-related drugs ADR, VCR, VBL and ACTD . Focusing on DDP and ADR, we found that the increased resistance of the bcl-2 transfectants to DDP was correlated to their ability to prevent apoptosis, while the enhanced sensitivity to ADR was associated with an increased ADR accumulation and a decreased ADR efflux . Moreover, while bcl-2 overexpression does not induce changes in P-170 glycoprotein expression, it did induce a reduction of the adenosine triphosphate (ATP) levels and basal protein kinase C (PKC) activity, both of which have a crucial role in the regulation of the MDR phenotype . In conclusion, the effect of bcl-2 on antineoplastic sensitivity observed in this study underscores the idea that bcl-2 may have distinct biological effects depending on the anticancer drug used. Eur J Cancer, 2002 Dec, 38(18), 2422 - 7 EVE/cyclosporin (etoposide, vincristine, epirubicin with high-dose cyclosporin)-chemotherapy selected for multidrug resistance modulation; Davidson A et al.; Sixteen children and young adults were treated with high-dose cyclosporin combined with a combination of cytotoxics (epirubicin, vincristine and etoposide) (EVE) known to be influenced by P-glycoprotein-mediated multidrug resistance (MDR) . Tumour types were neuroblastoma 3, Ewing's sarcoma 2, rhabdomyosarcoma 5, osteosarcoma 3, desmoplastic small round cell tumour 1, nephroblastoma 1, T-acute lymphoblastic leukaemia (ALL) 1 . All had progressed or relapsed following at least two of the drug types included in EVE . Acute reactions to cyclosporin and myelosuppression were the major toxicities documented . Renal and hepatic toxicity was rarely severe and always transient . Partial responses (PR) were observed in 2 patients (1 rhabdomyosarcoma, 1 Ewing's sarcoma) . We conclude that this combination is tolerable in heavily pretreated patients and may be suitable for further evaluation in untreated poor risk tumours. Eur J Cancer, 2002 Dec, 38(18), 2388 - 96 Phase I and pharmacological studies of the cryptophycin analogue LY355703 administered on a single intermittent or weekly schedule; Sessa C et al.; LY355703 is a synthetic derivative of the marine cryptophycins, cytotoxic agents which induce mitotic arrest by binding at the microtubule vinca binding domain . Promising preclinical features of LY355703 were the 40-400 greater potency than paclitaxel or vinca alkaloids, the broad spectrum of antitumor activity in xenografts and the antitumour activity in multidrug resistant (MDR)-expressing murine tumours . Aims of this study were to define the maximum tolerated dose (MTD) and the dose recommended for Phase II, the pattern of toxicity, the pharmacokinetic profile and to document hints of antitumour activity of LY355703 given as 2-h infusion on day 1 every 3 weeks (Study 1) or, later on, on days 1, 8 and 15 every 4 weeks (Study 2) . The latter weekly regimen was selected because of the acute dose-related toxicity reported in Study 1 . The dose was escalated using a modified Continual Reassessment Method . Pharmacokinetic studies were performed on day 1 of cycle 1 in both studies; LY355703 plasma concentrations were assessed by liquid chromatography with tandem mass spectrometry . A total of 35 adult patients with solid tumours entered Study 1; the dose was escalated from 0.1 to 1.92 mg/m(2); at this dose 2 of 5 patients presented grade 3 neuropathy and myalgias; 1.48 mg/m(2) was then recommended for Phase II study . A total of 8 patients were treated in Study 2 at 1 mg/m(2); cumulative long-lasting neuroconstipation and neurosensory toxicity precluded the completion of the cycle in 9 out of 15 cycles; the clinical development of the weekly regimen was then discontinued . Other toxicities included cardiac dysrhythmia and mild alopecia . Pharmacokinetics of LY355703 appeared to be linear over the dose range studied . The administration of LY355703 on a 3-week schedule is associated with an acute dose-dependent peripheral neuropathy and myalgia of high interpatient variability for which possible risk factors and pharmacokinetic correlates could not be identified. Jpn J Cancer Res, 2002 Nov, 93(11), 1230 - 6 Quantitative analysis of multidrug-resistance mdr1 gene expression in head and neck cancer by real-time RT-PCR; Tseng CP et al.; Progression of head and neck cancer is always associated with changes of gene expression profile . In this study, we characterized the expression of multidrug-resistance mdr1 gene, which may play a role in tumorigenesis and multidrug resistance in head and neck cancer . A TaqMan one-step RT-PCR with a linear range for quantification across at least a 5 log scale of concentration of mdr1 mRNA was designed to determine the level of mdr1 expression in 50 pairs of normal vs . malignant head and neck tissues . Both the absolute level of mdr1 mRNA in tumor (T) and the relative mdr1 expression between tumor and its normal counterpart (T/N) were measured and their associations with several clinical variables were analyzed . Among the clinical variables analyzed, only the clinical stage of tumor was found to be associated with mdr1 expression . The distribution of clinical stages differed significantly (P<0.01) among the 27 specimens that had a T/N>1, with 59.3%, 22.2%, 14.8% and 3.7% in stage IV, III, II, and I, respectively . In addition, 76% of stage IV and 75% of stage III tumors had a T/N>1 compared to 25% of stage II and 20% of stage I tumors (P=0.004) . Multivariate logistic regression analysis also indicated a significant difference of mdr1 expression between the early (I and II) and advanced (III and IV) stages tumors . The adjusted odds ratios (95% confidence intervals) were 1.477 (1.084 - 2.012) and 1.001 (1.000-1.002) for T/N (P<0.05) and T (P<0.05) treated as continuous variables, and 15.521 (3.414-70.550) and 5.074 (1.154-22.311) for T/N (P<0.001) and T (P<0.05) treated as binary variables, respectively . Taken together, the data presented here indicated that real-time RT-PCR provides a quantitative way to monitor mdr1 gene expression . The differential expression of mdr1 between early and advanced stages of head and neck cancer may shed light on the process of tumorigenicity and offer clues to the planning of new treatments. BJU Int, 2002 Dec, 90(9), 957 - 64 Intravesical pH: a potentially important variable affecting efficacy and the further development of anthracycline chemotherapy for superficial bladder cancer; Harris NM et al.; OBJECTIVE: To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro, the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy . MATERIALS AND METHODS: Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate . The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay . Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy . Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0 . The MDR-reversing agent verapamil was added at 100 micro g/mL to some incubations . RESULTS: Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0 . Flow cytometry results showed striking and consistent differences in epirubicin handling with pH . Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour . Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH . Adding verapamil to resistant cells restored the sensitive phenotype of drug handling . CONCLUSION: Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity . The potential for synergy at certain drug combinations will be enhanced by applying these findings . MDR reversal and fatty acid augmentation of drug uptake are discussed as examples. J Biol Chem, 2003 Feb 7, 278(6), 3599 - 605 Epub 2002 Nov 27. ATP binding, not hydrolysis, at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain; Hou YX et al.; Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP . We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J . R., and Chang, X . (2002) J . Biol . Chem . 277, 5110-5119) . These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2 . However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis . We now report the following . 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C . 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping . 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement . 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed . We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2. Eukaryot Cell, 2002 Oct, 1(5), 799 - 810 Dtrlp, a multidrug resistance transporter of the major facilitator superfamily, plays an essential role in spore wall maturation in Saccharomyces cerevisiae; Felder T et al.; The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast Saccharomyces cerevisiae . Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process . Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface . Here we present evidence that the sporulation-specific protein Dtrlp (encoded by YBR180w) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane . DTR1 was identified in a genome-wide screen for spore wall mutants . The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface . As a result, many mutant spores have aberrant surface structures . Dtrlp, a member of the poorly characterized DHA12 (drug:H+ antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation . Transport by Dtrlp may not be restricted to its natural substrate, bisformyl dityrosine . When expressed in vegetative cells, Dtrlp renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives . Dtrlp is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell. Int J Cancer, 2003 Jan 1, 103(1), 121 - 5 Modulation of P-glycoprotein but not MRP1- or BCRP-mediated drug resistance by LY335979; Shepard RL et al.; Our study examines the ability of LY335979 (Zosuquidar trihydrochloride) to modulate 3 distinct ABC transporters that are mechanisms of drug resistance: P-glycoprotein (Pgp, ABCB1), multidrug resistance associated protein (MRP1, ABCC2) and breast cancer resistance protein (BCRP, ABCG2) . Pgp-mediated resistance can be modulated by coadministration with the highly potent, selective inhibitor, LY335979 . Modulation of resistance by mitoxantrone and vinorelbine, 2 drugs used to treat certain solid tumors, was examined in a 3-day cytotoxicity assay using a panel of HL60 leukemia cell lines or MCF-7 breast cancer transfectants . LY335979, at 0.5 microM, substantially reversed mitoxantrone resistance and fully reversed vinorelbine resistance of Pgp-expressing HL60/Vinc cells . However, LY335979 did not modulate drug resistance in the MRP1-expressing HL60/ADR or drug-sensitive parental HL60 cells . To ascertain if LY335979 modulates BCRP-mediated drug resistance, the sensitivity of 26-fold mitoxantrone resistant, BCRP-transfected MCF-7 cells was evaluated . Addition of 5 microM LY335979, a concentration approximately 100-fold higher than the affinity of Pgp, had little to no effect on the BCRP transfectant . {(125)I}Iodomycin photolabeled Pgp in CEM/VLB(100) membranes and was inhibited by 5 microM LY335979 and GF120918 . No photolabeling of MRP or BCRP occurred in H69AR or MCF-7/BCRP membranes, respectively . These results further demonstrate that LY335979 is highly specific for Pgp and does not modulate MRP1- or BCRP-mediated resistance and can be used in combination with mitoxantrone and vinorelbine in tumor cells . Int J Cancer, 2003 Jan 1, 103(1), 29 - 37 Role of XIAP in the malignant phenotype of transitional cell cancer (TCC) and therapeutic activity of XIAP antisense oligonucleotides against multidrug-resistant TCC in vitro; Bilim V et al.; XIAP directly inhibits executor caspases, making it the most downstream antiapoptotic molecule . Here, we examined the expression and function of XIAP in normal urothelium and TCC . We also examined the therapeutic effect of xiap AS PODN on the cell cycle and apoptosis of multidrug-resistant T24 bladder cancer cells . XIAP was moderately expressed in normal transitional epithelium with prominent expression on the superficial layer cells . Seventy-nine of 108 (73.15%) tumor samples were positive for XIAP protein, but XIAP positivity was not correlated with tumor stage or grade . Moreover, 4 bladder cancer cell lines (SCaBER, HT1376, T24 and RT4) expressed similar levels of XIAP . xiap AS PODN dose-dependently reduced the XIAP protein level and induced apoptosis, leading to decreased cell viability by 87% . Combined administration with doxorubicin resulted in marked cytotoxicity due to escalation of apoptosis . Overexpression of XIAP in T24 cells resulted in a modest but statistically significant (p < 0.01) survival advantage compared to parental cells . Thus, XIAP expression may be critical for maintaining the viability and drug resistance of TCC, and endogenous XIAP levels are sufficient to protect cells from apoptosis . Our results suggest that XIAP may play an important role early in human TCC carcinogenesis . xiap AS may be a candidate for use as a cancer therapy for overcoming drug resistance in highly malignant TCC . Cytometry, 2002 Dec 1, 49(4), 135 - 42 Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors; Raaijmakers HG et al.; BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes . The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux . METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer . P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester {calcein-AM}) and specific inhibitors (verapamil and probenecid, respectively) . RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment . P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01) . P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells . In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively) . CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells . Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells . Leukemia, 2002 Dec, 16(12), 2388 - 94 The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia cells in xenotransplanted NOD-SCID mice; Lehne G et al.; Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies . Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids . Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention . In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro . This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice . The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24) . PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h . The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration . Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016) . This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy. J Clin Microbiol, 2002 Dec, 40(12), 4750 - 2 Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis; Park WG et al.; The drug susceptibility testing performance of a broth-based method with microscopic reading of bacillary growth, the microscopic observation drug susceptibility (MODS) assay, was compared to that of the reference 7H10 agar method of proportion by using 53 isolates of Mycobacterium tuberculosis from persons at risk for multidrug-resistant TB . For isoniazid (0.1 micro g/ml) and rifampin (2.0 micro g/ml), there was 100% agreement between MODS results read at day 11 and the reference method . Levels of agreement for ethambutol tested at 2.5 and 7.5 micro g/ml were 70 and 58%, respectively . Levels of agreement for streptomycin tested at 2.0 and 6.0 micro g/ml were 77 and 51%, respectively . For isoniazid and rifampin drug susceptibility testing, MODS is as accurate as and more rapid than the reference method. Ann Oncol, 2002 Dec, 13(12), 1925 - 34 Clinical phase I and pharmacokinetic study of S 16020, a new olivacine derivative: report on three infusion schedules; Awada A et al.; S 16020, a new 9-OH olivacine derivative, is a novel topoisomerase II inhibitor with activity in cell lines presenting the classical multidrug resistance phenotype . This report summarizes, in addition to pharmacokinetic data, the whole phase I clinical experience of S 16020 using three different infusion schedules . Asthenia and skin toxicity were the main side effects . In an attempt to understand the skin toxicity mechanism, experiments in animals were performed, the results of which are reported . S 16020 showed rapid tumor necrotizing activity in some patients, with soft tissue metastases of epidermoid tumors and pain at the tumor site . To document the side effects of S 16020 and tumor site reactions (pain, edema, inflammatory signs), inflammatory parameters and some cytokines were measured . In our patients there was no hemolysis and no detection of anti-S 16020 antibodies, confirming the absence of immunogenicity of the compound . Based on the overall data of the three infusion schedules of S 16020, the dose of 100 mg/m(2) over 3 h every 3 weeks was selected for phase II studies. Emerg Infect Dis, 2002 Nov, 8(11), 1320 - 6 Rifampin- and multidrug-resistant tuberculosis in Russian civilians and prison inmates: dominance of the beijing strain family; Drobniewski F et al.; Consecutive patient cultures (140) of Mycobacteriium tuberculosis were collected from five Russian civilian and prison tuberculosis laboratories and analyzed for rifampin (rpoB) and isoniazid resistance (inhA, katG, ahpC); transmission of Beijing family isolates; and the importance of prison and previous therapy in drug resistance . Rifampin, isoniazid, and multidrug resistance occurred in 58.2%, 51.6%, and 44.7% of cultures, respectively; 80% of prison cultures were rifampin resistant . Spoligotyping and variable number tandem repeat (VNTR) fingerprinting divided the isolates into 43 groups . Spoligotyping demonstrated that a high proportion (68.1%) of patients were infected with Beijing family strains and that most (69.1%) were rifampin resistant; the highest proportion (81.6%) occurred in prison . One VNTR subgroup (42435) comprised 68 (72.3%) of the Beijing isolates with a small number of IS6110 types; 50 (73.5%) were rifampin resistant . Rifampin-resistant Beijing isolates are dominant within the patient population, especially among prisoners, and threaten treatment programs. Emerg Infect Dis, 2002 Nov, 8(11), 1230 - 8 Molecular epidemiology of multidrug-resistant tuberculosis, New York City, 1995-1997; Munsiff SS et al.; From January 1, 1995, to December 31, 1997, we reviewed records of all New York City patients who had multidrug-resistant tuberculosis (MDRTB); we performed insertion sequence (IS) 6110-based DNA genotyping on the isolates . Secondary genotyping was performed for low IS6110 copy band strains . Patients with identical DNA pattern strains were considered clustered . From 1995 through 1997, MDRTB was diagnosed in 241 patients; 217 (90%) had no prior treatment history, and 166 (68.9%) were born in the United States or Puerto Rico . Compared with non-MDRTB patients, MDRTB patients were more likely to be born in the United States, have HIV infection, and work in health care . Genotyping results were available for 234 patients; 153 (65.4%) were clustered, 126 (82.3%) of them in eight clusters of >or=4 patients . Epidemiologic links were identified for 30 (12.8%) patients; most had been exposed to patients diagnosed before the study period . These strains were likely transmitted in the early 1990 s when MDRTB outbreaks and tuberculosis transmission were widespread in New York. Semin Perinatol, 2002 Oct, 26(5), 315 - 21 Risk factors for hospital-acquired infections in the neonatal intensive care unit; Saiman L; Infants in the neonatal intensive care unit (NICU) have many risk factors for infection . Compared with older children and adults, infants, particularly premature infants, are relatively immunocompromised . Patients in the NICU have intrinsic risk factors for infections due to immunological "deficiencies" or inadequate development of mechanical barriers such as skin and gastrointestinal tract mucosa . Like other ICU populations, NICU patients have extrinsic risk factors for infection such as prolonged hospitalization, invasive procedures, instrumentation, medical treatments and concomitant medical conditions . Compared with healthy full-term infants, patients in the NICU develop abnormal flora, which is generally acquired in the NICU from patient-to-patient transmission via hand carriage of healthcare workers . This flora is frequently multidrug-resistant as it has developed under the selective pressure of antibiotics and can cause invasive disease . An understanding of the risk factors that are associated with hospital-acquired infections is essential to design preventive strategies. Am J Trop Med Hyg, 2002 Oct, 67(4), 400 - 5 Genetics of drug-resistant Plasmodium falciparum malaria in the Venezuelan state of Bolivar; Contreras CE et al.; The state of Bolivar in Venezuela experiences episodic outbreaks of multidrug-resistant Plasmodium falciparum malaria . We obtained P . falciparum-infected blood samples in Bolivar in 1998-2000, and performed molecular assays for mutations conferring resistance to the antifolate combination of sulfadoxine-pyrimethamine (SP) and to chloroquine . All infections carried the dihydrofolate reductase (dhfr) S108A and N51I mutations, and 45% of the infections had the dhfr C50R mutation, which has been implicated in mid-level resistance to SP . Two dihydropteroate synthase (dhps) mutations also involved in SP resistance, A581G and K540E, were detected in 90% and 67% of the samples, respectively . The dhfr 1164L mutation, which confers high-level resistance, was not identified . The P . falciparum chloroquine resistance transporter (pfcrt) K76T mutation, which is critical for chloroquine resistance, was found in 167 of 168 infections . Six dhfr/dhps allelotypes and four pfcrt-resistant alleles were observed . Their interrelationships suggest a semi-clonal propagation of P . falciparum malaria in Bolivar, and an invasion of multi-resistant pathogens from Brazil . Despite national restrictions on the use of SP and chloroquine, genotypic resistance to these therapies remains widespread in Bolivar. Ai Zheng, 2002 Apr, 21(4), 430 - 2 {Influence of P-glycoprotein expression on chemotherapeutic response of metastatic breast carcinoma}; Li EX et al.; BACKGROUND & OBJECTIVES: The clinical study showed that the P-glycoprotein(P-gp) expression was closely associated with the chemotherapeutic effect, response rate, prognosis, and survival time . Until now, few clinical papers have reported about metastatic sites and its response to chemotherapy with P-gp expression in metastatic breast carcinoma . The current study was designed to investigate the role of P-gp expression and its clinical value of chemotherapy for the patients with different metastatic sites of this carcinoma . METHODS: P-gp expression in 46 postoperative patients with metastatic breast carcinoma was detected by SABC immunohistochemical method . 43 cases treated with combination regimen: Cyclophosphamide 600 mg/m2 i.v . on day 1, Pirarubicin 60 mg/m2 i.v . on day 1, 5-Fluorouracil 600 mg/m2 i.v . on days 1 and 8 . Repeat the cycle every 3 weeks for at least 2 cycles . The correlation between P-gp expression and chemotherapeutic response was analyzed . RESULTS: 1) P-gp expression positive rate was 56.5%, the P-gp expression in the patients with lung or liver viscera metastasis was higher than that in skin or lymph node metastasis (P = 0.049) . 2) The overall response rate was 58.1% in 43 patients . The response rate of the P-gp negative group was higher than the P-gp positive group(P < 0.01) . 3) The response rate in the patients with skin and lymph node metastasis was higher than the patients with lung and liver metastasis (P < 0.05) . 4) The postoperative patients who had received CAF(cyclophosphamide + adriamycin + 5-fluorouracil) or CMF(cyclophosphamide + methotrexate + 5-fluorouracil) regimen adjuvant chemotherapy previously, the response rate of metastatic diseases to chemotherapy had no significant difference (P > 0.05) . CONCLUSIONS: P-gp expression may be considered as an index for evaluating multidrug resistance, guiding drug use, and judging prognosis of the patients with metastatic breast carcinoma. Cancer Chemother Pharmacol, 2002 Dec, 50(6), 490 - 6 Epub 2002 Oct 16. Antitumor activity of troxacitabine (Troxatyl) against anthracycline-resistant human xenografts; Gourdeau H et al.; PURPOSE: We have recently identified a deoxycytidine nucleoside analogue, troxacitabine (beta- L-dioxolane cytidine, Troxatyl; Shire BioChem), which has potent antitumor activity against both leukemia and solid tumors . In contrast to the cytidine nucleoside analogues currently in clinical use (cytarabine and gemcitabine), troxacitabine is a poor substrate of nucleoside transporters and enters cells primarily by passive diffusion . This unusual property led us to evaluate the efficacy of troxacitabine in multidrug resistant (MDR) and multidrug resistance-associated protein (MRP) tumors . METHODS: The in vitro antiproliferative activity of troxacitabine was investigated in the human nasopharyngeal epidermoid carcinoma cell line, KB, and its vincristine-resistant derivative (KBV), as well as in human leukemia cell lines of myeloid and lymphoblastoid origin, HL60 and CCRF-CEM, respectively, and their MDR (HL60/R10 and CCRF-CEM/VLB) and MRP (HL60/ADR) derivatives, using the thymidine incorporation assay . For in vivo studies, we compared the antitumor efficacy of troxacitabine with that of doxorubicin and vinblastine in xenograft models of these solid and hematological human anthracycline-resistant tumor xenografts . RESULTS: Troxacitabine demonstrated potent antiproliferative activity against both P-glycoprotein-positive (KBV, HL60/R10, CCRF-CEM/VLB) and P-glycoprotein-negative (HL60/ADR) multidrug-resistant cell lines with IC(50) values ranging from 7 to 171 n M . Tumor regression was observed in the KBV xenograft following a 5-day treatment with 20, 50 and 100 mg/kg of troxacitabine, with percent total growth inhibition (TGI) of 81, 96 and 97, respectively, and some cures at the two highest dose levels . In the HL60, HL60/R10, HL60/ADR and CCRF-CEM/VLB xenografts, the effect of troxacitabine was evaluated on survival time . In the HL60 promyelocytic human xenograft models, troxacitabine treatment (25, 50 and 100 mg/kg per day for 5 days) was initiated 10 days after tumor cell inoculation, once animals had developed disseminated tumors . In all three promyelocytic leukemia xenografts, troxacitabine was quite potent, producing T/C values of 162% to 315% as well as complete cures at the higher dose levels . In the CCRF-CEM/VLB T-lymphoblastoid leukemia xenograft, troxacitabine treatment (10, 30 or 250 mg/kg total doses using different schedules) was initiated 20 days after tumor cell inoculation . Troxacitabine was not as potent in this model but did result in significant antileukemic activity (T/C of 131%) when administered at 10 mg/kg on days 20, 27 and 34 . CONCLUSIONS: These results indicate that troxacitabine has a potent in vivo antitumor activity associated with tumor regressions and complete cures in animals with tumors refractory to current chemotherapeutic agents. Biochemistry, 2002 Dec 3, 41(48), 14132 - 40 A positively charged amino acid proximal to the C-terminus of TM17 of MRP1 is indispensable for GSH-dependent binding of substrates and for transport of LTC4; Ren XQ et al.; MRP1 is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells . Our recent study demonstrated that GSH is required for the labeling of MRP1(932)(-)(1531) with a photoanalogue of agosterol A (AG-A) and suggested that GSH interacts with the L(0) region of MRP1 . In this study, we further characterized the GSH-dependent binding site of azido AG-A on MRP1 . Coexpression of the N- and C-terminal halves of MRP1 (residues 1-1222, TM1-16, and 1223-1531, TM17, respectively) in Sf21 insect cells reconstituted a functional drug transporter with a K(m) for LTC(4) (97 nM) similar to that of intact MRP1 . In membrane vesicles from those cells, GSH-dependent photolabeling of the MRP1 fragment (1-1222) required the coexpression of the C-terminal MRP1 fragment (1223-1531) . An MRP1 fragment extending from residue 1 to 1295 however could be photolabeled by azido AG-A in a GSH-dependent manner . These data indicate that amino acids 1223-1295 of MRP1 are required for AG-A binding to MRP1 in a GSH-dependent manner . However, cross-linking of the photolabel to MRP1 occurs at a more upstream site . An arginine residue at position 1249 of MRP1 was shown to be important for the GSH-dependent binding of AG-A to MRP1 . Mutation of this arginine to alanine (R1249A) resulted in a decreased level of GSH-dependent azido AG-A photolabeling of MRP1 . Furthermore, this mutant attenuated MRP1 function by decreasing the level of LTC(4) substrate transport and impairing resistance to the drug vincristine (VCR) . In summary, this study demonstrates that a region of MRP1 (amino acids 1223-1295), which includes TM helix 17, is required for azido AG-A binding to MRP1 in a GSH-dependent manner . A GSH-dependent drug binding site may exist in this region . Furthermore, our findings suggest that the charged amino acid Arg(1249) proximal to the C-terminus of TM helix 17 is indispensable for MRP1-substrate interaction and the function of MRP1. Lepr Rev, 2002 Sep, 73(3), 239 - 44 Drug susceptibility of Mycobacterium leprae: a retrospective analysis of mouse footpad inoculation results from 1983 to 1997; Sekar B et al.; We analysed the results of mouse foot pad (MFP) tests performed between 1983 and 1997 in our laboratory for the cases referred with clinical suspicion of relapse/drug resistance . A total of 214 cases, with clinical suspicion of relapse/drug resistance were investigated for susceptibility to the drugs of MDT by MFP inoculation . Among 96 inoculations that showed conclusive results, 81 (84%) were fully sensitive to dapsone, suggesting that most of the clinically suspected relapse is due to drug susceptible Mycobacterium leprae . Of the remaining 15 strains (16%) found resistant to dapsone, 13 (87%) were of high grade resistance and one strain each of intermediate grade and low grade dapsone resistance, suggesting that most of the dapsone resistance is secondary in nature . No case of rifampicin resistance was found . Only one case of combined dapsone and unconfirmed clofazimine resistance was found . No other combined multidrug resistance was observed in our analysis. Biol Reprod, 2002 Dec, 67(6), 1959 - 74 Structural requirements for potent anti-human immunodeficiency virus (HIV) and sperm-immobilizing activities of cyclohexenyl thiourea and urea non-nucleoside inhibitors of HIV-1 reverse transcriptase; D'Cruz OJ et al.; The current pandemic of sexually transmitted human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) has created an urgent need for a new type of microbicide, one that is both a spermicide and a virucide . In a systematic effort to identify a non-detergent-type antiviral spermicide, we have rationally designed and synthesized a series of cyclohexenyl thiourea (CHET) nonnucleoside inhibitors (NNIs) of HIV-1 reverse transcriptase (RT) with sperm-immobilizing activity (SIA) . To gain further insight into the structural requirements for the optimal activity of these dual-function NNIs, we compared the effects of thiazolyl, benzothiazolyl, and pyridyl ring substitutions and functionalization with electron-donating and electron-withdrawing groups as well as the importance of thiourea and urea moieties of 15 heterocyclic ring-substituted NNIs . RT activity and p24 antigen production in HIV-infected peripheral blood mononuclear cells were used as markers of viral replication . Computer-assisted sperm analysis was used for evaluating SIA of CHET compounds . The rabbit model was used for evaluation of in vivo mucosal toxicity and contraceptive activity of the lead NNIs . Three CHET-NNIs with a bromo, chloro, or methyl substitution at the 5 position of the pyridyl ring exhibited potent anti-HIV activity at nanomolar concentrations (IC(50) = 3-5 nM) and SIA at micromolar concentrations (EC(50) = 45-96 micro M) . The dual-function CHET-NNIs were potent inhibitors of drug-resistant HIV-1 strains with genotypic and phenotypic NNI resistance . Upon substitution of the sulfur atom of the thiourea moiety with an oxygen atom, the most striking difference noted was a 38-fold reduction in time required for 50% sperm immobilization (T(1/2)) . A quantitative structure-activity relationship (QSAR) analysis was used in deriving regression equations between 20 physicochemical properties and SIA of NNIs . QSAR analysis showed that the T(1/2) values positively correlated with values for molecular refractivity (r = 0.88), hydrophobicity (r = 0.72), atomic polarizability (r = 0.70), and principal moment of inertia (r = 0.63) of spermicidal NNIs . A stepwise multiple regression model to describe the relationship of T(1/2) values with these four regressors provided excellent predictability (r = 0.93) . Exposure of semen to thiourea/urea NNIs either alone or in combination at the time of artificial insemination led to marked or complete inhibition of pregnancy in rabbits as assessed by the number of embryo implants versus corpora lutea on Day 8 of pregnancy . Repeated intravaginal application of a gel-microemulsion with and without 0.5%, 1%, and 2% CHET-NNI or its urea analog either alone or in combination did not induce mucosal toxicity . We hypothesize that the gain of spermicidal function by CHET-NNIs is due to their metabolic oxidation to urea analogs by sperm . Three reaction pathways are discussed . The extremely rapid SIA of the urea analog as well as the broad-spectrum anti-HIV activity of spermicidal CHET-NNIs together with their lack of mucosal toxicity and the marked ability to reduce in vivo fertility is particularly useful for the clinical development of a dual-function spermicidal microbicide . The cyclohexenyl pyridyl NNIs, especially N-{2-(1-cyclohexenyl)ethyl} N'-{2-(5-bromopyridyl)}-thiourea in combination with the urea analog, show unique clinical potential as anti-HIV spermicides aimed at curbing the sexual transmission of multidrug-resistant HIV-1 while providing effective fertility control for women. Biol Reprod, 2002 Dec, 67(6), 1699 - 707 Multidrug resistance genes and p-glycoprotein in the testis of the rat, mouse, Guinea pig, and human; Melaine N et al.; Study of the multidrug resistance phenomenon in tumor cell lines has led to the discovery of the product of the multidrug resistance (MDR) type 1 genes, the plasma membrane P-glycoprotein (P-gp) that functions as an energy-dependent pump for the efflux of diverse anticancer drugs . P-gp was also recently identified in normal epithelial cells with secretory/excretory functions and in the endothelial cells of the capillary blood vessels in the brain and the testis . These endothelial cells are key elements of the blood-brain and blood-testis barriers, respectively . The aim of this study, in the rat, mouse, guinea pig, and human, was to determine whether testicular cells other than the capillary endothelial cells could express MDR type I genes . Immunohistochemistry on testicular sections revealed that P-gp is present in interstitial cells in the mouse, rat, and human testes, in early and late spermatids in guinea pig testis, and in late spermatids in the rat, mouse, and human . Reverse transcription-polymerase chain reaction analysis on isolated mouse, rat, and human cells showed that all somatic testicular cells (Leydig cells, macrophages, peritubular cells, and Sertoli cells) and the cytoplasmic lobes from rat late spermatids expressed MDR type I mRNAs, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not . An ontogenesis study in the mouse reveals that type I MDR gene expression begins at 13.5 days postcoitum at the time when the seminiferous cords and the blood vessels appear and are maintained thereafter . Finally, two functional tests on isolated rat cells, the doxorubicin and rhodamine uptake assays, demonstrated that rat testicular macrophages, Leydig cells, peritubular cells, and Sertoli cells displayed a multidrug-resistance activity, whereas spermatogonia, pachytene spermatocytes, and early spermatids did not . Western blot experiments have revealed that a P-gp of 175 kDa is present in the human testis as well as in the rat Leydig cells, testicular macrophages, peritubular cells, and Sertoli cells, but is absent in spermatogonia, spermatocytes, and early spermatids . We conclude that P-gp is involved in the self-protection of the somatic cells and is most probably one of the molecules that confers its functionality to the blood-testis barrier . The absence of expression of MDR type I genes in mitotic and meiotic germ cells probably explains their particular vulnerability to various anticancer drugs . In contrast, expression of the P-gp in the haploid cells most likely reflects the ability of spermatozoa to assume their own antidrug defense. Bioorg Med Chem Lett, 2002 Dec 16, 12(24), 3505 - 7 Synthesis and cytotoxic activity of a new potent daunomycinone derivative; Aligiannis N et al.; The preparation and cytotoxic activity of 4'-azido-3'-bromo-3'-deamino-4'-deoxydaunorubicin is described . The new compound was found to be less active in vitro than adriamycin against L1210 and the sensitive cell lines KB-3-1 and MES-SA, but retained interesting cytotoxicity against the adriamycin resistant subline KB-A1 and the multidrug resistant MES-SA/Dx5 subline. J HIV Ther, 2002 Aug, 7(3), 63 - 7 Support required for antiretroviral uptake in developing countries; Gold J; It is evident from the current global debate on improving care and treatment for people living with HIV that antiretroviral therapy will become increasingly available in resource-poor countries . As this happens, there is an imperative to determine which factors will promote optimal use of these complex therapies to minimise the potentially serious consequences of poor adherence and the development of a worse epidemic of multidrug-resistant HIV (MDRHIV) . In parallel with the process of allocating funds to purchase drugs, there needs to be a pro-active scaling up of healthcare worker training and infrastructure support systems and development of continuity of care from healthcare services into the community . The engagement of healthcare workers in the HIV epidemic has been largely ignored in favour of a focus on prevention which, understandably, works through the non-government sector as the vehicle for implementation . This upscaling and recruitment of healthcare workers needs involvement not only from our more eminent clinicians as spokespeople but also from experienced field workers, who will be caring for the majority of patients as they become ill . This article identifies the multidimensional approach necessary to implement a broad initiative for care and treatment and discusses the possible strategies and consequences of each factor. AIDS, 2002 Nov 22, 16(17), 2295 - 301 Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs; Huisman MT et al.; BACKGROUND: Various drug transporters of the ATP-binding cassette (ABC) family restrict the oral bioavailability and cellular, brain, testis, cerebrospinal fluid and fetal penetration of substrate drugs . MDRI P-glycoprotein (P-gp) has been demonstrated to transport most HIV protease inhibitors (HPI) and to reduce their oral bioavailability and lymphocyte, brain, testis and fetal penetration, possibly resulting in major limiting effects on the therapeutic efficacy of these drugs . OBJECTIVES: To investigate whether the ABC transporters MRP1, MRP2, MRP3, MRP5 and breast cancer resistance protein 1 (Bcrp1) are efficient transporters of the HPI saquinavir, ritonavir and indinavir . METHODS: Polarized epithelial non-human (canine) cell lines transduced with human or murine complementary DNA (cDNA) for each of the transporters were used to study transepithelial transport of the HPI . RESULTS: MRP2 efficiently transported saquinavir, ritonavir and indinavir and this transport could be enhanced by probenecid . Sulfinpyrazone was also able to enhance MRP2-mediated saquinavir transport . In contrast, MRP1, MRP3, MRP5, or Bcrp1 did not efficiently transport the HPI tested . CONCLUSIONS: Human MRP2 actively transports several HPI and could, based on its known and assumed tissue distribution, therefore reduce HPI oral bioavailability . It may also limit brain and fetal penetration of these drugs and increase their hepatobiliary, intestinal and renal clearance . MRP2 function and enhancement of its activity could adversely affect the therapeutic efficacy, including the pharmacological sanctuary penetration, of HPI . In vivo inhibition of MRP2 function might, therefore, improve HIV/AIDS therapy . Cell Mol Life Sci, 2002 Sep, 59(9), 1577 - 83 Multidrug-resistance-associated protein MGr1-Ag is identical to the human 37-kDa laminin receptor precursor; Shi Y et al.; We report the isolation and functional characterization of the gene encoding MGr1-Ag, a multidrug-resistance-associated protein . A lambdagt11 cDNA library derived from colorectal carcinoma SW480 cells was screened with monoclonal antibody MGr1 . DNA homology analysis of 22 positive clones (designated R1-R22) suggested human 37-kDa laminin receptor precursor (37LRP, R7/R9/R15/R16/R19/R20) and a novel gene (R22) as candidate genes encoding MGr1-Ag . Western blot analysis showed that anti-R20 serum reacted with a unique protein band that was consistent with MGr1-Ag, while anti-R22 serum could not react with MGr1-Ag . The coding gene for MGr1-Ag was amplified using reverse transcription-PCR . Sequence analysis revealed that the MGr1-Ag and 37LRP genes shared the same coding sequence . An in vitro drug sensitivity assay indicated that down-regulation of 37LRP by an antisense technique could significantly enhance the cytotoxicity of anticancer drugs to gastric cancer cells . Thus we draw the conclusion that MGr1-Ag is identical to 37LRP. World J Gastroenterol, 2002 Dec, 8(6), 1029 - 34 Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel(7402); Jin J et al.; AIM: To investigate the resistance mechanism of 5-fluorouracil (5-FU) in Bel(7402)/5-FU cells which was established in our lab by in vitro continuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel(7402) to 5-FU . METHODS: The expression of multidrug resistance-associated protein (MRP) and thymidylate synthase (TS) in Bel(7402) cells was detected by immonocytochemistry . The fluorescein (FLU) accumulation, an index of MRP functional activity, was determined by flow cytometry . The distribution of FLU was observed by confocal laser scanning microscope . The spectrofluorometry was used to show the intracelluar content of glutathione (GSH) . Cell growth inhibition was determined by MTT assay . The activity of glutathione S-transferases (GSTs) was determined by spectrophotometry . RESULTS: A higher expression of MRP in the Bel(7402)/5-FU cells was observed by using monoclonal mouse anti-MRP antibody, MRPr-1, in comparison with Bel(7402) cells . Bel(7402)/5-FU cells also showed a significant decrease of FLU accumulation . FLU mainly accumulated in the nucleus with a high nuclear/cytoplasmic ratio in Bel(7402) cells, whereas there was no difference of FLU accumulation between the nucleus and cytoplasm in Bel(7402)/5-FU cells . The intracellular GSH content in Bel(7402)/5-FU cells was almost 3 folds higher than that in Bel(7402) cells . Addition of D, L-buthione-S, R-sulfoximine (BSO) dose-dependently reduced the GSH content in Bel(7402)/5-FU cells, however, only a weak enhancement on the cytotoxicity of 5-FU and doxorubicin (Dox) to Bel(7402)/5-FU cells was observed . Bel(7402)/5-FU cells also exhibited 29.1 % higher total GSTs activity than Bel(7402) cells . Immunocytochemical staining by using anti-TS monoclonal antibody TS 106 showed that the level of TS in Bel(7402)/5-FU cells elevated markedly as compared with Bel(7402) cells . CONCLUSION: The continuous exposure of Bel(7402) cells to 5-FU led to overexpression of TS and MRP, as well as increased intracellular GSH content and total GST activity. Neuroreport, 2002 Nov 15, 13(16), 2059 - 63 Localisation of breast cancer resistance protein in microvessel endothelium of human brain; Cooray HC et al.; Movement of substrates between blood and brain is known to be influenced by P-glycoprotein (P-gp) at the luminal surface of the endothelium lining brain microvessels and by multidrug resistance associated protein 1 (MRP1) at the basolateral surface of the choroid plexus epithelium . Here, using RT-PCR and Western blotting, we investigate other ABC transporters in both normal and tumour human brain tissue and demonstrate the presence of breast cancer resistance protein (BCRP) . Immunofluorescence confocal microscopy demonstrates that BCRP is located at the blood-brain barrier, mainly at the luminal surface of microvessel endothelium . This localization closely resembles that of P-gp . BCRP has several substrates in common with P-gp and may pose an additional barrier to drug access to the brain. Saudi Med J, 2002 Oct, 23(10), 1227 - 31 Mycobacterium tuberculosis susceptibility in Saudi Arabia; Alrajhi AA et al.; OBJECTIVE: To present the available susceptibility data of Mycobacterium tuberculosis (M . tuberculosis) isolates from the Kingdom of Saudi Arabia (KSA) published in peer-reviewed journals . METHODS: In a meta-analysis, studies published between 1966 and 2001 were included . Publication sites include Medline-indexed and non-indexed . Numbers of grown and resistant isolates were tabulated for first-line anti-tuberculosis agents . RESULTS: Twelve studies met the pre-set criteria . Data on 6,316 isolates between 1979 and 2000 were available . Resistance to at least one agent of the first-line anti-tuberculosis agents was 18.4% . Monoresistance to a single first-line agent was found in 10.9%, while polyresistance was noted in 7.6% . Multidrug-resistant M . tuberculosis was noted in 5.7% of all isolates . Resistance to isoniazid was most common noted in 11% of isolates . Resistance rates to other agents were: rifampin 9.7%, streptomycin 9.1%, pyrazinamide 3.1%, and ethambutol 2.5% . The overall resistance rate to at least one agent was not statistically different in isolates grown between 1979-1991 (18.5%) and 1989-2000 (18.3%) . There were large regional variations and higher resistance rates in the Western and Southern regions . CONCLUSION: Mycobacterium tuberculosis resistance rates to first-line antituberculosis agents and multidrug-resistant M . tuberculosis are high in KSA . A survey and monitoring program for drug-resistant tuberculosis will determine resistance rates at the community level. Mol Pharmacol, 2002 Dec, 62(6), 1321 - 31 Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells; Wielinga PR et al.; Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG) . Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters . We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5 . Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate . In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP) . During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4 . Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside . This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate . Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail. Mol Pharmacol, 2002 Dec, 62(6), 1288 - 98 Characterization of two pharmacophores on the multidrug transporter P-glycoprotein; Garrigues A et al.; The multidrug transporter P-glycoprotein is a plasma membrane protein involved in cell and tissue detoxification and the multidrug resistance (MDR) phenotype . It actively expels from cells a number of cytotoxic molecules, all amphiphilic but chemically unrelated . We investigated the molecular characteristics involved in the binding selectivity of P-glycoprotein by means of a molecular modeling approach using various substrates combined with an enzymological study using these substrates and native membrane vesicles prepared from MDR cells . We determined affinities and mutual relationships from the changes in P-glycoprotein ATPase activity induced by a series of cyclic peptides and peptide-like compounds, used alone or in combination . Modeling of the intramolecular distribution of the hydrophobic and polar surfaces of this series of molecules made it possible to superimpose some of these surface elements . These molecular alignments were correlated with the observed mutual exclusions for binding on P-glycoprotein . This led to the characterization of two different, but partially overlapping, pharmacophores . On each of these pharmacophores, the ligands compete with each other . The typical MDR-associated molecules, verapamil, cyclosporin A, and actinomycin D, bound to pharmacophore 1, whereas vinblastine bound to pharmacophore 2 . Thus, the multispecific binding pocket of P-glycoprotein can be seen as sites, located near one another, that bind ligands according to the distribution of their hydrophobic and polar elements rather than their chemical motifs . The existence of two pharmacophores increases the possibilities for multiple chemical structure recognition . The size of the ligands affects their ability to compete with other ligands for binding to P-glycoprotein. Antimicrob Agents Chemother, 2002 Dec, 46(12), 3947 - 53 Therapeutic efficacies of artesunate-sulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations; Tjitra E et al.; Artemisinin-derivative combination therapies (ACT) are highly efficacious against multidrug-resistant Plasmodium falciparum malaria . Few efficacy data, however, are available for vivax malaria . With high rates of chloroquine (CQ) resistance in both vivax and falciparum malaria in Papua Province, Indonesia, new combination therapies are required for both species . We recently found artesunate plus sulfadoxine-pyrimethamine (ART-SP) to be highly effective (96%) in the treatment of falciparum malaria in Papua Province . Following a preliminary study of CQ plus sulfadoxine-pyrimethamine (CQ-SP) for the treatment of Plasmodium vivax infection, we used modified World Health Organization criteria to evaluate the efficacy of ART-SP for the treatment of vivax malaria in Papua . Nineteen of 22 patients treated with ART-SP could be evaluated on day 28, with no early treatment failures . Adequate clinical and parasitological responses were found by day 14 in all 20 (100%) of the patients able to be evaluated and by day 28 in 17 patients (89.5%) . Fever and parasite clearance times were short, with hematological improvement observed in 70.6% of the patients . Double (at positions 58 and 117) and quadruple (at positions 57, 58, 61, and 117) mutations in the P . vivax dihydrofolate reductase (PvDHFR) were common in Papuan P . vivax isolates (46 and 18%, respectively) . Treatment failure with SP-containing regimens was significantly higher with isolates with this PvDHFR quadruple mutation, which included a novel T-->M mutation at residue 61 linked to an S-->T (but not an S-->N) mutation at residue 117 . ART-SP ACT resulted in a high cure rate for both major Plasmodium species in Papua, though progression of DHFR mutations in both species due to the continued use of SP monotherapy for clinically diagnosed malaria threatens the future utility of this combination. Antimicrob Agents Chemother, 2002 Dec, 46(12), 3900 - 6 ABC transporters and azole susceptibility in laboratory strains of the wheat pathogen Mycosphaerella graminicola; Zwiers LH et al.; Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both . Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole . No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14alpha-demethylase gene, CYP51 . ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M . graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels . However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level . We propose that variations in azole susceptibility in laboratory strains of M . graminicola are mediated by multiple mechanisms. Biochem J, 2003 Feb 15, 370(Pt 1), 185 - 93 Inhibitors of vacuolar H+-ATPase impair the preferential accumulation of daunomycin in lysosomes and reverse the resistance to anthracyclines in drug-resistant renal epithelial cells; Ouar Z et al.; It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance . The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PR(col50) cells and their drug-sensitive PKSV-PR cell counterparts . Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6-5.7), but that PKSV-PR(col50) cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N -acetyl-beta-hexosaminidase and beta-glucuronidase than their parent PKSV-PR cells . Concanamycin A (CCM A), a potent inhibitor of the vacuolar H(+)-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N -acetyl-beta-hexosaminidase in both drug-sensitive and drug-resistant cells . Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PR(col50) cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments . CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PR(col50) cells . In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin . These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment. Clin Chem Lab Med, 2002 Sep, 40(9), 918 - 25 Overcoming multidrug resistance in taxane chemotherapy; Geney R et al.; Paclitaxel (Taxol) and docetaxel (Taxotere) are currently two of the most important anticancer drugs in cancer chemotherapy . However, clinical treatment with these taxane agents often encounters undesirable side effects and multidrug resistance (MDR) caused by overexpression of P-glycoprotein (Pgp) . Photoaffinity labeling of Pgp using photoreactive radiolabeled paclitaxel analogs along with molecular modeling has revealed a unique binding region for paclitaxel on the C-terminal half of Pgp . Highly efficient taxane-based MDR reversal agents (TRAs) have been developed . Extensive structure-activity relationship (SAR) studies have led to the development of new generation taxanes that possess 2-3 orders of magnitude higher potencies against human cancer cell lines expressing the MDR phenotype . One of these taxanes, SB-T-1 10131 (IDN5109, BAY59-8862), exhibits excellent activity against a variety of drug-sensitive and drug-resistant cancer cell lines as well as human tumor xenografts in mice . This taxane is orally active with excellent bioavailability, and is currently undergoing phase II human clinical trials . Novel taxane-antibody immunoconjugates have shown very promising results for tumor-specific delivery and release of an extremely cytotoxic taxane, wherein epidermal growth factor receptor is used as the specific antigen on the tumor surface of human squamous cancer xenograft in SCID mice. Clin Chem Lab Med, 2002 Sep, 40(9), 863 - 8 Genetic susceptibility to tuberculosis; Malik S et al.; Tuberculosis, caused by the human pathogen Mycobacterium tuberculosis (M . tuberculosis), affects an estimated 8 million people annually, resulting in approximately 2 million deaths . Human genetic variability is an important modulator of tuberculosis susceptibility . This review will discuss candidate susceptibility genes that have been implicated in tuberculosis susceptibility across various ethnic groups and epidemiological settings . Evaluating the genetic variants of tuberculosis susceptibility genes will provide us with a better understanding of the disease mechanisms in tuberculosis . Ultimately, such genetic studies may lead to the development of effective alternative treatments to cope with the growing problem of tuberculosis infections due to the AIDS pandemic, the emergence of multidrug resistant M . tuberculosis, and the limited efficacy of Mycobacterium bovis (BCG) vaccination. J Pharm Sci, 2002 Dec, 91(12), 2511 - 9 Kinetic characterization of secretory transport of a new ciprofloxacin derivative (CNV97100) across Caco-2 cell monolayers; Ruiz-Garcia A et al.; The kinetics of transport of a new fluoroquinolone antibiotic (CNV97100) and its analogs were characterized using the Caco-2 cell culture model . Unidirectional permeabilities of these analogs were greater (p < 0.05) than that of ciprofloxacin . The absorptive permeabilities (P(AB)) of 4'-N-substituted analogs (CNV97101-104) were 400-600% greater, whereas the secretory permeability (P(BA)) was 25-80% greater than unsubstituted analogs because CNV97101-104 were poor substrates for efflux transporters (efflux ratio approximately 1) . The transport of compounds without 4'-N-substitution (i.e., ciprofloxacin and CNV97100) favored secretion (efflux ratio approximately 4) . Further characterization of CNV97100 transport revealed that it was concentration dependent (apparent K(m) = 0.484 mM, and apparent V(max) = 17.5 nmol x cm(-2) x h(-1)), and temperature dependent (E(a) = 20.57 for P(AB) and 31.45 kcal/mol for P(BA), respectively) . p-Glycoprotein (p-gp)inhibitors, such as verapamil (100 microM) and cyclosporin A (CsA, 20 microM) significantly (p < 0.05) inhibited P(BA) but significantly (p < 0.05) enhanced P(AB) . Multidrug resistance related protein (MRP) inhibitor leukotriene C(4) only decreased (p < 0.05) P(BA) of ciprofloxacin but not that of CNV97100 . In the presence of increasing concentrations of verapamil, the P(BA) of CNV97100 decreased significantly (p < 0.05), with an IC(90) value of 96.5 microM . Taken together, these results suggested that 4'-N-alkylation of fluoroquinolones improves their absorptive permeability . Secretion of CNV97100 is dominated by p-gp, whereas the secretion of ciprofloxacin is via a combination of efflux transporters . Drug Metab Dispos, 2002 Dec, 30(12), 1300 - 10 RLIP76, a novel transporter catalyzing ATP-dependent efflux of xenobiotics; Awasthi S et al.; Transport of xenobiotics and their metabolites by ATP-binding cassette (ABC) transporters particularly P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP1) has been extensively studied during last decade . Our recent studies demonstrate that RLIP76, a previously known GTPase-activating protein catalyzes ATP-dependent, uphill transport of anionic glutathione conjugates as well as of weakly cationic anthracyclines including doxorubicin (Adriamycin), a widely used drug in cancer chemotherapy . RLIP76 has inherent ATPase activity, which is stimulated by doxorubicin and glutathione conjugates . RLIP76 does not meet the criteria for classical ABC proteins such as MRP1 or Pgp, but similar to ABC proteins, it has two ATP-binding sequences, (69)GKKKGK(74) and (418)GGIKDLSK(425) . Mutations in these sequences abrogate its ATP-binding, ATPase activity, and transport function . Purified RLIP76 when reconstituted in proteoliposomes mediates ATP-dependent saturable transport of doxorubicin and glutathione conjugates . Transfection of K562 cells with RLIP76 confers these cells resistance to doxorubicin and 4-hydroxynonenal . Cells enriched with RLIP76 also acquire resistance to radiation toxicity . RLIP76 also catalyzes the transport of physiologic ligands such as leukotrienes (LTC4) and the conjugate of 4-hydroxynonenal and glutathione . In some cells (e.g., erythrocytes and lung cancer cells), the majority of transport activity for Adriamycin and glutathione conjugates including LTC4 is accounted for by RLIP76 . These studies strongly suggest that RLIP76-mediated transport of organic ions has physiological and toxicological relevance and that it may play an important role in the mechanism of drug resistance. J Med Chem, 2002 Nov 21, 45(24), 5330 - 9 Effects of isoprenoid analogues of SDB-ethylenediamine on multidrug resistant tumor cells alone and in combination with chemotherapeutic drugs; Sidorova TA et al.; Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) remains the major obstacle for successful treatment of cancer . Inhibition of Pgp transport is important for higher efficacy of anticancer drugs . Lipophilic cationogenic amines with at least one tertiary N atom, such as verapamil, are classical PgP-blocking agents . In a search for novel accessible compounds potent against MDR tumor cells, we synthesized a series of arylalkylamines that contain isoprenoid side chains of different length . Two out of seven new analogues of the known N,N'-bis(3,4-dimethoxybenzyl)-N-solanesylethylenediamine (SDB-ethylenediamine), namely, compounds with C10 and C15 side chains, at low micromolar concentrations were preferentially toxic for several mammalian tumor cell lines that acquired MDR during prolonged drug selection . Moreover, at noncytotoxic concentrations, these compounds potently sensitized MDR cells to Pgp substrates vinblastine and adriamycin . We conclude that these analogues of SDB-ethylenediamine may have dual therapeutic advantage because (i) they are preferentially toxic for MDR cells when administered alone and (ii) they potentiate the cytotoxicity of Pgp-transported anticancer drugs. Int J Hematol, 2002 Aug, 76 Suppl 2, 206 - 11 Biology and modulation of multidrug resistance (MDR) in hematological malignancies; Hirose M; Drug resistance is one of the most significant impediments in the treatment of hematological malignancies . There have been a number of studies on the incidence of P-GP expression in tumor cells or tissues, where detectable level of P-GP has been found in all types of hematological malignancies . P-GP expression and significance in the patients varies widely between reported studies on patients with different ages and different disease types . Some of this validation can be accounted for by the threshold used to consider a sample positive for P-GP . However, mdr-1 is likely important in determining therapeutic outcome in patients with AML, NHL, and MM, although there is a suggestion of a different "behavior" between adult and childhood AML . In contrast, the significant prognostic association with expression of MRP and LRP is not consistent with disease types and disease stages . Clinical trials of modulation of MDR have been limited by following major factors . One is inability of achieving adequate blood levels of the modulator to reverse MDR, and the other is presence of other resistance mechanisms in addition to P-GP . The fact that P-GP modulators alter the pharmacokinetics of anti-cancer drugs can potentially increase toxicities if the dose of anticancer drugs is not appropriately reduced . Recently, MDR modulators such as valspodar have demonstrated substantial inhibition of P-GP . In this presentation, a number of characteristics in VCR-resistant cells are reported . We demonstrate that acquisition of MDR or recovery from MDR phenotypes differ in one cell type to another, a marked correlation between P-GP and susuceptibility to oxygen radicals, and altered gene expression of cell membrane antigen and apoptosis cascade genes . The efficacy of immunotherapies depends on the altered or unchanged target molecules of MDR cells . Thus, immunotherapies or reversal agents that aim at these substances in tumor cells should be useful to overcome MDR phenotypes. Biochem Pharmacol, 2002 Dec 1, 64(11), 1569 - 78 Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man; Goh LB et al.; The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport . Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties . We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities . Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines . MRP2 mRNA was expressed in all cell lines except MDCK . Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1 . Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps . Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells . OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells . In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATP's . Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver . The findings presented are relevant to the use of porcine and canine models for drug disposition. J Eur Acad Dermatol Venereol, 2002 Sep, 16(5), 486 - 7 Watering can perineum--a forgotten complication of gonorrhoea; Pandhi D et al.; In the modern era of broad spectrum antibiotics, urethral fistulae (watering can perineum) is one of the forgotten sequelae of chronic gonococcal infection . We report a 20-year-old unmarried male with gonococcal urethritis and two sinuses in the scrotum (watering can perineum) . The micturating and retrograde urethrogram revealed mucosal irregularity and extravasation of contrast medium at the junction of bulbous and membranous urethra . Recent worldwide emergence of multidrug resistant strains of gonococci give rise to alarm . In the present scenario of HIV pandemic, ineffective treatment of patient or partner with gonorrhoea may result in development of these complications. Mol Med, 2002 Jun, 8(6), 318 - 25 Gene expression of ABC proteins in hepatocellular carcinoma, perineoplastic tissue, and liver diseases; Bonin S et al.; BACKGROUND: The development of hepatocellular carcinoma (HCC) is a frequent event during the natural history of cirrhosis . Effective treatment is, however, hampered by drug resistance related to the expression of multidrug resistance (MDR) proteins belonging to the ABC family transporters . Studying expression of genes coding for these proteins may help to explain the potential sensitivity of HCC to chemotherapy . MATERIAL AND METHODS: The expression of MRP1, MRP2, MRP3, MDR1, and MDR3 was investigated by quantitative RT-PCR analyses in paraffin-embedded tissues obtained from 9 cases of HCC, 16 cases of cirrhosis, 10 cases of chronic extrahepatic cholestasis, and 16 cases of normal liver . In HCC cases, gene expression was assessed both in neoplastic and perineoplastic tissue after microscopically assisted microdissection . RESULTS: MRP1 was significantly and similarly overexpressed in HCC and perineoplastic tissue . MRP2 and MDR1 were also increased in HCC, but the level of expression did not correlate with that of perineoplastic tissue . The level of expression was either reduced or normal in cirrhotic liver and during chronic cholestasis . Expression of MDR3 was unchanged in all conditions investigated . CONCLUSIONS: The genetic expression of multi-drug resistance proteins, in particular MRP1, MRP2, and MDR1, is increased during HCC . In the case of MRP1, the extent of expression is similar in neoplastic and perineoplastic tissue, but this is not the case for MRP2 and MDR1 . The assessment of ABC protein expression pattern may provide important information for the diagnosis and treatment of HCC. Cell Microbiol, 2002 Nov, 4(11), 725 - 37 Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression; Marchat LA et al.; Here, we show the relevance of promoter regions (-74 to +24, -167 to -75 and -259 to -168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays . We also demonstrate that both CCAAT/enhancer binding protein sites (-54 to -43 bp and -198 to -186 bp) are cis-activating elements of gene expression in the drug-resistant (clone C2) and -sensitive (clone A) trophozoites . Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti-human C/EBPbeta antibodies . UV cross-linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively . The nuclear factors that bind to C/EBP sites were semi-purified by affinity chromatography . They were immunodetected by anti-human C/EBPbeta antibodies and formed a specific complex with the C/EBP probe . The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments . Based on our results, we propose that the C/EBP site at -54 bp stabilizes the transcription pre-initiation complex, whereas the other site at -198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription. Clin Pharmacol Ther, 2002 Nov, 72(5), 572 - 83 The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol; Siegmund W et al.; BACKGROUND AND OBJECTIVES: A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes . Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance . METHODS: In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression . Moreover, the disposition of the probe drug talinolol was evaluated in 55 subjects after oral administration (100 mg) and in 23 subjects after intravenous administration(30 mg) . RESULTS: Duodenal MDR1 messenger ribonucleic acid and P-glycoprotein, as assessed by real-time polymerase chain reaction (TaqMan) and immunostaining, were not influenced by any MDR1 polymorphism studied . Talinolol disposition was not affected by the exon 26 mutation C3435T . In carriers of the TT/TA variants of G2677T/A, the area under the serum concentration-time curve values of oral talinolol were slightly but significantly elevated compared with those in carriers of at least 1 wild-type allele (P <.05, Kruskal-Wallis test; P =.014, Mann-Whitney U test) . However, multiple comparisons with combinations of putative functional SNPs did not confirm a significant influence of the MDR1 genotype on talinolol disposition . CONCLUSIONS: We did not identify any influence of MDR1 genotypes on duodenal expression of P-glycoprotein and disposition of talinolol in humans. Cells Tissues Organs, 2002, 172(2), 133 - 44 Gene therapy for sarcoma; Fruehauf S et al.; Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy . Recent studies suggest a higher response rate with an increased doxorubicin dosage . However, this was parallel with a profound hematotoxicity in 75% of patients . Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy . To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy . In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05) . While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors . On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma . Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting . Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g . by transfer of suicide genes . We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible . The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages . Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks . These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts . Environ Health Perspect, 2002 Oct, 110 Suppl 5, 801 - 6 Cysteine-modifying agents: a possible approach for effective anticancer and antiviral drugs; Casini A et al.; Modification of cysteine residues in proteins, due to a) the participation of the thiol moiety of this amino acid in oxido-reduction reactions, b) its ability to strongly coordinate transition metal ions, or c) its nucleophilic nature and facile reaction with electrophiles, may be critically important for the design of novel types of pharmacological agents . Application of such procedures recently led to the design of novel antivirals, mainly based on the reaction of zinc finger proteins with disulfides and related derivatives . This approach was particularly successful for developing novel antiviral agents for human immunodeficiency virus and human papilloma virus . Several new anticancer therapeutic approaches, mainly targeting tubulin, have also been reported . Thus, this unique amino acid offers very interesting possibilities for developing particularly useful pharmacological agents, which generally possess a completely different mechanism of action compared with classic agents in clinical use, thus avoiding major problems such as multidrug resistance (for antiviral and anticancer agents) or high toxicity. Environ Health Perspect, 2002 Oct, 110 Suppl 5, 689 - 94 Transport of toxic metals by molecular mimicry; Ballatori N; Intracellular concentrations of essential metals are normally maintained within a narrow range, whereas the nonessential metals generally lack homeostatic controls . Some of the factors that contribute to metal homeostasis have recently been identified at the molecular level and include proteins that mediate import of essential metals from the extracellular environment, those that regulate delivery to specific intracellular proteins or compartments, and those that mediate metal export from the cell . Some of these proteins appear highly selective for a given essential metal; however, others are less specific and interact with multiple metals, including toxic metals . For example, DCT1 (divalent cation transporter-1; also known as NRAMP2 or DMT1) is considered to be a major cellular uptake mechanism for Fe(2+) and other essential divalent metals, but this protein also mediates uptake of Cd(2+), Pb(2+), and possibly of other toxic divalent metals . The ability of nonessential metals to interact with binding sites for essential metals is critical for their ability to gain access to specific cellular compartments and for their ability to disrupt normal biochemical or physiological functions . Another major mechanism by which metals traverse cell membranes and produce cell injury is by forming complexes whose overall structures mimic those of endogenous molecules . For example, it has long been known that arsenate and vanadate can compete with phosphate for transport and metabolism, thereby disrupting normal cellular functions . Similarly, cromate and molybdate can mimic sulfate in biological systems . Studies in our laboratory have focused on the transport and toxicity of methylmercury (MeHg) and inorganic mercury . Mercury has a high affinity for reduced sulfhydryl groups, including those of cysteine and glutathione (GSH) . MeHg-l-cysteine is structurally similar to the amino acid methionine, and this complex is a substrate for transport systems that carry methionine across cell membranes . Once MeHg has entered the cell, some of it binds to GSH, and the resulting MeHg-glutathione complex appears to be a substrate for proteins that mediate cellular export of glutathione S-conjugates, including the apically located MRP2 (multidrug resistance-associated protein 2) transporter, a member of the adenosine triphosphate-binding cassette protein superfamily . Because other toxic metals also form complexes with endogenous molecules, comparable mechanisms may be involved in their membrane transport and disposition. Pharm Res, 2002 Oct, 19(10), 1509 - 15 Effect of organic isothiocyanates on the P-glycoprotein- and MRP1-mediated transport of daunomycin and vinblastine; Tseng E et al.; PURPOSE: Organic isothiocyanates (ITCs), or mustard oils, are non-nutrient components present in the diet, especially in cruciferous vegetables . The purpose of this investigation was to examine the effect of ITCs on P-glycoprotein (P-gp)- and multidrug resistance-associated Protein (MRP1)-mediated transport in multidrug resistant (MDR) human cancer cell lines . METHODS: The direct effect of ITCs on the 2-h cellular accumulation of daunomycin (DNM) and vinblastine (VBL), substrates for both P-gp and MRP1, were measured in sensitive and resistant MCF-7 cells and in PANC-1 cells . Resistant MCF-7 cells (MCF-7/ADR) overexpress P-gp whereas PANC-1 cells overexpress MRP1 . The following compounds were evaluated: allyl-, benzyl-(BITC), hexyl-, phenethyl-(PEITC), phenyl-, 1-naphthyl-(NITC), phenylhexyl-, phenylpropyl-, and phenylbutyl-ITC, sulforaphane, erucin, and erysolin . RESULTS: NITC significantly increased the accumulation of DNM and VBL in both resistant cell lines, but had no effect on DNM accumulation in sensitive MCF-7 cells . VBL accumulation in resistant MCF-7 cells was increased 40-fold by NITC whereas that in PANC-1 cells was increased 5.5-fold . Significant effects on the accumulation of DNM and VBL in resistant MCF-7 cells were also observed with benzyl-isothiocyanate whereas PEITC, erysolin, phenylhexyl-ITC, and phenylbutyl-ITC increased the accumulation of DNM and/or VBL in PANC-1 cells . Overall, the inhibitory activities of these compounds in MCF-7 cells and PANC-1 cells were significantly correlated (r2 = 0.77 and 0.86 for DNM and VBL, respectively) . Significant effects on accumulation were generally observed with the ITCs at 50 microM concentrations, but not at 10 microM concentrations . CONCLUSIONS: One strategy to enhance the effectiveness of cancer chemotherapy is to reverse the MDR phenomena . Our results indicate that certain dietary ITCs inhibit the P-gp- and the MRP1-mediated efflux of DNM and VBL in MDR cancer cells and suggest the potential for diet-drug interactions. Braz J Med Biol Res, 2002 Oct, 35(10), 1127 - 31 Epub 2002 Oct 13. Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the mycobacteria growth indicator tube (MGIT) system; Telles MA et al.; The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has increased the need for rapid drug susceptibility tests, which are needed for adequate patient treatment . The objective of the present study was to evaluate the mycobacteria growth indicator tube (MGIT) system to detect multidrug-resistant M . tuberculosis strains . The MGIT system was compared with two standard methods (proportion and resistance ratio methods) . One hundred clinical M . tuberculosis isolates {25 susceptible to isoniazid (INH) and rifampicin (RIF), 20 resistant to INH, 30 resistant to INH-RIF, and 25 resistant to INH-RIF and other drugs} obtained in the State of S o Paulo were tested for INH and RIF susceptibility . Full agreement among the tests was found for all sensitive and all INH-resistant strains . For RIF-resistant strains results among the tests agreed for 53 (96.4%) of 55 isolates . Results were obtained within 6 days (range, 5 to 8 days), 28 days and 12 days when using MGIT, the proportion method and the resistance ratio methods, respectively . The MGIT system presented an overall agreement of 96% when compared with two standard methods . These data show that the MGIT system is rapid, sensitive and efficient for the early detection of multidrug-resistant M . tuberculosis. Med Princ Pract, 2002 Oct-Dec, 11(4), 202 - 5 Primary antituberculosis drug resistance at Turkish military chest diseases hospital in Istanbul; Kartaloglu Z et al.; OBJECTIVE: The aim of this study was to investigate the prevalence of primary drug resistance to tuberculosis . METHODS: We evaluated the clinical data, radiological features and sputum samples from 365 newly diagnosed patients with a positive culture of pulmonary tuberculosis at the Turkish Military Chest Diseases Hospital, Istanbul, Turkey . No patients had taken antituberculosis drugs previously . The Bactec method was used to perform drug susceptibility testing for isoniazid, rifampicin, ethambutol, and streptomycin . RESULTS: Primary resistance to one or more drugs was detected in 87 (23.8%) patients; resistance to isoniazid was most common (54 patients) followed by resistance to ethambutol (n = 39), rifampicin (n = 11), and streptomycin (n = 9) . One-drug resistance was detected in 69 patients; two-drug resistance in 11, three-drug resistance in 6, and four-drug resistance in 1 . Multidrug resistance (resistance to at least isoniazid and rifampicin) was detected in 10 patients . In logistic-regression analysis, primary drug resistance was associated with radiological advanced tuberculosis (p < 0.001) . CONCLUSION: Primary resistance to one or more drugs used in treating tuberculosis is relatively high . It is necessary to regularly screen for and treat drug resistance among those who live in close quarters, such as army barracks, school dormitories and prisons . Regular surveillance of drug sensitivity patterns should be maintained to determine appropriate alternate drug regimens and detect the spread of resistant stains in the population . J Biol Chem, 2003 Jan 31, 278(5), 3347 - 56 Epub 2002 Nov 06. Intermediate structural states involved in MRP1-mediated drug transport . Role of glutathione; Manciu L et al.; Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4) . The transport of chemotherapeutic drugs requires the presence of reduced GSH . By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed . Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium . The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity . Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment . Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis . This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains . On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis . Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium . No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis . Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release. Blood, 2003 Mar 15, 101(6), 2125 - 31 Epub 2002 Nov 07. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML); Del Poeta G et al.; The inability to undergo apoptosis is a crucial mechanism of multidrug resistance in acute myeloid leukemia (AML), and the analysis of mitochondrial apoptotic proteins may represent a significant prognostic tool to predict outcome . Bcl-2 and Bax oncoproteins were evaluated in 255 de novo AML patients (pts) by flow cytometry using an anti-bcl-2 monoclonal antibody (MoAb) and an anti-bax MoAb . The results were expressed as an index (bax/bcl-2) obtained by dividing bax mean fluorescence intensity (MFI) and bcl-2 MFI . Lower bax/bcl-2 ratio was associated with French-American-British (FAB) M0-M1 classes (P =.000 01) and CD34 more than 20% (P <.000 01) . There were striking inverse correlations between CD34 or CD117 MFI and bax/bcl-2 values (r = -.40, P <.000 001 and r = -.29, P =.000 002), confirming that immaturity is consistent with this index . Moreover, lower bax/bcl-2 levels were correlated with poor-risk cytogenetics (P =.0002) . A significant higher complete remission (CR) rate was found in pts with higher bax/bcl-2 levels (79% versus 45%; P =.000 01) . Also, both a longer overall survival (OS) and disease-free survival (DFS) were observed in pts with higher bax/bcl-2 levels (P =.000 01 and =.019) . Noteworthy, bax/bcl-2 levels accurately predicted the clinical response and outcome of pts with normal or unknown cytogenetics . Indeed, within this subset of 147 pts, higher bax/bcl-2 ratio was significantly associated both with a higher CR rate (86% versus 42%; P <.000 01) and a longer OS (P =.0016) . The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis . Therefore, mitochondrial oncoproteins, such as bcl-2 and bax, represent both sensitive indicators of clinical outcome and potential targets of novel proapoptotic molecules in order to circumvent chemoresistance. Exp Hematol, 2002 Nov, 30(11), 1302 - 8 Impact of the expression of P glycoprotein, the multidrug resistance-related protein, bcl-2, mutant p53, and heat shock protein 27 on response to induction therapy and long-term survival in patients with de novo acute myeloid leukemia; Kasimir-Bauer S et al.; OBJECTIVE: Resistance to chemotherapy-induced apoptosis and a multidrug-resistance phenotype is the major problem in the treatment of acute myeloid leukemia (AML) . PATIENTS AND METHODS: We recently demonstrated that the coexpression of at least two proteins, including P glycoprotein, multidrug resistance-related protein, bcl-2 (flow cytometry), p53 (luminometric immunoassay), and heat shock protein 27 (Western blotting), was predictive for response to induction therapy in de novo AML comparing leukemic blasts of 20 responders with 20 nonresponders . After long-term follow-up, we now present our evaluation on the prognostic significance of these proteins in leukemic blasts of 124 untreated AML patients with regard to the probability of remission (PoR) and overall survival (OS) . RESULTS: Analyzing leukemic blasts obtained from bone marrow samples, we found that no single protein significantly correlated with PoR or OS . In contrast, the coexpression of at least two of these proteins was predictive for reduced OS in univariate as well as multivariate analysis . Although we could not identify any particular protein combination predictive for reduced OS, those patients with no or only one protein expressed in their leukemic blasts had a survival probability of 48% in contrast to 24% in those patients with the coexpression of two or more proteins . Among the clinical markers, only response to chemotherapy had a significant effect on OS and age was of prognostic relevance for PoR . CONCLUSION: We conclude that overexpression of only one protein possibly involved in resistance, is not sufficient to influence the prognosis for long-term survival in AML, whereas the expression of more than one protein is predictive for reduced OS . Protein combination seems to be individually different, and targeting only one protein in further clinical trials may not help to overcome multifactorial resistance. Epilepsia, 2002 Nov, 43(11), 1318 - 23 Regional expression of multidrug resistance genes in genetically epilepsy-prone rat brain after a single audiogenic seizure; Kwan P et al.; PURPOSE: The multidrug resistance (mdr) gene family encodes the drug transport macromolecule P-glycoprotein (P-gp), which contributes to the functionality of the blood-brain barrier . Recent evidence suggests that P-gp-mediated drug extrusion may play a facilitatory role in refractory epilepsy . We investigated the regional expression of mdr genes in genetically epilepsy-prone rat (GEPR) brain after a single audiogenic seizure . METHODS: Three groups of adult male GEPRs (n = 5/group) were exposed to a seizure-inducing audiogenic stimulus and killed at 4 h, 24 h, and 7 days thereafter . A further group (n = 5) served as a stimulus-naive control . Expression of mdr1a and mdr1b in distinct anatomic brain regions (cortex, midbrain, pons/medulla, hippocampus) was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the presence of competitive internal standards . RESULTS: When compared with control, mdr1a expression in cortex and midbrain was significantly (p < 0.05) increased at 24 h after a single audiogenic seizure . Cortical mdr1a expression remained elevated at 7 days after stimulus . In contrast, mdr1a expression in pons/medulla and hippocampus was unchanged . The mdr1b isoform was quantifiable in hippocampus alone and not influenced by seizure activity . CONCLUSIONS: These findings suggest that acute seizures in the GEPR can induce the expression of mdr genes . The pattern of increased expression appears to follow the anatomic pathway of audiogenic seizures in these animals, with initiation in the midbrain and propagation to the cortex . Further studies are required to investigate the effects of recurrent seizure activity and to characterise mdr expression in other experimental seizure models. J Org Chem, 2002 Nov 15, 67(23), 8284 - 6 A modified synthesis of iodoazidoaryl prazosin; Andrus MB et al.; The antihypertension agent iodoazidoaryl prazosin (IAAP) has been made using a convergent route involving addition of an acylated piperazine 7 to 2-chloroquinazoline 5 . IAAP has been shown to function as a multidrug resistance (MDR) reversal agent and bind to P-glycoprotein, a transmembrane transport protein . A study is also reported involving palladium-catalyzed substitution with amine heterocycles . With N,N-bis(2,6-diisopropyl)dihydroimidazolium chloride (10) as the ligand (2 mol %) for palladium(II) acetate (2 mol %) in THF at room temperature, morpholine added to 5 in 81% yield. AAPS PharmSci . 2002;4(3):E15. Cloning and characterization of the rat multidrug resistance-associated protein 1; Yang Z et al.; Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs . MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates . An ortholog of MRP1 in the mouse has been cloned and characterized . Significant functional differences between murine and human MRP1 have been noted . Since drug disposition and pharmacology studies often are conducted in rats, there is a need to clone and characterize the rat ortholog of MRP1 . We isolated a rat MRP1 (rMRP1) cDNA from rat brain astrocytes, characterized its coding sequences, and verified the transport activity of the protein expressed in MRP1 cDNA-transfected Madin-Darby canine kidney (MDCK) cells . Our results showed that rMRP1 has a coding sequence of 4599 bp, which predicts a polypeptide of 1533 amino acids with an apparent molecular weight of 190 kd by Western immunoblot analysis . rMRP1-transfected MDCK cells are capable of efflux transport of a fluorescent MRP1 marker - calcein - that is inhibitable by known MRP1 inhibitors, indomethacin, and MK571 . Sequence analysis indicates that rMRP1 is more closely related to mouse MRP1 than human MRP1. AAPS PharmSci . 2002;4(3):E14. Role of MRP4 and MRP5 in biology and chemotherapy; Sampath J et al.; Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades . However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles . Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents) . We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines . MRP5, a close structural homologue of MRP4 also transported PMEA . MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents . The ability of MRP4 and MRP5 to transport 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides . Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression . Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response. J Biol Chem, 2003 Jan 17, 278(3), 1575 - 8 Epub 2002 Nov 05. Drug binding in human P-glycoprotein causes conformational changes in both nucleotide-binding domains; Loo TW et al.; The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell . It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) . Recently, we showed that the "LSGGQ" motif in one NBD ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) in NBD1) in the other NBD (Loo, T . W., Bartlett, M . C., and Clarke, D . M . (2002) J . Biol . Chem . 277, 41303-41306) . Drug substrates can stimulate or inhibit the ATPase activity of P-gp . Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively) . Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) were tested for their effect on oxidative cross-linking . Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate . By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) decreased the rate of cross-linking . These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis . Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences. Parasitol Int, 2002 Dec, 51(4), 353 - 9 Multidrug resistance in the protozoan parasite Entamoeba histolytica; Orozco E et al.; In this review we discuss the mechanisms and molecules involved in the multidrug resistance (MDR) of the protozoan parasite Entamoeba histolytica . Drug resistant mutants exhibited the main characteristics presented by the MDR mammalian cells . They showed cross-resistance to several unrelated drugs that is reverted by calcium channel blockers . MDR phenotype in E . histolytica is regulated at a transcriptional level by the EhPgp1 gene, which is constitutively expressed and by the EhPgp5 gene, whose expression is induced in the presence of the drug . Transcription factors participate in the expression regulation of these genes . After over transcription, the EhPgp genes are amplified, cooperating to produce the MDR phenotype . Post-transcriptional mechanisms such as mRNA stability seem to be involved in this phenomenon . As for other mdr gene products, the EhPGP5 protein functions as a chloride current inductor or as a regulator of cellular regulatory volume decrease. An Med Interna, 2002 Sep, 19(9), 477 - 85 {P-glycoprotein, a membrane pump that represents a barrier to chemotherapy in cancer patients}; Ruiz Gomez MJ et al.; Multidrug resistance (MDR) in oncology is considered to be the main cause of chemotherapy failure in the treatment of patients with cancer . The resistance mechanism consists in decrease intracellular drug accumulation by P-glycoprotein (Gp-P) overexpression . This protein acts as a drug-extracting pump that needs energy in the process . The efflux takes place by mean of a pore in the cell membrane that consist in twelve segments . The activity of this pump is regulated by protein kinase C and shows homology with other transport systems . The analysis of the presence of Gp-P and the characterization of MDR phenotype in biopsy material could be important in the overcome of the resistance to cancer chemotherapy. Biol Pharm Bull, 2002 Nov, 25(11), 1391 - 400 MDR1 genotype-related pharmacokinetics and pharmacodynamics; Sakaeda T et al.; The multidrug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily of membrane transporters . MDR1 acts as an energy-dependent efflux pump that exports its substrates out of cells . MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multidrug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites . A number of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics . In 2000, Hoffmeyer et al . performed a systemic screening for MDR1 polymorphisms and detected 15 single nucleotide polymorphisms (SNPs) . They also indicated that a polymorphism in exon 26 at position 3435 (C3435T), a silent mutation, affected the expression level of MDR1 protein in duodenum, and thereby the intestinal a