World J Gastroenterol, 2005 Feb 7, 11(5), 726 - 8 Effect of mutated IkappaBalpha transfection on multidrug resistance in hilar cholangiocarcinoma cell lines; Chen RF et al.; AIM: To explore the expression effect of mutated IkappaBalpha transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-kappaB (NF-kappaB) . METHODS: We used the mutated IkappaBalpha plasmid to transfect QBC(939)HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-kappaB DNA and the effect of the transfrecting mutated IkappaBalpha plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP) . RESULTS: Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes . The radioactivity accumulation of QBC(939)HCVC+ and QBC939 cells transfected with mutated IkappaBalpha plasmid was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid . Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IkappaBalpha plasmid could inhibit the binding activity of NF-kappaB DNA in hilar cholangiocarcinoma cells . Compared to control group not transfected with m IkappaBalpha plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha plasmid was lower . The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid . CONCLUSION: The mutated IkappaBalpha plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells . Interruption of NF-kappaB activity may become a new target in gene therapy for hilar cholangiocar-cinogenesic carcinoma. J Invest Dermatol, 2005 Jan, 124(1), 28 - 37 Interleukin-6-Type Cytokines Upregulate Expression of Multidrug Resistance-Associated Proteins in NHEK and Dermal Fibroblasts; Dreuw A et al.; Normal human epidermal keratinocytes (NHEK) and dermal fibroblasts express a cell-specific pattern of efflux transport proteins . Since regulatory mechanisms for these transporters in cells of the human skin were unknown, we analyzed the influence of inflammatory cytokines on the expression of multidrug resistance-associated proteins (MRP1, 3, 4, 5) . Using real-time PCR, RT-PCR, cDNA microarray, immunostaining and efflux assays we demonstrated that stimulation of NHEK and primary human dermal fibroblasts with interleukin-6 (IL-6), in combination with its soluble alpha-receptor, or oncostatin M (OSM) for 24-72 h resulted in an upregulation of MRP expression and activity . Both cytokines induced a strong activation of signal transducer and activator of transcription (STAT)1 and STAT3 as well as the mitogen-activated protein kinase (MAPK) Erk1/2 . OSM additionally activated proteinkinase B strongly . Using the MAPK/extracellular signal-regulated kinase kinase 1-specific inhibitor U0126 we could exclude a stimulatory effect of MAPK on MRP gene expression . Inhibition of the phosphatidylinositol 3-kinase, however, indicated that this pathway might be involved of OSM-mediated upregulation of MRP4 in dermal fibroblasts . Several inflammatory skin diseases show an enhanced expression of IL-6-type cytokines . Correspondingly, upregulation of MRP expression was found in lesional skin taken from patients with psoriasis and lichen planus. Trans R Soc Trop Med Hyg, 2005 Mar, 99(3), 234 - 7 Absence of nucleotide polymorphism in a Plasmodium vivax multidrug resistance gene after failure of mefloquine prophylaxis in French Guyana; Picot S et al.; Vivax malaria is widespread and resistance has been described for chloroquine and sulfadoxine-pyrimethamine . We report on evidence of failure of mefloquine prophylaxis in a French soldier who contracted Plasmodium vivax in French Guyana, South America . Despite regular weekly mefloquine prophylaxis (250mg/d), the patient presented with a first episode of vivax malaria, which was treated by chloroquine alone, then experienced a second crisis in France . The reappearance of the parasites occurred one day after the end of prophylaxis, confirming parasitological and clinical resistance in a non-immune patient . Mefloquine was detected by a liquid chromatography assay in plasma at a level of 1062ng/ml, which was higher than the expected concentration after five months of weekly prophylaxis . This isolate had no single nucleotide polymorphisms of the pvmdr1 gene at seven allele positions: pvmdr1 N91, Y189, Y976, S1071, F1076, N1079 and D1291, corresponding to codons 86, 184, 939, 1034, 1039, 1042 and 1246 in P . falciparum . This observation of failure of mefloquine prophylaxis against P . vivax, when added to previously reported chloroquine and atovaquone-proguanil failure, strengthens the case for re-evaluating drug policies for vivax malaria and the need for continuous research on molecular markers of drug resistance. Biochem Pharmacol, 2005 Feb 1, 69(3), 531 - 9 Epub 2004 Dec 15. Mechanisms involved in spironolactone-induced choleresis in the rat Role of multidrug resistance-associated protein 2; Ruiz ML et al.; The mechanisms involved in spironolactone (SL, 200mumol/kg body weight, 3 days i.p.)-induced choleresis were explored in vivo by evaluating bile salt export pump (Bsep)-, multidrug resistance-associated protein 2 (Mrp2)-, and anion exchanger 2 (AE2)-mediated secretory processes in rat liver . Hepatic bile salt metabolism was also analyzed . Total bile flow was significantly increased by SL, primarily due to an increase in bile salt-independent bile flow, whereas bile salt secretion was decreased . SL did not produce any choleresis in TR(-) rats . SL decreased the de novo bile salt synthesis rate in concordance with impaired microsomal cholesterol 7alpha-hydroxylase activity, thus leading to a decrease in endogenous bile salt pool size . In contrast, the maximum secretory rate of tauroursodeoxycholate as well as expression of Bsep protein detected by Western blotting were not affected . Thus, decreased bile salt availability for canalicular transport rather than transport capability itself likely explains reduced biliary secretion of bile salts . Biliary secretion of glutathione, an endogenous substrate of Mrp2, and HCO(3)(-), the AE2 substrate, were increased by SL, as a main factor explaining enhanced bile salt-independent bile flow . Western blot studies revealed increased expression of Mrp2 in response to SL whereas AE2 content remained unchanged . Enhanced activity and expression of Mrp2 was confirmed by analyzing the excretion rate of dinitrophenyl S-glutathione, an exogenous substrate of Mrp2, in isolated hepatocytes and by immunofluorescence microscopy, respectively . We conclude that SL increased bile flow mainly by increasing the biliary secretion of glutathione species and HCO(3)(-); increased expression of Mrp2 is also involved. Altern Lab Anim, 2004 Oct, 32(4), 391 - 9 A New Human Breast Carcinoma Cell Line Resistant to DNA-damaging Drugs; Levina VV et al.; To investigate the phenomenon of active dissociation of the vital dye, Hoechst 33342 (Ho342), from DNA (DNA clearing), a new MCF7HoeR-7 human breast carcinoma cell line was isolated from parent MCF7 cells by step-wise selection with increasing concentrations of Ho342 . This cell line possesses an enhanced ability for DNA clearing . The MCF7HoeR-7 line is characterised in detail and compared with the parental MCF7 line and a typical P-glycoprotein-mediated multidrug resistant (MDR) cell line, MCF7/Adr . MCF7HoeR-7 cells have an increased population growth rate, a lower DNA content and a reduced number of chromosomes . Enhanced DNA clearing in MCF7HoeR-7 cells is associated with the high resistance of the cells to the toxic effects of Ho342 and cross-resistance to etoposide, a topoisomerase II inhibitor in clinical use . The MCF7HoeR-7 and parent MCF7 cell lines have similar expression levels of transport proteins . The results obtained confirm that DNA clearing is an atypical MDR mechanism in tumour cells. Antivir Ther, 2004 Dec, 9(6), 929 - 35 Influence of single-nucleotide polymorphisms in the multidrug resistance-1 gene on the cellular export of nelfinavir and its clinical implication for highly active antiretroviral therapy; Zhu D et al.; Protease inhibitors (PIs) such as nelfinavir (NFV) suppress HIV replication . PIs are substrates of P-glycoprotein (P-gp), the product of the multidrug-resistance-1 (MDR1) gene . Three single-nucleotide polymorphisms (SNPs) are present in exons of the MDR1 gene: MDR1 1236, MDR1 2677 and MDR1 3435 . We speculated that these genetic polymorphisms affected PI concentration in the cell . To verify this hypothesis, we first genotyped these SNPs in 79 Japanese patients by the SNaPshot method and found incomplete linkage disequilibrium between the SNPs . Because the SNP at MDR1 3435 has been reported to be associated with P-gp expression, we evaluated the effect of that SNP on the export of NFV from HIV-positive patients' lymphoblastoid cell lines by measuring time-dependent decrease in the amount of intracellular NFV by high-performance liquid chromatography . We found the intracellular concentration of NFV in lymphoblastoid cell lines (LCLs) with the homozygous T/T genotype at MDR1 3435 were higher than that with C/C genotype with statistical significance . This suggests that the activity of P-gp in patients' LCL cells with the MDR1 3435 T/T genotype was lower . In a retrospective study we evaluated the effect of the SNPs on CD4 cell count recovery in response to antiretroviral treatment with PIs, and obtained statistically significant evidence that suggested marginal association of the SNP at MDR1 1236 but not at MDR1 2677 or MDR1 3435 . As in vitro results were not consistent with the clinical evaluation, clinical importance of MDR1 genotyping for antiretroviral therapy remains to be investigated in a larger, case-controlled study. Oncology (Huntingt), 2004 Nov, 18(13 Suppl 10), 21 - 4 Bcl-2 antisense therapy in multiple myeloma; Chanan-Khan AA; Most malignant plasma cells overexpress Bcl-2, which contributes to resistance against apoptosis induced by dexamethasone and other anticancer agents . Oblimersen sodium (Genasense, previously known as G3139), an antisense oligonucleotide that specifically binds to bcl-2 messenger RNA, decreases production of Bcl-2 protein in both human myeloma cell lines, as well as in ex vivo purified myeloma cells, and enhances the cytotoxicity of dexamethasone and doxorubicin . Combining oblimersen with other anticancer agents represents a therapy-enhancing strategy to reverse the multidrug resistance seen in multiple myeloma (MM) . Phase II trials are evaluating the potential role of oblimersen in reversing resistance to standard therapies . Preliminary results from these trials in patients with refractory or relapsed MM indicate that the combination of oblimersen with dexamethasone/thalidomide (Thalomid) or vincristine/doxorubicin/dexamethasone is active and well tolerated and that oblimersen may help overcome chemotherapy resistance and restore sensitivity to MM cells . A randomized phase III clinical trial comparing dexamethasone plus oblimersen with dexamethasone alone in patients with relapsed or refractory myeloma has completed enrollment, with results expected to be available in 2004 . Future studies will focus on the role of oblimersen in combination with novel biologic agents such as bortezomib (Velcade). Am J Physiol Gastrointest Liver Physiol . 2005 Jan 13; {Epub ahead of print} Short-term Regulation of Multidrug Resistance-Associated Protein 3 (Mrp3/MRP3) in Rat and Human Hepatocytes; Chandra P et al.; The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and protein kinase C (PKC) was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers . The modulators glucagon (500 nM) and the phorbol ester, PMA (0.1 microM), were utilized to increase intracellular cAMP and PKC levels, respectively . In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy- 2',7'dichlorofluorescein (CDF) increased ~1.5 fold, and in the presence of the Oatp inhibitor sulfobromophthalein (BSP), the same increase in CDF efflux was observed relative to BSP treatment alone . Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment . PMA had no effect on either Mrp3 activity or localization in sandwich-cultured rat hepatocytes . Glucagon and PMA treatment in isolated perfused rat livers resulted in a 3-fold increase (14 +/- 4.6 microl/min/g liver) and 4-fold decrease (1.3 +/- 0.3 microl/min/g liver) in CDF basolateral clearance, respectively, compared to control livers (4.7 +/- 2.3 microl/min/g liver), while CDF biliary clearance was not statistically different . In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane . In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3 as demonstrated by alterations in both activity and localization in rat and human hepatocytes. Mol Endocrinol . 2005 Jan 13; {Epub ahead of print} The Pregnane X Receptor Regulates Gene Expression in a Ligand- and Promoter-selective Fashion; Masuyama H et al.; Recent studies have revealed that pregnane X receptor (PXR) can function as a master regulator to control the expression of phase I and phase II drug-metabolizing enzymes, as well as members of the drug transporter family, including multiple drug resistance 1 (MDR1), which has a major role in multidrug resistance . Previously, we have demonstrated that steroid/xenobiotics metabolism by tumor tissue through the PXR-CYP3A pathway might play an important role in endometrial cancer . In this study, we examined which endocrine-disrupting chemicals (EDCs) and anticancer agents might be ligands for PXR and whether these chemicals enhanced PXR-mediated transcription through two different PXR responsive elements (PXRE), CYP3A4 and MDR1, in endometrial cancer cell lines . Some steroids/EDCs strongly activated PXR-mediated transcription through the CYP3A4-responsive element compared with the MDR1-responsive element, while these steroids/EDCs also enhanced the CYP3A4 expression compared with the MDR1 expression . In contrast, the anticancer agents, cisplatin and paclitaxel, strongly activated PXR-mediated transcription through the MDR1-responsive element compared with the CYP3A4-responsive element, while these drugs also enhanced the MDR1 expression compared with the CYP3A4 expression . We also analyzed how these ligands regulated PXR-mediated transcription through two different PXREs . In the presence of PXR ligands, there was no difference in the DNA binding affinity of the PXR/RXR heterodimer to each PXRE, but there were different interactions of the coactivator to each PXR/PXRE complex . These data suggested that PXR ligands enhanced PXR-mediated transcription in a ligand- and promoter-dependent fashion, which in turn differentially regulated the expression of individual PXR targets, especially CYP3A4 and MDR1. Virus Res, 2005 Feb, 107(2), 151 - 64 Cell entry and export of nucleoside analogues; Pastor-Anglada M et al.; Some nucleoside analogues currently used as antiretroviral agents might promote mutagenesis besides their putative ability to interfere with endogenous nucleotide metabolism and/or inhibit viral transcription . The intracellular concentration of nucleosides and nucleobases is to some extent the result of the metabolic background of the specific cell line used for infection studies, its particular suit of enzymes and transporters . This review focuses on the transporter-mediated pathways implicated in either the uptake or the efflux of nucleoside- and nucleobase-derivatives . From a biochemical point of view, four different types of transport processes for nucleoside-related antiviral drugs have been described: (1) equilibrative uniport, (2) substrate exchange, (3) concentrative Na(+)- or H(+)-dependent uptake and finally, (4) substrate export through primary ATP-dependent active efflux pumps . These mechanisms are mainly related to the following set of transporter families: Concentrative Nucleoside Transporter (CNT), Equilibrative Nucleoside Transporter (ENT), Organic Anion Transporter (OAT) and Organic Cation Transporter (OCT), Peptide Transporter (PEPT) and Multidrug Resistance Protein (MRP) . The basic properties of these carrier proteins and their respective role in the transport across the plasma membrane of nucleoside-derived antiviral drugs are reviewed. Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 866 - 70 Effects of dietary chemopreventive phytochemicals on P-glycoprotein function; Nabekura T et al.; The effects of dietary phytochemicals on P-glycoprotein function were investigated using human multidrug-resistant carcinoma KB-C2 cells and the fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123 . The effects of natural chemopreventive compounds, capsaicin found in chilli peppers, curcumin in turmeric, {6}-gingerol in ginger, resveratrol in grapes, sulforaphane in broccoli, 6-methylsulfinyl hexyl isothiocyanate (6-HITC) in Japanese horseradish wasabi, indole-3-carbinol (I3C) in cabbage, and diallyl sulfide and diallyl trisulfide in garlic, were examined . The accumulation of daunorubicin in KB-C2 cells increased in the presence of capsaicin, curcumin, {6}-gingerol, and resveratrol in a concentration-dependent manner . The accumulation of rhodamine 123 in KB-C2 cells was also increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by these phytochemicals . Sulforaphane, 6-HITC, I3C, and diallyl sulfide and diallyl trisulfide had no effect . These results suggest that dietary phytochemicals, such as capsaicin, curcumin, {6}-gingerol, and resveratrol, have inhibitory effects on P-glycoprotein and potencies to cause drug-food interactions. Br J Biomed Sci, 2004, 61(4), 206 - 10 Reversal of multidrug resistance of gastric cancer cells by down-regulation of ZNRD1 with ZNRD1 siRNA; Hong L et al.; The over-expression of a new zinc ribbon (ZNRD1) gene has been shown previously to promote a multidrug-resistant phenotype in gastric cancer cells through the up-regulation of permeability-glycoprotein (P-gp) . In the present study, siRNA eukaryotic expression vectors of ZNRD1 are constructed and transfected into SGC7901/VCR cells to examine whether or not down-regulation of ZNRD1 increases cell sensitivity towards chemotherapeutic drugs . After transfection, ZNRD1 expression decreased dramatically in ZNRD1 siRNA transfectants compared with that in parental cells and empty vector control cells . Down-regulation of ZNRD1 significantly enhanced the sensitivity of SGC7901/VCR cells to vincristine, adriamycin and etoposide, but not to 5-fluorouracil and cisplatin . Cell capacity to efflux adriamycin decreased markedly in ZNRD1 siRNA transfectants, and correlation between ZNRD1 down-regulation and increased multidrug resistance 1 (MDR1) gene transcriptional activity was observed . These results suggest that the ZNRD1 siRNA constructs down-regulate the expression of ZNRD1 effectively and reverse the resistant phenotype of gastric cancer cells . Furthermore, ZNRD1 might influence transcription of the MDR1 gene and thus play an important role in multidrug resistance in gastric carcinoma. Curr Opin Investig Drugs, 2004 Dec, 5(12), 1340 - 7 MS-209 Schering; Robert J; MS-209, a quinolone-derived sphingomyelin synthase inhibitor that blocks P-glycoprotein and multidrug resistance-associated protein-1, is under development by Schering for the potential treatment of multidrug resistant tumors . By March 2003, phase II trials in breast cancer and non-small-cell lung cancer had been initiated. New Microbiol, 2004 Apr, 27(2 Suppl 1), 63 - 9 Host factors and efficacy of antiretroviral treatment; Turriziani O et al.; It has been proposed that some host factors may affect the intracellular drug concentration leading to the inability of drug regimens to inhibit human immunodeficiency virus (HIV) replication in cells . Among them, two factors, whose description is the main aim of this review, have been considered during the last years with particular emphasis . They are: i) altered uptake and reduced activation of nucleoside reverse transcriptase inhibitors (NRTIs) in target cells, and ii) efflux of NRTIs and protease inhibitors (PIs) by cellular transporter molecules . In fact, several authors have shown that: changes in the activity of various purine and pyrimidine biosynthetic enzymes may occur in lymphocytes of HIV-infected patients; HIV-infected patients on prolonged treatment with nucleoside analogs, such as zidovudine, show significantly decreased activity of thymidine kinase compared to untreated HIV-infected persons; NRTI and PIs are substrates for the so-called multidrug membrane transporters . With regard to the latter issue, it is known that the ATP-binding cassette transporter proteins such as the P glycoprotein, and the newly discovered family of multidrug resistance-associated proteins (MRP 1-9) promote the active extracellular efflux of a wide variety of therapeutics and overexpression of some of them lowers intracellular concentration of PIs . In the very near future such mechanisms, called by most authors "cellular drug-resistance", might be taken into account, together with other immunological, virological and behavioral factors, to explain "drug failure" and/or the variability of response in HIV patients undergoing an antiretroviral treatment. Int J Cancer . 2005 Jan 11; {Epub ahead of print} Intrinsic chemotherapy resistance to the tubulin-binding antimitotic agents in renal cell carcinoma; Ferguson RE et al.; Renal cancer is one of the most chemoresistant tumor types . Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin-binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P-glycoprotein (PgP), multidrug resistance associated protein-2 (MRP2) and major vault protein (MVP) proteins . The IC(50) values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance . A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013) . MVP expression did not correlate with sensitivity to any of the drugs examined . All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer . In tissue samples from chemotherapy-naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen . Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples . This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies . (c) 2004 Wiley-Liss, Inc. Rev Bras Psiquiatr, 2004 Sep, 26(3), 189 - 201 Epub 2004 Nov 17. {The hypothalamic pituitary adrenal axis, glucocorticoid receptor function and relevance to depression}; Juruena MF et al.; OBJECTIVES: Changes in the hypothalamic-pituitary-adrenocortical (HPA) system are characteristic of depression . Because the effects of glucocorticoids are mediated by intracellular receptors including, most notably, the glucocorticoid receptor (GR), several studies have examined the number and/or function of GRs in depressed patients . METHODS: Review scientific evidences have consistently demonstrated that GR function is impaired in major depression, resulting in reduced GR-mediated negative feedback on the HPA axis and increased production and secretion of CRF in various brain regions postulated to be involved in the causality of depression . RESULTS: This article summarizes the literature on GR in depression and on the impact of antidepressants on the GR in clinical and preclinical studies, and supports the concept that impaired GR signalling is a key mechanism in the pathogenesis of depression, in the absence of clear evidence of decreased GR expression . The data also indicate that antidepressants have direct effects on the GR, leading to enhanced GR function and increased GR expression . Although the effects of antidepressants on glucocorticoid hormones and their receptors are relevant for the therapeutic action of these drugs, the molecular mechanisms underlying these effects are unclear . We propose that antidepressants in humans could inhibit steroid transporters localised on the blood-brain barrier and in neurones, like the multidrug resistance p-glycoprotein, and thus increase the access of cortisol to the brain and the glucocorticoid-mediated negative feedback on the HPA axis . CONCLUSION: Enhanced cortisol action in the brain might prove to be a successful approach to maximise therapeutic antidepressant effects . Hypotheses regarding the mechanism of these receptor changes involve non-steroid compounds that regulate GR function via second messenger pathways . Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders. Can J Physiol Pharmacol, 2004 Dec, 82(12), 1084 - 1090 Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells; Kothan S et al.; Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments . However, their modes of action are still unclear . In this study, the effects of quercetin on mitochondrial membrane potential (&Delta;&Psi;m) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated . Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 +/- 2.0 micromol/L and 5.0 +/- 0.4 micromol/L for K562 and K562/adr, respectively . Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI . Quercetin (10 micromol/L, 3 h) and etoposide (100 micromol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells . Quercetin induces an increase followed by a decrease in |&Delta;&Psi;m| value depending on its concentration . A decrease in the |&Delta;&Psi;m| value is associated with an increase in the percentage of early apoptotic cells . It is clearly shown that quercetin results in a spontaneous &Delta;&Psi;m change during apoptotic induction . Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level. ILAR J, 2005, 46(1), 62 - 4 Veterinary medicine in the 21st century: the challenge of biosecurity; Kelly AM; The veterinary profession is presently challenged with developing and maintaining on-farm biosecurity protocols to protect the nation's food supply from acts of bioterrorism, from the growing threat of foreign animal diseases, and from multidrug resistance among pathogenic organisms . This challenge comes at a time when the supply of food animal veterinarians in the United States is progressively in decline, and raises the possibility that the profession is not adequately prepared to fulfill its responsibilities to the health and productivity of the US livestock and poultry populations . Causes of the decline in demand for veterinary services are discussed . They include consolidation of the food animal industries and a trend toward transferring performance of tasks traditionally carried out by veterinarians to the province of lay staff . This development potentially reduces veterinary surveillance of food animal populations . It also runs the risk of delay in recognizing and controlling serious health problems when they arise . Several remedies are proposed, including profound changes in the curriculum for educating food animal veterinarians to serve the consolidated but vulnerable livestock and poultry industries suitably . Also advocated is the initiation of training programs for herdsmen on the symptoms of foreign animal diseases, together with advice on when to call a veterinarian . Significant investment of federal or state resources will be required if these changes are to become reality. J Pharmacol Exp Ther . 2005 Jan 11; {Epub ahead of print} TRIMERS OF N-ALKYLGLYCINES ARE POTENT MODULATORS OF THE MULTIDRUG RESISTANCE PHENOTYPE; Abad-Merin MJ et al.; The multidrug resistance (MDR) phenotype is considered a major cause of the failure of cancer chemotherapy . The acquisition of MDR is usually mediated by the overexpression of drug-efflux pumps such as glycoprotein P (P-gp) or multidrug resistance-related protein 1 (MRP1) . Thus, the identification, validation and development of compounds that mitigate the MDR phenotype by modulating the activity of these transport proteins is an important, yet elusive, target . Here, we have addressed this issue and screened an N-trialkylglycine-based combinatorial library composed of 5,120 compounds to search for modulators of the MDR phenotype . The screening identified 20 trimers of N-alkylglycine that increased the intracellular accumulation of daunomycin (DNM) in drug-resistant L1210R tumor cells that overexpressed the P-gp . These compounds appear to act as P-gp antagonists as evidenced by the augment of DNM accumulation in the cell line L1210(P-gp), a drug sensitive L1210 cell stably expressing the murine P-gp protein . Similarly, several of the active N-trialkylglycines also produced an increment in DNM uptake in human HL-60R cells, which primarily express the MRP1 protein . Trialkylglycines notably sensitized L1210R and HL60R tumor cells to DNM with a potency that rivaled that of verapamil (VRP) . These findings provide new molecular scaffolds for the development of effective chemosensitizers against the MDR phenotype that, in due turn, could be used as adjuvant drugs in cancer chemotherapy. Oncol Rep, 2005 Feb, 13(2), 217 - 22 Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human non-small cell lung cancer cells; Ikuta K et al.; Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy . Recently, the constitutive activation of signal transducer and activator of transcription (STAT) factors has been found in a variety of human cancers . In the present study, the mRNA expression of STATs in various human NSCLC cell lines was investigated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine whether STATs can be implicated in cisplatin resistance and apoptosis inducibility . Cisplatin triggered apoptosis in Ma-46 based on biochemical and morphological findings, but not in Ma-31 . The mRNA expression of STAT3 was highest in cisplatin-resistant Ma-31 and lowest in cisplatin-sensitive Ma-46 . A 6-hour exposure of cancer cells to cisplatin failed to stimulate STAT3 mRNA expression . Therefore, an increased transcriptional level of constitutive STAT3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemo-resistant NSCLC cells. Haematologica, 2005 Jan, 90(1), 54 - 9 Differences in anti-apoptotic and multidrug resistance phenotypes in elderly and young acute myeloid leukemia patients are related to the maturation of blast cells; Suarez L et al.; BACKGROUND AND OBJECTIVES: Elderly patients with acute myeloid leukemia (AML) have a less favorable outcome, which has been related, among other factors, to multidrug resistance (MDR) phenotypes . DESIGN AND METHODS: Freshly obtained erythrocyte-lysed bone marrow samples from 150 elderly patients (> 65 years) with de novo AML and 30 younger AML patients were analyzed using a 4-color immunofluorescence technique for quantitative expression of proteins associated with apoptosis (bcl-2, bax, APO2.7) and MDR (P-gp, MRP, LRP) in 3 blast cell subpopulations, defined according to their maturation stage . RESULTS: Although a homogeneous CD34+ blast cell population was more frequent in the elderly patients, (25% vs 7%, p=0.02), no statistically significant differences were detected between the two age groups in the expression of either apoptosis- or MDR-associated proteins, except for slightly higher quantities of LRP protein in the more immature CD34+ blast cell subset in the elderly AML cases (p=0.04) . Interestingly, when different blast cell populations were compared, immature (CD34+) blast cells were characterized by higher levels of bcl-2 in both age groups and lower levels of APO2.7 in the elderly group . In addition, higher P-gp levels were found in CD34+ blast cells than in CD34-- ones in elderly AML patients . Reactivity for LRP was low in both elderly and younger patients . INTERPRETATION AND CONCLUSIONS: In summary, our results suggest that the higher resistance to chemotherapy observed in elderly AML patients could be related to a higher incidence of cases with a CD34+ homogeneous blast cell population, since these blast cells frequently display a more pronounced anti-apoptotic and MDR1 phenotype. Ai Zheng, 2005 Jan, 24(1), 53 - 7 {Relationships among Expressions of hTERT, MDR1, MRP mRNA, and C-myc Protein in Non-small Cell Lung Cancer.}; Li L et al.; BACKGROUND & OBJECTIVE: The latest researches showed that myc protein could up-regulate the expression of human telomerase reverse transcriptase (hTERT), multidrug resistance gene 1(MDR1), multidrug resistance-related protein (MRP) in some kinds of tumors, and hTERT is correlated with efficiency of anti-tumor chemotherapy . This study was to investigate relations among expressions of hTERT, MDR1, MRP mRNA, and C-myc protein in non-small cell lung cancer (NSCLC) . METHODS: Expressions of hTERT, MDR1, MRP mRNA in 113 cases of NSCLC tissues were detected by in situ hybridization, expression of C-myc protein was detected by SP immunohistochemistry, their correlations with clinicopathologic features of NSCLC were statistically analyzed . RESULTS: Positive rates of hTERT, MDR1, MRP mRNA, and C-myc protein in NSCLC tissues were 80.5%, 51.3%, 80.5% and 68.1%, repectively . Expressions of MDR1, MRP mRNA, and C-myc protein were significantly related to that of hTERT mRNA (P<0.05) . Expression of C-myc protein did not correlate with expression of MDR1 or MRP mRNA . All 4 factors have no correlation with clinicopathologic features of NSCLC (P>0.05) . CONCLUSION: Expression of hTERT mRNA may be related to those of MDR1, MRP mRNA, and C-myc in NSCLC . Overexpression of C-myc protein may be one of the molecular regulatory mechanisms of hTERT mRNA. Biochemistry, 2005 Jan 18, 44(2), 643 - 55 Interaction of LDS-751 with P-Glycoprotein and Mapping of the Location of the R Drug Binding Site; Lugo MR et al.; One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products . The protein appears to interact with its substrates within the membrane environment . Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively . In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site . A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform . LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 muM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye . Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites . The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet . The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane. Cancer Lett, 2005 Jan 31, 218(1), 15 - 20 4'-O-Alkyaloenin derivatives and their sulfates directed toward overcoming multidrug resistance in tumor cells; Jin GZ et al.; The cytotoxic effects on HCT 116, Hep G2 and HCT 116/VCR 100-1-1 cell lines of synthetic 4'-O-alkylaloenins (2-17), 4'-O-benzylaloenin (18) and 4'-O-allylaloenin (19) were examined by MTT assay, and compared with that of aloenin (1) isolated from Aloe arborescens Mill . Var . natalensis Berger which showed no marked effect (IC(50) value: >100muM) . The cytotoxic effects of 4'-O-alkylaloenin sulfates (21-29) were also examined on the same cell lines . The introduction of a longer alkyl group at the O-4' position of 1 resulted in a higher cytotoxic action on HCT 116 and Hep G2 cells . Among 4'-O-alkylaloenins 2-17, 4'-O-tetradecylaloenin 14 was the most cytotoxic to both on HCT 116 cells (IC(50) value: 5.3+/-2.3muM) and Hep G2 cells (IC(50) value: 4.0+/-0.6muM) . Also among 4'-O-alkylaloenin sulfates 21-29, 4'-O-dodecylaloenin sulfate 29 was the most cytotoxic to both on HCT 116 (IC(50) value: 4.8+/-0.2muM) and Hep G2 cells (IC(50) value: 4.0+/-0.5muM) . 4'-O-Alkylaloenins 7-14 and 4'-O-alkylaloenin sulfates 24-29 were also cytotoxic to Hep G2 and HCT 116/VCR 100-1-1 cell lines, which overexpress P-glycoprotein, as well as HCT 116 cell lines which scarcely express it. Curr Med Chem, 2005 Jan, 12(2), 127 - 51 The Search of DNA-Intercalators as Antitumoral Drugs: What it Worked and What did not Work; Martinez R et al.; The discovery of new compounds with antitumoral activity has become one of the most important goals in medicinal chemistry . One interesting group of chemotherapeutic agents used in cancer therapy comprises molecules that interact with DNA . Research in this area has revealed a range of DNA recognizing molecules that act as antitumoral agents, including groove binders, alkylating and intercalator compounds . DNA intercalators (molecules that intercalate between DNA base pairs) have attracted particular attention due to their antitumoral activity . For example, a number of acridine and anthracycline derivatives are excellent DNA intercalators that are now on the market as chemotherapeutic agents . Commercially available acridine and anthracycline derivatives have been widely studied from a variety of viewpoints, such as physicochemical properties, structural requirements, synthesis and biological activity . However, the clinical application of these and other compounds of the same class has encountered problems such as multidrug resistance (MRD), and secondary and/or collateral effects . These shortcomings have motivated the search for new compounds to be used either in place of, or in conjunction with, the existing compounds . Unfortunately, the results of this search have not met expectations . The vast majority of candidate intercalator compounds tested for use as anticancer agents have shown little or no biological activity . Research in this area has not been without benefits, however, for it has produced much information on the synthesis and antitumoral properties of hundreds of compounds, which have been tested on diverse tumoral cell lines . This review considers the structural and biological considerations relevant to the use of DNA intercalators and bis-intercalators as antitumoral agents, with an emphasis on the relationship between structure and activity, produced in last decade. Gen Physiol Biophys, 2004 Sep, 23(3), 357 - 66 Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy; Orlicky J et al.; Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp) . Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp . Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker . To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors . Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters . Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS) . Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal . Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3 . Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro . These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells. Int J Tuberc Lung Dis, 2004 Dec, 8(12), 1448 - 57 Molecular epidemiology of drug-resistant Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis in Poland: a 1-year study; Sajduda A et al.; OBJECTIVE: To characterise drug-resistant Mycobacterium tuberculosis strains isolated in Poland and to estimate the amount of recent transmission in the population . DESIGN: M . tuberculosis strains isolated from 251 patients with resistant pulmonary tuberculosis in Poland in 2000 were analysed by spoligotyping and IS6110 DNA fingerprinting . Part of the strains was also characterised by sequencing of the rpoB, katG and/or the regulatory region of the inhA gene . RESULTS: Using combined spoligotyping/IS6110-RFLP defined clusters, 29% of the strains were clustered, suggesting possible recent transmission . In some cases, transmission links among strains in clusters could be confirmed by epidemiological data and in addition, for most of the strains, by analysis of the mutations associated with resistance to rifampicin and/or isoniazid . Younger age, sex, immigration and history of previous treatment were not associated with clustering, whereas multidrug-resistant disease was more likely to cluster . Strains of the Beijing family could also be found in Poland, although with a much lower frequency than in the neighbouring countries . CONCLUSION: Transmission of drug-resistant M . tuberculosis strains was demonstrated, which might contribute to the emergence of drug-resistant tuberculosis in Poland. Epidemiol Infect, 2004 Dec, 132(6), 1099 - 108 Explaining risk factors for drug-resistant tuberculosis in England and Wales: contribution of primary and secondary drug resistance; Conaty SJ et al.; Drug-resistant tuberculosis can be transmitted (primary) or develop during the course of treatment (secondary) . We investigated risk factors for each type of resistance . We compared all patients in England and Wales with isoniazid- and multidrug-resistant tuberculosis in two time-periods (1993-1994 and 1998-2000) with patients with fully sensitive tuberculosis, examining separately patients without and with previous tuberculosis (a proxy for primary and secondary drug-resistant tuberculosis) . Patients with previous tuberculosis smear positivity and arrival in the United Kingdom <5 years were strongly associated with multidrug resistance and isoniazid resistance . In patients with no previous tuberculosis HIV infection, residence in London and foreign birth were risk factors for multidrug resistance, and non-white ethnicity, residence in London and HIV infection for isoniazid resistance . Risk factors for each type of resistance differ . Elevated risks associated with London residence, HIV positivity, and ethnicity were mainly seen in those without previous tuberculosis (presumed transmission). Vnitr Lek, 2001 Jan, 47(1), 25 - 9 {Multidrug resistance of the leukemia cells--the functional fluorescent dye efflux assay evaluated by the flow cytometry}; Jankovicova K et al.; The functional fluorescent dye efflux assays are used in the study of the multidrug resistance of the malignant cells to the cytotoxic drugs which is caused by the overexpression of P-glycoprotein (P-gp) or another membrane transport system--a multidrug resistance related protein (MRP) . P-glycoprotein and a multidrug resistance related protein are involved in the efflux of cytotoxic drugs out of the cell and are responsible for the resistance . Fluorescent dye efflux mediated by these proteins could be evaluated by the flow cytometry . This test seems to be an optimal approach to study the multidrug resistance . With help of the specific inhibitors such as verapamil and cyclosporine A, the functional capacity of these proteins and the possibility to overcome the multiresistant phenotype can be revealed . The cell line K562 with transfected P-glycoprotein gene serves as a model system for the studying of the transport function with the use of the fluorescent substrates and P-glycoprotein inhibitors. Biol Pharm Bull, 2005 Jan, 28(1), 138 - 42 Effects of 19 Herbal Extracts on the Sensitivity to Paclitaxel or 5-Fluorouracil in HeLa Cells; Takara K et al.; The popularity of traditional herbal medicine (THM) being used as complementary medicines or alternative medicines is increasing . On the other hand, the development of multidrug resistance (MDR) remains a major hurdle to successful cancer chemotherapy . Some THMs capable of reversing MDR may contribute to the improvement of clinical outcomes in cancer chemotherapy . Herein, 19 kinds of herb were chosen from the ingredients of major THMs, and their effects on the sensitivity to anticancer drugs of tumor cells were investigated using the human cervical carcinoma HeLa cells . Focusing on the major mechanism for MDR, i.e., MDR1/P-glycoprotein, the effects of herbal extracts on its transport function were also examined using a MDR1 substrate Rhodamine123 . Glycyrrhizae Radix, Rhei Rhizoma, Scutellariae Radix, Poria, Zizyphi Fructus, Zingiberis Rhizoma (dry), Coptidis Rhizoma, Ephedrae Herba and Asiasari Radix significantly enhanced the sensitivity to a MDR1 substrate paclitaxel, whereas none of the herbal extracts used had any effect on the sensitivity to 5-fluorouracil, which is not a substrate for MDR1 . Rhodamine123 uptake was significantly increased by Rhei Rhizoma, Poria or Ephedrae Herba among nine herbal extracts sensitized to paclitaxel . This suggests that the increase in paclitaxel sensitivity by Glycyrrhizae Radix, Rhei Rhizoma, Poria or Ephedrae Herba was caused, in part, by the inhibition of MDR1 function, and the change in paclitaxel sensitivity by the other herbal extracts was not always dependent on this . Collectively, these findings indicate that the combination of anticancer drugs with some herbal extracts contributes to the enhancement of clinical outcomes in cancer chemotherapy. J Clin Microbiol, 2005 Jan, 43(1), 406 - 13 Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis; Palmer S et al.; To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis . Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen . Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product . For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced . Sequences from 15 to 46 single viral genomes were obtained from each plasma sample . Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied . Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86) . Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes . For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods . Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype . In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis . These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations . In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance . Such linkage cannot be detected by standard genotype analysis. J Clin Microbiol, 2005 Jan, 43(1), 306 - 13 Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family; Kam KM et al.; Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis and may represent a feasible approach to study global M . tuberculosis molecular epidemiology . To evaluate the use of MIRU typing in discriminating drug-resistant M . tuberculosis strains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing . Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype . MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index {HGI} of 0.8827) compared with the IS6110 RFLP method (HGI = 0.9979) . For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961) . There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains of M . tuberculosis . Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype . Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%) . This supported the potential use of this method to analyze the global genetic diversity of MDR M . tuberculosis strains that may be at different levels of evolutionary divergence. J Clin Microbiol, 2005 Jan, 43(1), 208 - 13 How evolution of mutations conferring drug resistance affects viral dynamics and clinical outcomes of cytomegalovirus-infected hematopoietic cell transplant recipients; Springer KL et al.; Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality among hematopoietic cell transplant (HCT) recipients . We describe two pediatric HCT recipients who developed persistent and severe drug-resistant CMV infections . CMV resistance to foscarnet and ganciclovir was detected after only 6 and 11 weeks of therapy, respectively . Viral pol mutations associated with drug resistance in these patients included T838A (a novel mutation) and D588N, which were shown by marker transfer to confer foscarnet and multidrug resistance, respectively . Each of these mutations significantly reduced in vitro replication of CMV, suggesting that they may decrease viral fitness . This finding was further supported by the disappearance of mutations upon withdrawal of antiviral pressure in one patient . Novel antivirals or combination therapy may be required for the treatment of drug-resistant CMV in HCT recipients and perhaps in other severely immunocompromised patients. J Pharmacol Exp Ther . 2005 Jan 5; {Epub ahead of print} Efflux of Depsipeptide FK228 (FR091228, NSC-630176) is Mediated by Both P-glycoprotein and MRP1; Xiao JJ et al.; Depsipeptide FK228, a novel histone deacetylase (HDAC) inhibitor, was previously reported to be a P-glycoprotein (Pgp) substrate . We now expand the investigation to demonstrate that FK228 is a substrate for both Pgp and multidrug resistance-associated protein 1 (MRP1) . Transport of FK228 across the Caco-2 cell monolayer in both apical to basolateral (AP-->BL) and basolateral to apical (BL-->AP) directions in the absence and presence of Pgp and MRP inhibitors were investigated . An in vitro uptake study in human red blood cells (RBC) and a cytotoxicity assay in MRP1(-) HL60 and MRP1(+) HL60Adr cells were conducted to show that FK228 is a MRP1 substrate . A FK228 resistant cell line (HCT-15R) was developed from HCT-15 colon carcinoma, and characterized using a 70-oligomer cDNA microarray, RT PCR, western blot, histone acetyltransferase (HAT) and HDAC activity assays, and cytotoxicitiy assays . FK228 showed a nearly unidirectional flux across the Caco-2 cell monolayer, with the BL-->AP apparent permeability coefficient 32x that of AP-->BL without apparent saturation . Pgp inhibition decreased the BL-->AP Papp and increased the AP-->BL Papp . RBC showed a concentration-dependent uptake and a saturable efflux of FK228 . HL60Adr cells were 4-fold more resistant to FK228 than HL60 cells and the resistance was reversed by MRP inhibition . Upregulation of Pgp, but not changes of MRPs or HAT/HDAC enzymatic activities, was the major mechanism for the acquired FK228 resistance . These studies demonstrate that FK228 is a substrate for both Pgp and MRP1, and reversible Pgp upregulation is predominantly involved in FK228 resistance in vitro. Mol Cancer Ther, 2004 Dec, 3(12), 1631 - 7 The ex vivo characterization of XR5944 (MLN944) against a panel of human clinical tumor samples; Di Nicolantonio F et al.; XR5944 (MLN944) is a novel DNA targeting agent with potent antitumor activity, both in vitro and in vivo, against several murine and human tumor models . We have used an ATP-tumor chemosensitivity assay to assess the ex vivo sensitivity of a variety of solid tumors (n = 90) and a CCRF-CEM leukemia cell line selected with XR5944 . Differences in gene expression between the parental CCRF-CEM and the resistant subline were investigated by quantitative reverse transcription-PCR . Immunohistochemistry for topoisomerases I and IIalpha and multidrug resistance (MDR1) protein was done on those tumors for which tissue was available (n = 32) . The CCRF-CEM XR5944 line showed increased mRNA levels of MDR1, major vault protein, and MDR-associated protein 1 compared with the parental line, whereas the expression of topoisomerases I, IIalpha, and IIbeta was essentially unchanged, suggesting that XR5944 is susceptible to MDR mechanisms . The median IC(90) and IC(50) values for XR5944 in tumor-derived cells were 68 and 26 nmol/L, respectively, 6-fold greater than in resistant cell lines . XR5944 was 40- to 300-fold more potent than the other cytotoxics tested, such as doxorubicin, topotecan, and paclitaxel . Breast and gynecologic malignancies were most sensitive to XR5944, whereas gastrointestinal tumors showed greater resistance . A positive correlation (r = 0.68; P < 0.0001) was found between the IC(50) values of XR5944 and P-glycoprotein/MDR1 staining but not with either topoisomerase I or IIalpha immunohistochemistry index . These data support the rapid introduction of XR5944 to clinical trials and suggest that it may be effective against a broad spectrum of tumor types, especially ovarian and breast cancer. Mol Cancer Ther, 2004 Dec, 3(12), 1577 - 84 Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference; Ee PL et al.; Overexpression of the breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy . To circumvent BCRP-mediated MDR, a common approach is the use of potent and specific inhibitors of BCRP transport such as fumitremorgin C, novobiocin, and GF120918 . Here, we evaluated a new approach using RNA interference for the specific knockdown of BCRP . We designed and synthesized small interfering RNA (siRNA) using T7 RNA polymerase and showed that siRNAs markedly down-regulated both exogenous and endogenous expression of BCRP . As a functional consequence, knockdown of BCRP by siRNAs increased the sensitivity of human choriocarcinoma BeWo cells to mitoxantrone and topotecan by 10.5- and 8.2-fold, respectively . Using flow cytometry, we found that introduction of siRNAs also enhanced the intracellular accumulation of topotecan . We have previously identified an estrogen response element in the BCRP promoter and have shown that 17beta-estradiol increased BCRP mRNA expression . Furthermore, in the present study, we found that expression of BCRP protein was inducible by 17beta-estradiol and that this effect was ameliorated by the introduction of siRNAs . These studies indicate that siRNAs could modulate MDR in vitro and may present a new approach to overcome BCRP-mediated drug resistance. Zhonghua Zhong Liu Za Zhi, 2004 Oct, 26(10), 601 - 5 {Effect of arsenic trioxide on drug transporting molecules in acute promyelocytic leukemia cell line.}; Qian XP et al.; OBJECTIVE: To study the effect of arsenic trioxide (As2O3) on expression of drug transporting molecules in APL MR(2) cell line . METHODS: MR(2) resistant to all-trans retinoic acid (ATRA) and non-ATRA resistant APL cell line NB(4) was used in this in vitro study . Expression of P-glycoprotein (Pgp), multidrug resistance protein (MRP) and lung resistance-related protein (LRP) was detected by immunocytochemical assay . RESULTS: The expression of Pgp was significantly higher in MR(2) (30% - 40%) than in NB(4) (10% - 20%) (P < 0.001), and that of MRP was also higher in MR(2) (56.9 +/- 3.4 approximately 21.2 +/- 1.1) than in NB(4) (20.6 +/- 5.3 approximately 16.7 +/- 1.2) (P < 0.001) . As2O3 at concentrations ranging from 0.5 approximately 2.0 micromol/L could significantly decrease the expression of Pgp and MRP, but not that of LRP . The decrease in the expression of Pgp and MRP in MR(2) cell line was negatively correlated with the dose and duration of action of As2O3 . CONCLUSION: Pgp and MRP, but not LRP, may be the sensitive targets of As2O3 to overcome drug-resistance . ATRA might be the substrates of Pgp and MRP. Yi Chuan Xue Bao, 2004 Dec, 31(12), 1332 - 6 {Molecular mutations of rpoB gene of multidrug resistant Mycobacterium tuberculosis isolates from China}; Yue J et al.; To characterize rpoB mutations of multidrug resistant Mycobacterium tuberculosis clinical isolates from China, mutations in the 81 bp rifampin resistance determining region (RRDR) and mutation V176F locating in the beginning of the rpoB gene were analyzed by DNA sequencing . Eighty six Mycobacterium tuberculosis clinical isolates, including 72 rifampin resistant strains and 14 rifampin sensitive strains were sequenced . Six five mutations of 22 distinct kinds, 21 point mutations and one insertion, were observed in 65 of 72 resistant isolates . The most frequent mutations were in codon 531 (41%), 526 (40%), and 516 (4%) . Mutations were not found in seven (10%) of the resistant isolates . Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported . None of isolates contained the V176 mutation. Microbiology, 2005 Jan, 151(Pt 1), 99 - 111 Acetaminophen toxicity and resistance in the yeast Saccharomyces cerevisiae; Srikanth CV et al.; Acetaminophen (paracetamol), one of the most widely used analgesics, is toxic under conditions of overdose or in certain disease conditions, but the mechanism of acetaminophen toxicity is still not entirely understood . To obtain fresh insights into acetaminophen toxicity, this phenomenon was investigated in yeast . Acetaminophen was found to be toxic to yeast cells, with erg mutants displaying hypersensitivity . Yeast cells grown in the presence of acetaminophen were found to accumulate intracellular acetaminophen, but no metabolic products of acetaminophen could be detected in these extracts . The toxicity response did not lead to an oxidative stress response, although it did involve Yap1p . The cytochrome P450 enzymes of yeast, Erg5p and Erg11p, did not appear to participate in this process, unlike the mammalian systems . Furthermore, we could not establish a central role for glutathione depletion or the cellular glutathione redox status in acetaminophen toxicity, suggesting differences from mammalian systems in the pathways causing toxicity . Investigations of the resistance mechanisms revealed that deletion of the glutathione-conjugate pumps Ycf1p (a target of Yap1p) and Bpt1p, surprisingly, led to acetaminophen resistance, while overexpression of the multidrug resistance pumps Snq2p and Flr1p (also targets of Yap1p) led to acetaminophen resistance . The Yap1p-dependent resistance to acetaminophen required a functional Pdr1p or Pdr3p protein, but not a functional Yrr1p . In contrast, resistance mediated by Pdr1p/Pdr3p did not require a functional Yap1p, and revealed a distinct hierarchy in the resistance to acetaminophen. Am J Pathol, 2005 Jan, 166(1), 39 - 48 Expression, localization, and function of MRP5 (ABCC5), a transporter for cyclic nucleotides, in human placenta and cultured human trophoblasts: effects of gestational age and cellular differentiation; Meyer Zu Schwabedissen HE et al.; The placenta functions both as site for nutrition and protection of the fetus . Transport proteins, including members of the multidrug resistance protein (MRP)/ABCC subfamily, have been recognized to contribute to the latter function . MRP5 (ABCC5) was identified as transmembrane transport protein for cyclic nucleotides, especially 3',5'-cyclic GMP (cGMP), indicating an additional role in signal transduction and a potential role in placenta development . We therefore studied expression, localization, and function of MRP5 in placenta of different gestational ages . Quantitative real-time polymerase chain reaction revealed expression of MRP5 in all 60 samples from pre-term and term placenta, with a decreasing mean expression with gestational age (MRP5/18S-ratio x 1000; < 32 weeks: 2.91 +/- 0.73, n = 15; 32 to 37 weeks: 2.10 +/- 0.87, n = 15; > 37 weeks: 0.46 +/- 0.08, n = 30; P < 0.01) . Immunofluorescence microscopy with an anti-MRP5 antibody indicated localization of MRP5 preferentially in the basal membrane of syncytiotrophoblasts and in and around fetal vessels . ATP-dependent {(3)H}cGMP transport as evidence for MRP5 function could be demonstrated in isolated basal membrane vesicles . Moreover, the influence of cellular differentiation on MRP5 expression was studied in isolated trophoblasts, revealing an increase of the MRP5 expression in parallel with the hCG production (MRP5/18S-ratio x 1000 was 2.4 +/- 0.5 at day 5 of culture and 1.45 +/- 0.5 at day 0 of culture, n = 3 preparations, significant difference with P < 0.05) . In conclusion, MRP5 expression depends on gestational age and varies throughout the differentiation process . In view of the important role of cGMP for cellular differentiation, MRP5 may play a role in placental development in context with a specific need for cellular cGMP export. Zhongguo Zhong Yao Za Zhi, 2004 Oct, 29(10), 970 - 3 {Study on ligustrazine in reversing multidrug resistance of HepG2/ADM cell in vitro}; Mei Y et al.; OBJECTIVE: To study the reverse effect of ligustrazine (TMP) on HepG2/ADM, a herd of hepatocellular carcinoma cell, multidurg resistance (MDR) and the influence of P-gp170 expression . METHOD: The reverse effect of ligustrazine on HepG2/ADM cell was observed, with the methods of cell culture, MTT's analyze, RT-PCR and Flow cytometric, etc . RESULT: Ligustrazine could make MDR of cell line of HepG2/ADM reduce the expression of P-gp170, enhance the density of adriamycin in cell and increase the adriamycin's cytotoxicity . With the Flow cytometric, the results of RT-PCR showed the transcriptional activity of the MDR1 decreased . CONCLUSION: Ligustrazine can reverse MDR of HCC cell line of HepG2/ADM and has prospect in clinical use. J Clin Invest, 2005 Jan, 115(1), 76 - 85 Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy; Hauber I et al.; The introduction of highly active antiretroviral therapy (HAART) has significantly decreased morbidity and mortality among patients infected with HIV-1 . However, HIV-1 can acquire resistance against all currently available antiretroviral drugs targeting viral reverse transcriptase, protease, and gp41 . Moreover, in a growing number of patients, the development of multidrug-resistant viruses compromises HAART efficacy and limits therapeutic options . Therefore, it is an ongoing task to develop new drugs and to identify new targets for antiretroviral therapy . Here, we identified the guanylhydrazone CNI-1493 as an efficient inhibitor of human deoxyhypusine synthase (DHS) . By inhibiting DHS, this compound suppresses hypusine formation and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), a cellular cofactor of the HIV-1 Rev regulatory protein . We demonstrate that inhibition of DHS by CNI-1493 or RNA interference efficiently suppressed the retroviral replication cycle in cell culture and primary cells . We show that CNI-1493 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and viral strains with high-level resistance to inhibitors of viral protease and reverse transcriptase . Moreover, no measurable drug-induced adverse effects on cell cycle transition, apoptosis, and general cytotoxicity were observed . Therefore, human DHS represents a novel and promising drug target for the development of advanced antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses. Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 437 - 45 Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand; Winstone TL et al.; Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations . To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent . EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM . The purified protein was analyzed by SEC-HPLC to estimate the monodispersity and to determine the amount of bound detergent . The results show that EmrE is homogeneous in DM with a Stokes radius of 3.6nm compatible with that of a monomer . Sedimentation velocity experiments indicated that the EmrE preparation was monodisperse and supports the fact that the organic extracted protein solubilized in DM is monomeric . This monomeric form of the protein analyzed here is also shown to bind substrate in the micromolar range. Biochemistry, 2005 Jan 11, 44(1), 340 - 51 The Leucotriene C(4) Binding Sites in Multidrug Resistance Protein 1 (ABCC1) Include the First Membrane Multiple Spanning Domain; Karwatsky J et al.; The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites . Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1 . In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)) . The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody . Moreover, binding with radioiodinated {(125)I}AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH) . Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1 . Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1 . Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1 . Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site . Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222) . MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4) . These results confirmed that sequences in MSD0 interact with IAALTC(4) . Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s) . Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0. Oligonucleotides, 2004, 14(3), 191 - 8 The reduction of P-glycoprotein expression by small interfering RNAs is improved in exponentially growing cells; Stierle V et al.; Small interfering RNAs (siRNAs) are powerful tools in specifically silencing gene expression . Nevertheless, their efficiency can be limited when targeting proteins with an unusually long half-life, such as P-glycoprotein (P-gp), which is involved in the multidrug resistance phenomenon . P-gp is characterized by a long half-life, which may vary depending on the cell line and, for some of them, on serum deprivation or high cell density . In the present paper, involvement of an exponential cell growth phase in the improvement of siRNA efficiency has been suggested . The doxorubicin-selected human line MCF7-R was shown to be a more adapted model than NIH-MDR-G185 cells stably transfected with human mdr1 . Nonspecific effects occurring at moderate (100 nM) siRNA concentration have been shown . Two efficient siRNAs led to a very satisfactory P-gp extinction (only 20% P-gp expression remaining) with siRNA concentration as low as 20 nM. Zhonghua Xue Ye Xue Za Zhi, 1997 Aug, 18(8), 425 - 8 {Effects of antisense oligodeoxynucleotides targeting multidrug resistance gene on resistant cell line K562/ AO2}; Hu B et al.; OBJECTIVE: To investigate the reversal effect of antisense oligodeoxynucleotide on human multidrug-resistant leukemic cell line K562/AO2 . METHODS: Antisense oligodeoxynucleotides (AOD) targeting-6 approximately 9 sites of exon 2 in human multidrug resistance gene(mdr-1), one of which is sequence-strict-complied and linked with polyethyleneglycol (PEG) at 5' end (AP, 15 mer), the other lacks nucleotide complied site-1 (AP', 14mer), were synthesized . AP, AP' and verapamil were simultaneously added to human mdr-1-mRNA positive leukemia cell line K562/AO2 and, mdr-1-mRNA and p170 were detected . AS' and AP'were labelled by FITC and designated as ASF' and APF', respectively . In addition, the intracellular concentration of them was detected by FACS . RESULTS: AP' significantly enhanced the sensitivity of K562/AO2 to DOX, down-regulated the expression of mdr-1 and MRP-mRNA and p170, elevated the intracellular concentration of the two AOD, while AP had no effect . The uptake of APF' was significantly higher than that of ASF' in K562/AO2, and the fluorescence was observed in the plasma and nuclear of K562/AO2 cells . CONCLUSION: (AOD targeting mdr-1 promoted the drug sensitivity of drug-resistant tumor cells . 2 AOD had no cytotoxicity to tumor cells . 5 Low molecule PEG enhanced significantly the uptake of AOD by tumor cells. Zhonghua Xue Ye Xue Za Zhi, 1997 Dec, 18(12), 646 - 8 {Effects of cytokines on multidrug-resistance in K562/A02 cells}; Liu L et al.; OBJECTIVE: To explore the effects of cytokines on human leukemic cell line K562/S and its multidrug-resistant counterpart K562/A02 . METHODS: The toxicities of cytokines and the IC50 (the concentration causing 50% inhibition of cell growth) of DNR were assayed by MTT method; intracellular drug concentration was measured by fluorometry; p-glycoprotein (p-gp) expression was detected by APAAP and mdr-1 mRNA was assayed by RT-PCR . RESULTS: The IC50 of DNR for K562/A02 and K562/S cells were 45.08 microg/ml and 0.607 microg/ml, respectively . Pretreating K562/A02 cells with rhu IFN (500 U/ml) or rhu IL-2 (250 U/ml) for 24 hours partially restored the sensitivity of K562/A02 cells to DNR (IC50 were 16.39 and 11.96 microg/ml, respectively) but had not effect on K562/S cells, and it elevated the intracellular DNR accumulation in K562/A02 from 2151 ng/mg x protein to 2570 and 2503ng/mg x protein, respectively . p-gp and mdr-1 mRNA were not down regulated . By contrast, rhu G-CSF and rhu GM-CSF had no effect on either K562/A02 or K562/S . CONCLUSION: rhu IFN or rhu IL-2 could partially restore the sensitivity of K562/A02 to DNR and elevate the intracellular DNR accumulation via a mechanism independent of p-gp or mdr-1 mRNA down-regulation. Zhonghua Xue Ye Xue Za Zhi, 1997 Dec, 18(12), 627 - 9 {Study of antisense oligodeoxynucleotide in reversing multidrug resistance and inducing apoptosis of tumor cells}; Zhang F et al.; OBJECTIVE: T o explore an approach to the reversal of multidrug resistance(MDR) of tumor cells . METHODS: Antisense oligodeoxynucleotide (mdr-1-AS PS-ODN) was used . RESULTS: A large amount of DNA fragments was found in mdr-1-AS PS-ODN treated K562/ADM cells and almost all the mdr-1+ K562/ADM cells underwent apoptosis . CONCLUSION: mdr-1-AS PS-ODN could inhibit specifically mdr-1 gene expression, effectively reverse MDR of the tumor cells and promote ADM induced apoptosis of mdr-1+ K562/ADM. Radiat Res, 2004 Nov, 162(5), 527 - 35 Fractionated irradiation leads to restoration of drug sensitivity in MDR cells that correlates with down-regulation of P-gp and DNA-dependent protein kinase activity; Ryu JS et al.; We showed that the drug sensitivity of multidrug-resistant (MDR) cells could be enhanced by fractionated irradiation . The molecular changes associated with fractionated radiation-induced chemosensitization were characterized . Irradiated cells of the multidrug-resistant CEM/MDR sublines (CEM/MDR/IR1, 2 and 3) showed a loss of P-glycoprotein (P-gp) and concurrent reduction of Ku DNA binding and DNA-PK activities with decreased level of Ku70/80 and increased level of DNA-PKcs, and these changes were followed by an increased susceptibility to anticancer drugs . These irradiated MDR cells also exhibited the reduction of other chemoresistance-related proteins, including BCL2, NF-kappaB, EGFR, MDM2 and Ku70/80, and the suppression of HIF-1alpha expression induced by hypoxia . In contrast, fractionated irradiation increased the levels of these proteins and induced drug resistance in the parental drug-sensitive CEM cells . These results suggest that the chemoresistance-related proteins are differentially modulated in drug-sensitive and MDR cells by fractionated irradiation, and the optimized treatment with fractionated radiation could lead to new chemoradiotherapeutic strategies to treat multidrug-resistant tumors. Indian Pediatr, 2004 Dec 7, 41(12), 1246 - 1251 Safety Profile of Ciprofloxacin used for Neonatal Septicemia; Chaudhari S et al.; We conducted a case matched control study to observe the adverse effects of ciprofloxacin used in neonatal septicemia We enrolled 30 neonates with multidrug-resistant septicemia who were treated with intravenous ciprofloxacin for 14 days . Thirty matched neonates with septicemia treated with other antibiotics were enrolled as controls There was no difference in the mean serum electrolytes, hepatic, renal and hematologic parameters of the two groups . Serial ultrasonographic measurements of the cartilage of the knee after 1 and 6 months showed no difference in the two groups . The femoral cartilage showed an increase of 78.8 percnt in the mean longitudinal area after 6 months in the study group . In the control group, the femoral cartilage showed a 78.4 percnt increase after 6 months . Similarly, the tibial cartilage showed no difference in the percentage increase in size of the study and control group at the end of 6 months . When controlled for birth weight and gestation, cartilage size was not affected by ciprofloxacin. Clin Cancer Res, 2004 Dec 15, 10(24), 8656 - 64 A selective retinoid X receptor agonist bexarotene (Targretin) prevents and overcomes acquired paclitaxel (Taxol) resistance in human non-small cell lung cancer; Yen WC et al.; PURPOSE: Paclitaxel is an important anticancer agent for the treatment of non-small cell lung cancer (NSCLC) . However, its use in cancer therapy is limited by development of acquired drug resistance . The goal of this study was to determine the effect of bexarotene on development of acquired paclitaxel resistance in NSCLC . EXPERIMENTAL DESIGN: Human NSCLC Calu3 cells were repeatedly treated in culture with intermittent paclitaxel alone or in combination with continuous bexarotene for 3 months . Thereafter, cells were isolated and characterized for their drug sensitivity in vitro and in vivo . RESULTS: Repeat exposure to paclitaxel alone resulted in development of paclitaxel resistance with cross-resistance to multidrug resistance P-glycoprotein substrates, whereas the bexarotene/paclitaxel combination prevented the development of drug resistance and the cells remained chemosensitive . Furthermore, paclitaxel resistance could be overcome when the resistant cells were treated with the combination regimen . Fluctuation analysis showed that treatment with bexarotene decreased the rate of spontaneous development of paclitaxel resistance . In vivo, the bexarotene/paclitaxel combination regimen produced a statistically significant decrease in tumor growth in a Calu3 NSCLC xenograft model compared with the single agents (two-tailed, P < 0.05) . In addition, paclitaxel-resistant Calu3 tumors treated with the bexarotene/paclitaxel combination showed greater delay in tumor growth compared with those treated with paclitaxel alone . CONCLUSIONS: Our results suggest that bexarotene may offer a novel approach to prevent and overcome paclitaxel resistance in patients with NSCLC. Sci China C Life Sci, 2004 Oct, 47(5), 425 - 33 The relationship between MRP1 activities and its NBD conformational changes; Huang Z et al.; MIANS, a sulfhydryl-reactive fluorescence, was used to label the cysteines of MRP1 (multidrug resistance protein), and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS, which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment . The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs, of which one lies close to NBD1, and the other near NBD2 . ATP, ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS . The collisional quenchers, acrylamide, l-, and Cs+ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged . Both MIANS and NEM, which are sulfhydryl-reactive reagents, inhibited MRP1 ATPase activity, whereas anticancer drugs activated it . These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1 . Nucleotides can bind directly to NBDs, but drugs may react first with TMDs, which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity, which is coupled with the transport of its substrates . Taken together, the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1. Zhonghua Xue Ye Xue Za Zhi, 1997 Feb, 18(2), 76 - 9 {Reversal of drug resistance in multidrug resistant tumor cells by an oligomer complementary to the MDR1 gene}; Li H et al.; OBJECTIVE: To overcome the multidrug resistance (MDR) in tumor cells . METHODS: Human drug resistant cell line KB-8-5 was transfected with a synthetic oligodeoxynucleotide complementary to the 5' end region of MDR1 gene (ODN) . In vitro drug sensitivity was measured by MTT assay . Intracellular drug concentration was assessed by flow cytometric analysis and expression of P-glycoprotein (Pgp) was determined by immunohistochemistry method . RESULTS: The administration of ODN for 72 hours increased daunorubicin (DNR) accumulation and decreased Pgp expression in KB-8-5 cells . Incomplete reversal of MDR in the KB-8-5 cells was observed when lipofectin was used to deliver ODN to the cells . Lipofectin enhanced the reversing activity, and approximately 74.43% of the cells lost their resistance to DNR . CONCLUSION: ODN circumvents resistance in the KB-8-5 cells may be ascribed to the reduced expression of Pgp in the cells. Zhonghua Xue Ye Xue Za Zhi, 1997 Feb, 18(2), 73 - 5 {Clinical study on reversal of multidrug resistance in refractory and relapsed acute leukemias by cyclosporin A}; Li X et al.; OBJECTIVE: To explore the clinical implication of reversal of multidrug resistance (MDR) by cyclosporin A (CsA) in refractory and relapsed acute leukemias . METHODS: The expression of p170 was assayed by immunocytochemical method (Avidin-Biotin Complex, ABC) using a monoclonal antibody JSB-1 against P-170 . The P-170 positive cases were randomly divided into two groups:trial group (with CsA as revertant) and control group (without CsA), and the blood concentration of CsA was detected by HPLC . RESULTS: The complete remission rates were 53% and 20% in trial and control group, respectively (P<0.05), and the reversing effect was positively correlated with blood concentration of CsA . CONCLUSION: CsA might be a safe and effective revertant, and can be applied in the treatment of refractory and relapsed acute leukemia. Zhonghua Xue Ye Xue Za Zhi, 1997 May, 18(5), 254 - 6 {Effect of cyclosporin A on intracellular accumulation and efflux of drug in K562/DOX cells}; Meng H et al.; OBJECTIVE: To explore measures to overcome multidrug resistance of tumor cells . METHODS: The effects of cyclosporin A (CsA) on drug sensitivity, intracellular drug accumulation and drug efflux in multidrug-resistant human leukemic cell line K562/DOX were studied by MTT colorimetric assay and spectrofluorimetry . RESULTS: CsA enhanced the cytotoxicity of doxorubicin (DOX) to K562/DOX, and when CsA > or = 2microg/ml the sensitivity of K562/DOX to DOX increased significantly . The efflux of DOX markedly decreased when K562/DOX cells were incubated with 2microg/ml CsA, while CsA had no effect on efflux of DOX in K562 cells . CONCLUSION: CsA could markedly decrease the efflux and enhance the intracellular accumulation of DOX in K562/DOX cells. Biol Trace Elem Res, 2004 Dec, 102(1-3), 91 - 104 Decreased Zinc Toxicity Resulting from Doxorubicin Without Increased GSSG Export in Three Human Lung Cell Lines; Walther UI et al.; Zinc-mediated cytotoxicity is recognized, at least in part, by a decrease of reduced glutathione (GSH) and an increase in the oxidized form of glutathione (GSSG) . Doxorubicin is a common inducer of multidrug-resistance-associated proteins and such proteins might, furthermore, be associated by an increased GSSG export rate . Therefore, zinc-mediated toxicity should be abolished after doxorubicin pretreatment . In the present study, zinc toxicity was characterized by methionine incorporation, glutathione content, and the GSSG/GSH ratio . Experiments were performed in three established lung cell lines comparing doxorubicin-pretreated cells with controls . Zinc-mediated toxicity was significantly decreased after pretreatment with doxorubicin as assessed by methionine-incorporation inhibition, GSH depletion, and/or GSSG increase in the two nonmalignant cell lines . Unexpectedly, zinc-associated GSSG export was not increased after doxorubicin pretreatment . This inconsistency might be explained as a result of a decreased zinc content in these cells, probably because of an increased export rate of zinc . The findings are in contradiction to the opinion of metal excretion by multidrug-resistance-associated proteins, matched to GSH conjugate excretion, as it is discussed for cadmium, for example. Leuk Lymphoma, 2005 Jan, 46(1), 63 - 70 Daunorubicin efflux assay in determining multidrug resistance of patients with acute myeloid leukemia; Kim DH et al.; We evaluated the predictive value of mdr1 mRNA RT-PCR, P-glycoprotein (Pgp), and Daunorubicin efflux assays as regards achieving complete remission (CR), overall survival (OS), and leukemia-free survival (LFS) in 72 patients with AML . mdr1 mRNA, Pgp, and Efflux were expressed in 55.6%, 36.1%, and 33.3% . Efflux(+) was associated with a lower CR rate (P = 0.006) . A multivariate analysis of OS identified 3 prognostic factors: WBC (P = 0.028), age (P = 0.002), and Efflux (P = 0.005), while those of LFS identified 2 prognostic factors: age (P = 0.021) and Efflux (P < 0.001) . Efflux was the most reliable method in predicting an achievement of CR and stratifying the patients according to the prognosis in terms of OS and LFS in AML. J Drug Target, 2004, 12(8), 517 - 26 Mixed polymer micelles of amphiphilic and cationic copolymers for delivery of antisense oligonucleotides; Vinogradov SV et al.; Cationic copolymers were synthesized by conjugation of branched 2 kDa polyethylenimine (PEI) and Pluronic block copolymers (F38, P85, P123) . Compositions of these copolymers mixed with corresponding free Pluronics at weight ratio 1:9 were used to complex phosphorothioate oligonucleotides (ODN) . As a result stable suspensions of small micelle-like particles (<220 nm) were obtained . Incorporation of ODN in these formulations increased uptake of ODN in KBv cells and increased sequence specific activity of antisense ODN targeted against MDR gene in multidrug resistant cells resulting in inhibition of the functional activity of P-glycoprotein (P-gp) in these cells . Furthermore, these formulations increased transport of ODN across model intestinal barrier, Caco-2 cell monolayers, suggesting that they could be useful for oral delivery of biologically active ODN. Nucleosides Nucleotides Nucleic Acids, 2004, 23(10), 1595 - 607 An antisense oligodeoxynucleotide-doxorubicin conjugate: preparation and its reversal multidrug resistance of human carcinoma cell line in vitro; Ren Y et al.; An antisense oligodeoxynucleotide-doxorubicin conjugate was synthesized by an aminocaproic acid linker . The synthetic conjugate was identified by HPLC analysis and UV-vis spectra . Properties of the conjugate in vitro conditions were investigated . The results demonstrated that the conjugate was remarkably stabilized by doxorubicin . When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37 degrees C, the conjugate was more stable than doxorubicin or the mixture of doxorubicin and antisense oligodeoxynucleotide . When incubated in 10% fetal serum at 37 degrees C, the conjugate showed a remarkable stabilization as compared to the unmodified oligodeoxynucleotide . Melting experiments demonstrated that the covalent attachment of doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its complementary sequence . In addition, in vitro reversion of multidrug resistance by the conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxorubicin . The result showed that the conjugate displayed very high reversal multdrug resistance activity in KB-A-1 cells in vitro . The conjugate lowered the IC50 value from 21.5 microM to 2.2 microM with a fold-reversal factor of 10 . In contrast, a slight decrease of the IC50 value was observed when they combined with the "free" antisense oligodeoxynucleotide: the IC50 value was down from 21.5 microM to 16.8 microM . This study suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be helpful in multidrug resistance reversal. Drug Metab Pharmacokinet, 2003, 18(6), 381 - 9 Involvement of Multiple Transport Systems in the Disposition of an Active Metabolite of a Prodrug-type New Quinolone Antibiotic, Prulifloxacin; Yagi Y et al.; Prulifloxacin is a prodrug-type new quinolone . The purpose of this study is to clarify the mechanism of biliary excretion and brain distribution of its active metabolite, UFX . UFX was efficiently excreted into the bile in rats, with its concentration in the bile being 30-60 times higher than that in plasma . The in vivo disposition study revealed that multidrug resistance-associated protein 2 (MRP2) was involved in the biliary excretion of glucuronide metabolite, but not of the unchanged UFX . A transport study using a P-glycoprotein (P-gp) overexpressing cell line, LLC-GA5-COL150, showed that UFX was a substrate of P-gp . Nevertheless, the biliary clearance (CLbile) of UFX in P-gp-gene-deficient mice was not different from that in the normal mice, although the concentration in the liver was slightly higher than that in the normal mice . These observations suggest that multiple transport systems are involved in the biliary excretion of UFX, with minor contribution of P-gp . The distribution of UFX in the rat brain was quite low, and its tissue to plasma concentration ratio (Kp) in the brain was much less than the unity and was increased by cyclosporin A . The Kp in the brain of mdr1a/1b(-/-) mice was higher than that in the normal mice, suggesting that efflux by P-gp played a major role in the limited brain distribution of UFX. Drug Metab Pharmacokinet, 2003, 18(4), 238 - 44 Mechanism of Active Secretion of Phenolsulfonphthalein in the Liver via Mrp2 (abcc2), an Organic Anion Transporter; Itagaki S et al.; Phenolsulfonphthalein (PSP) has been selected as a model drug that is eliminated from both the kidney and liver in rats . Although the renal PSP transport system has been studied, few details of the biliary excretion of PSP have been reported . We investigated the biliary excretion system for PSP in rats . It has been reported that the biliary excretion of many organic anions from hepatocytes into bile is mediated by a primary active transporter, referred to as multidrug resistance-associated protein 2 (Mrp2/abcc2) . The biliary excretion of PSP in SD rats was significantly decreased in the presence of Mrp2 inhibitors . The biliary excretion of PSP in Eisai hyperbilirubinemic rats (EHBR), hereditarily Mrp2-defective rats, was significantly lower than that in SD rats . Moreover, an efflux experiment using Caco-2 cells was carried out to confirm Mrp2-mediated PSP transport . Mrp2 inhibitors significantly decreased PSP efflux from Caco-2 cells . These results suggest that Mrp2 contributes to the biliary excretion of PSP in SD rats. Drug Metab Pharmacokinet, 2003, 18(1), 16 - 22 Potential cholestatic activity of various therapeutic agents assessed by bile canalicular membrane vesicles isolated from rats and humans; Horikawa M et al.; The active transport of solutes mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance associated protein-2 (MRP2/ABCC2) are thought to involve bile acid-dependent and -independent bile formation, respectively . To evaluate the potential of therapeutic agents as inhibitors of such transporters on bile canalicular membranes, we examined the inhibition of the primary active transport of typical substrates by 15 drugs, clinically known to cause cholestasis in canalicular membrane vesicles . The inhibition by most of the compounds in rat canalicular membrane vesicles (CMVs) was minimal or observed at much higher concentrations than obtained in clinical situations . However, cloxacillin, cyclosporin A and midecamycin inhibited BSEP, and cyclosporin A and midecamycin inhibited MRP2 with an inhibition constant close to the clinical concentration . By comparing the inhibition potential between rat and human CMVs, the inhibition of BSEP- and MRP2-mediated transport by midecamycin and cyclosporin A was relatively similar whereas the inhibitory effect on BSEP-mediated transport by cloxacillin and glibenclamide was more marked in humans than in rats . These results suggest that the majority of cholestasis-inducing drugs have a minimal inhibitory effect on rat BSEP and MRP2 although species differences in inhibitory potential should be considered, especially in the case of BSEP. Drug Metab Pharmacokinet, 2002, 17(5), 479 - 81 Polymorphism of MDR1 Gene in Healthy Japanese Subjects: A Novel SNP with an Amino Acid Substitution (Glu108Lys); Honda T et al.; We discovered a novel single nucleotide polymorphism (SNP) at position 325 (G325A) in exon 5 of the multidrug-resistance 1 (MDR1) gene in a study of 37 healthy Japanese subjects . Details are as follows . SNP, 020614Honda001; GENE NAME, human P-glycoprotein (MDR1); ACCESSION NUMBER, M29427; LENGTH, 25 bases; 5'-ATGAATCTGGAGG/AAAGACATGACCA-3' . This SNP is expected to cause an amino acid substitution (Glu108Lys) . In this study, one homozygote and one heterozygote for G325A were identified. Drug Metab Pharmacokinet, 2002, 17(4), 367 - 73 mRNA Expression and Amino Acid Transport Characteristics of Cultured Human Brain Microvascular Endothelial Cells (hBME); Umeki N et al.; An in vitro cell culture system for estimating the human blood-brain barrier (BBB) permeability of drugs is required for the development of drugs with effects on the central nervous system . In this study, cultured human brain microvascular endothelial cells (hBME) were characterized . hBME cells exhibited concentration-dependent uptake of L-Leu, L-Glu and L-Lys with K(m) values of 51.1+/-23.1 muM, 163.3+/-79.8 muM and 72.4+/-56.6 muM, respectively . The cellular accumulation of rhodamine123 in hBME cells was unaffected by P-glycoprotein (P-gp) substrates (cyclosporin A, quinidine and verapamil), while the accumulation in human P-gp-overexpressing cells was significantly increased in the presence of these P-gp substrates . RT-PCR revealed that hBME cells expressed large neutral amino acid transporter 1 (LAT1) and its associated molecule (4F2hc), excitatory amino acid transporter 3 (EAAT3), cationic amino acid transporter 1 (CAT1), glucose transporter 1 (GLUT1), monocarboxylic acid transporter 1 (MCT1) and multidrug resistance-associated protein 1 (MRP1) . However, no expression of multidrug resistance protein 1 (MDR1) was detected . The results suggest that these amino acid transporters are functionally expressed at the human BBB, and that hBME cells retain the in vivo BBB transport functions and expression characteristics . Consequently, hBME cells should be a useful tool for studies of the human BBB. Drug Metab Pharmacokinet, 2002, 17(1), 23 - 33 The Potential for an Interaction between MRP2 (ABCC2) and Various Therapeutic Agents: Probenecid as a Candidate Inhibitor of the Biliary Excretion of Irinotecan Metabolites; Horikawa M et al.; Irinotecan hydrochloride (CPT-11) is an anticancer agent with unpredictable bouts of diarrhea as a dose-limiting toxic side-effect . Since the biliary excretion of its active metabolite (SN-38) and SN-38 glucuronide (SN38-Glu), which are mediated by the multidrug resistance associated protein-2 (MRP2/ABCC2), has been proposed to be related to this gastrointestinal toxicity, we have attempted here to examine the potential of various therapeutic agents to interact with the biliary excretion in order to identify MRP2 inhibitors to prevent this toxicity . The inhibition constants (K(i)) of 26 compounds were examined for the transport of a typical MRP2 substrate in isolated canalicular membrane vesicles . Of these, 13 compounds inhibited the transport with K(i) values from 0.0461 to 281 muM . Three inhibitors (probenecid, sulfobromophthalein and glycyrrhizin) were also found to inhibit the biliary excretion of SN-38 and SN38-Glu in rats in vivo, and the degrees of inhibition were compatible with the estimated values based on the ratios of K(i) and unbound concentrations in circulating plasma . A similar estimation of the potential inhibitory effect in human was also examined by considering both the K(i) of each therapeutic agent and its unbound concentration both in circulating plasma and the inlet to the liver . The predicted degrees of inhibition by most compounds were minimal whereas approximately 75% inhibition was predicted for probenecid . Thus, probenecid may be a candidate which can be used clinically to inhibit the biliary excretion of CPT-11 metabolites, whereas an interaction between most of the other compounds and MRP2 is more unlikely. Plant Physiol, 2005 Jan, 137(1), 104 - 16 Epub 2004 Dec 23. Analysis of detergent-resistant membranes in Arabidopsis . Evidence for plasma membrane lipid rafts; Borner GH et al.; The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains . The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes . A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes . A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins . They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains . We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells . Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes . The DRMs were also 5-fold increased in sphingolipid-to-protein ratio . Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition . Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains . The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes. Cancer Lett, 2005 Jan 20, 217(2), 181 - 90 Potentiation by alpha-tocopheryl succinate of the etoposide response in multidrug resistance protein 1-expressing glioblastoma cells; Kang YH et al.; Multidrug resistance protein 1 (MRP1) is one of the representative members of the ATP-binding cassette superfamily of transporters that is involved in resistance to chemotherapeutic agents in cancer patients . MRP1 functions as an efflux pump of drugs, primarily those conjugated to glutathione (GSH) . Decreases in the intracellular concentration of GSH have been shown to enhance the response of MRP1-overexpressing cells to MRP1-substrate drugs by limiting the available drug-GSH conjugates . We report here that alpha-tocopheryl succinate (TOS), a vitamin E analogue, decreased intracellular GSH concentration and blocked MRP1 function in glioblastoma cells . Functional blockade by TOS of MRP1 was confirmed by the enhanced accumulation of etoposide (VP-16), an MRP1-substrate drug . As a result, co-treatment of TOS with VP-16 or treatment with liposomes containing both TOS and VP-16 greatly enhanced the response of MRP1-expressing glioblastoma cells to VP-16 . TOS may be a promising adjuvant for enhancing the therapeutic efficacy of VP-16 in patients with MRP1-expressing glioblastomas. Cancer Lett, 2005 Jan 20, 217(2), 171 - 80 A novel multidrug resistance phenotype of bladder tumor cells grown on Matrigel or SIS gel; Hurst RE et al.; We have previously shown that growth of bladder carcinoma cell lines onto matrices such as Matrigel and small intestinal submucosal (SIS) gel cause distinct changes in cellular morphology and motility . In these studies, we found that bladder cells grown on Matrigel showed increased resistance to either doxorubicin or mitomycin-C whereas growth of cells in SIS gel caused either significant increases or little difference in drug resistance, depending on both the cells and the drug . Finally, it was found that this altered drug sensitivity is reversible with a finite half-life and is likely due to altered drug accumulation and/or cell cycle kinetics. BMC Infect Dis . 2004 Dec 23;4(1):63 {Epub ahead of print} First documented cure of a suggestive exogenous reinfection in polymyositis with same but multidrug resistant M . tuberculosis; Mukhopadhyay C et al.; First documented cure of a suggestive exogenous reinfection in polymyositis with same but multidrug resistant M . tuberculosis Chiranjoy Mukhopadhyay, Ankita Garg, Archana Ayyagari BACKGROUND: MDR Mycobacterium tuberculosis is the major cause of treatment failure in tuberculosis patients, especially in immunosuppressed . We described a young polymyositis patient on immunosuppressive therapy who was started with antituberculosis therapy as a susceptible strain of M . tuberculosis was isolated from a single cutaneous abscess in his neck and from regional lymph nodes . Case Presentation He had non-reactive miliary tuberculosis and multiple cutaneous abscesses 6 months later with the same strain, which was resistant this time to 9 antituberculosis drugs . We described clinical presentation, radiological and laboratory work-up, treatment and follow-up as the patient was cured after 1.5 years with 6 antituberculosis drugs . CONCLUSION: To our knowledge, this is the first reported case where an immunosuppressed patient with suggestive exogenous reinfection within 6 months with the same but MDR strain of M . tuberculosis was cured . Intense management and regular follow up were important since the patient was a potent source of MDR M . tuberculosis infection and there was limited choice for therapy. Hepatology . 2004 Dec 22;41(1):55-63 {Epub ahead of print} Genetic polymorphisms influencing xenobiotic metabolism and transport in patients with primary biliary cirrhosis; Kimura Y et al.; Epidemiological data suggest that environmental factors may trigger autoimmunity in genetically susceptible individuals . In primary biliary cirrhosis (PBC), it has been postulated that halogenated xenobiotics can modify self-molecules, facilitating the breakdown of tolerance to mitochondrial antigens . The transport and metabolism of xenobiotics is highly dependent on key genetic polymorphisms that alter enzymatic phenotype . We analyzed genomic DNA from 169 patients with PBC and 225 geographically and sex-matched healthy subjects for polymorphisms of genes coding for cytochromes P450 (CYPs) 2D6 (CYP2D6*4, CYP2D6*3, CYP2D6*5, and CYP2D6*6) and 2E1 (c1/c2), multidrug resistance 1 (MDR1 C3435T) P-glycoprotein, and pregnane X receptor (PXR C-25385T, C8055T, and A7635G) . We compared the genotype frequencies in patients and controls and also correlated polymorphisms with PBC severity . The distributions of the studied genotypes did not significantly differ between patients and controls . However, when clinical characteristics of patients with PBC were compared according to genotype, the CYP2E1 c2 allele was associated with signs of more severe disease . In conclusion, genetic polymorphisms of CYP 2D6 and 2E1, PXR, and MDR1 do not appear to play a role in the onset of PBC . (HEPATOLOGY 2005;41:55-63.). Birth Defects Res B Dev Reprod Toxicol . 2004 Dec 22;71(6):380-394 {Epub ahead of print} Developmental toxicity of artesunate and an artesunate combination in the rat and rabbit; Clark RL et al.; The artemisinins are playing an increasingly important role in treating multidrug-resistant malaria . The artemisinin, artesunate, is currently in use in Southeast Asia and is advocated for use in Africa . In these areas, more than one million people die of malaria each year, with the highest mortality occurring in children and pregnant women . To test the developmental toxicity in ICH-compliant animal studies, embryofetal development studies were conducted in rats and rabbits treated with artesunate alone or a three-drug combination (CDA) consisting of chlorproguanil hydrochloride, Dapsone, and artesunate in the ratio 1.00:1.25:2.00 . Developmental toxicity seen with CDA could be attributed to the administered dose of artesunate . The hallmark effect of artesunate exposure was a dramatic induction of embryo loss, apparent as abortions in rabbits and resorptions in both rats and rabbits . In addition, low incidences of cardiovascular malformations and a syndrome of skeletal defects were induced at or close to embryolethal doses of artesunate in both rats and rabbits . The cardiovascular malformations consisted of ventricular septal and vessel defects . The skeletal syndrome consisted of shortened and/or bent long bones and scapulae, misshapen ribs, cleft sternebrae, and incompletely ossified pelvic bones . These developmental effects were observed largely in the absence of any apparent maternal toxicity . The no or low adverse effect levels were in the range of 5 to 7 mg/kg/day artesunate . Encouragingly, no adverse drug-related developmental effects have been observed in a limited number of pregnant women (more than 100 first trimester and 600 second and third trimester) treated with artemisinins, primarily artesunate . Investigations of the mechanism of developmental toxicity are ongoing to attempt to determine whether rats and rabbits are more sensitive to artemisinins than humans . Birth Defects Res B 71:380-394, 2004 . (c) 2004 Wiley-Liss, Inc. Antimicrob Agents Chemother, 2005 Jan, 49(1), 425 - 7 In-house phage amplification assay is a sound alternative for detecting rifampin-resistant Mycobacterium tuberculosis in low-resource settings; Simboli N et al.; An in-house mycobacteriophage amplification assay for detecting rifampin-resistant Mycobacterium tuberculosis showed 100% sensitivity, 97.7% specificity, and 95.2% predictive value for resistance in a test of 129 isolates from a hot spot area of multidrug-resistant M . tuberculosis . The applicability of the test was demonstrated in the routine work flow of a low-resource reference laboratory. Antimicrob Agents Chemother, 2005 Jan, 49(1), 421 - 4 Increased expression of two multidrug transporter-like genes is associated with ethidium bromide and ciprofloxacin resistance in Mycoplasma hominis; Raherison S et al.; Two genes, md1 and md2, coding for multidrug resistance ATP-binding cassette transporters were identified in Mycoplasma hominis PG21 . Expression of these two genes, quantified by quantitative competitive reverse transcription-PCR, was significantly increased in ethidium bromide-resistant strains of M . hominis compared to that in M . hominis PG21. Drug Metab Dispos . 2004 Dec 22; {Epub ahead of print} DIFFERENTIAL INTERACTION OF HMG-CoA REDUCTASE INHIBITORS WITH ABCB1, ABCC2, AND OATP1B1; Chen C et al.; ABSTRACT The present study examined the interaction of four 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (atorvastatin, lovastatin, and simvastatin in acid and lactone forms and pravastatin in acid form only) with multidrug resistance gene 1 (MDR1, ABCB1) P-glycoprotein, multidrug resistance associated protein 2 (MRP2, ABCC2), and organic anion transporting polypeptide 1B1 (OATP1B1, SLCO21A6) . P-glycoprotein substrate assays were performed using Madin-Darby Canine Kidney (MDCK) cells expressing MDR1 and the efflux ratio {the ratio of the ratio of basolateral-to-apical (B-A) apparent permeability and A-B between MDR1 and MDCK) was 1.87, 2.32/4.46, 2.17/3.17, 0.93/2.00 for pravastatin, atorvastain (lactone/acid), lovastatin (lactone/acid), and simvastatin (lactone/acid), respectively, indicating that these compounds are weak or moderate substrates of P-glycoprotein . In the inhibition assays (MDR1, MRP2, Mrp2, and OATP1B1), the IC50 values (microM) for efflux transporters (MDR1, MRP2 and Mrp2) were >100 for all statins in acid form except lovastatin acid (>33) and the IC50 values were up to 10-fold lower for the corresponding lactone forms . In contrast, the IC50 values (microM) for the uptake transporter OATP1B1 were 3- to 7-fold lower for statins in the acid form compared to the corresponding lactone form . These data demonstrate that lactone and acid forms of statins exhibit differential substrate and inhibitor activities towards efflux and uptake transporters . The intercoversion between the lactone and acid forms of most statins exists in the body and will potentially influence drug transporter interactions and may ultimately contribute to the differences in pharmacokinetic profiles observed between statins. J Virol, 2005 Jan, 79(2), 860 - 8 Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains; Yang OO et al.; Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development . Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain . Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting . A low set point of viremia (approximately 1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (approximately 40,000 HIV-1 copies/ml) manifested about 5 months after infection . Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B) . Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype . Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes . An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B . In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development. Crit Rev Toxicol, 2