Multidrug resistant

J Clin Oncol, 2003 Nov 15, 21(22), 4239 - 47
Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis; Yang HH et al.; Drug resistance remains a major clinical challenge for cancer treatment . Early studies suggested that overexpression of P-glycoprotein was a major contributor to the chemotherapy resistance of myeloma cells and other tumor cells . Attempts in several clinical studies to reverse multidrug resistance protein (MDR) by using MDR modulators have not yet generated promising results . Recently, the emerging knowledge about the importance of overcoming antiapoptosis and drug resistance in treating a variety of malignancies, including multiple myeloma (MM), raises new hope of improving the treatment outcome for patients with cancer . The therapeutic value of targeting therapies that aim to reverse the antiapoptotic process in MM cells has been explored in a number of experimental systems, and the results have been promising . The proteasome inhibitor PS-341 is a new specifically targeted proapoptotic therapy that has been tested in clinical studies . The results indicate that PS-341 alone is an effective therapy for patients with MM who experience disease relapse . Recent in vitro data also demonstrate that PS-341 can markedly sensitize chemotherapy-resistant MM cells to various chemotherapeutic agents . On the basis of these encouraging results, clinical studies are underway to test the efficacy of PS-341 and chemotherapeutic agents as combination therapy in treating patients with refractory and relapsed MM.

Clin Cancer Res, 2003 Nov 1, 9(14), 5221 - 7
Phase I and pharmacokinetic trial of the novel taxane BMS-184476 administered as a 1-hour intravenous infusion in combination with cisplatin every 21 days; Sun W et al.; BMS-184476 is a 7-methylthiomethyl ether derivative of paclitaxel that displays potency superior to paclitaxel against tumor cells in culture and human tumor xenografts . It also inhibits the growth of paclitaxel-resistant human tumor cell lines with multidrug resistance mediated by either P-glycoprotein or mutated tubulin . Given the known synergy between taxanes and cisplatin in vitro and their clinical activity in combination, we performed a Phase I trial of BMS-184476 as a 1-h i.v . infusion followed by cisplatin every 21 days . Twenty-seven patients with a variety of solid tumors and good performance status received 116 cycles of therapy at BMS-184476 doses of 40-60 mg/m(2) together with cisplatin at 75 mg/m(2) . The early observation of hypersensitivity reactions required prophylactic premedication in all patients . At the planned highest dose of BMS-184476 (60 mg/m(2)) and cisplatin (75 mg/m(2)), we observed dose-limiting toxicity in the form of neutropenia and diarrhea . Also at this level, five patients experienced grade 3 or worse nausea and vomiting . Aggressive prophylactic treatment eliminated the gastrointestinal toxicity . Mild to moderate peripheral neuropathy was infrequent, as was alopecia . Patient benefits included three partial responses in patients with mesothelioma, esophageal cancer, and head and neck cancer, and two additional minor responses . Plasma pharmacokinetic data are available for 23 patients treated at 40-60 mg/m(2) . The mean maximum plasma concentrations and areas under the curves increased in a dose-related manner . The pharmacokinetics of BMS-184476 appeared independent of dose . The mean (+/- SE) values for clearance, volume of distribution at steady state, and the apparent terminal half-lives of the three dose groups during cycle 1 were 243 +/- 5 ml/min/m(2), 423 +/- 58 l/m(2), and 32.2 +/- 4.5 h, respectively . BMS-184476 60 at mg/m(2) with cisplatin at 75 mg/m(2) with appropriate supportive therapy is the dose recommended for further evaluation.

Clin Cancer Res, 2003 Nov 1, 9(14), 5171 - 7
The role of breast cancer resistance protein in acute lymphoblastic leukemia; Plasschaert SL et al.; PURPOSE: Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin . This study analyzes the ABCG2 expression and activity in 46 human de novo acute lymphoblastic leukemia B- and T-lineage (ALL) samples . EXPERIMENTAL DESIGN: ABCG2 expression was measured flow cytometrically with the BXP-34 monoclonal antibody . ABCG2 functional activity was determined flow cytometrically by measuring mitoxantrone accumulation in combination with the ABCG2 inhibitor fumitremorgin C (FTC) . To determine a possible effect of the transporters P-glycoprotein and multidrug resistance-associated protein (MRP1 and MRP2) on mitoxantrone accumulation, the accumulation was investigated in the presence of the P-glycoprotein inhibitor PSC 833 and MRP inhibitor MK-571 . The ABCG2 gene was sequenced to investigate the amino acid at position 482 . RESULTS: In B-lineage ALL (n = 23), the median BXP-34:IgG1 ratio was higher, namely 2.4 (range, 1.7-3.7), than in T-lineage ALL (n = 23; 1.9; range, 1.2-6.6; P = 0.003) . The addition of FTC to mitoxantrone treatment caused a median increase in mitoxantrone accumulation of 21% (range, 0-140%) in B-lineage ALL . In T-lineage ALL, this FTC effect was less pronounced (5%; range, 0-256%; P = 0.013) . The influence of FTC on mitoxantrone accumulation correlated with ABCG2 protein expression (r = 0.52; P < 0.001; n = 43) . The increase in mitoxantrone accumulation, when FTC was added to cells treated with both PSC 833 and MK-571, correlated with the ABCG2 expression in B-lineage ALL but not in T-lineage ALL . Sequencing the ABCG2 gene revealed no ABCG2 mutation at position 482 in patients who accumulated more rhodamine after FTC . CONCLUSIONS: This study shows that ABCG2 is expressed higher and functionally more active in B-lineage than in T-lineage ALL.

Toxicol Appl Pharmacol, 2003 Nov 15, 193(1), 66 - 72
Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via pregnane X receptor; Mikamo E et al.; Pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of genes for cytochrome P450 3A (CYP3A), multidrug resistance 1 (MDR1), and organic anion-transporting peptide 2 (OATP2) . These genes control the metabolism (CYP3A subfamily) and aspects of the pharmacokinetics (MDR1 and OATP2) of both endogenous and xenobiotic compounds . Since PXR is important in understanding the actions of endocrine disruptors (EDs), we determined the ability of suspected EDs to interact with PXR . In our study, 7 of 54 xenobiotics compounds interacted with PXR, including methoxychlor and benzophenone . All of the chemicals activated PXR in vitro and induced CYP3A mRNA in the male rat liver . In addition, CYP2C11 was also induced by some PXR agonists and converted methoxychlor into xenoestrogen . These findings suggest that some EDs affect sex hormone receptor indirectly by induction of metabolic enzyme via PXR, to produce rapidly higher concentrations of effective metabolites, leading to disturbance of the endocrine system.

Int J Oncol, 2003 Dec, 23(6), 1505 - 13
Voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, enhances the cytotoxic effect of doxorubicin on multidrug-resistant tumor cells; Meschini S et al.; Multidrug-resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the chemotherapy . In order to overcome MDR, substances, such as verapamil and cyclosporin A (CsA), were employed . As these P-gp modulating agents did not seem promising in clinical practice, new compounds with a low degree of undesirable side effects, were introduced . In this study, bisindolic alkaloid voacamine was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on drug resistant cells . Two different pairs of tumor cell lines were analyzed: the parental lymphoblastoid cell line CEM-WT and its MDR derivative CEM-R, the parental osteosarcoma cell line U-2 OS-WT and its resistant counterpart U-2 OS-R . These cell lines were characterized for their morphological features by scanning electron microscopy (SEM) and for the expression of the main drug transporters by flow cytometric analysis . The effects of voacamine on the cell survival and on both accumulation and efflux of DOX were then investigated . The intracellular distribution of DOX, given alone or in association with CsA or voacamine, was observed by laser scanning confocal microscopy . A differential effect of voacamine between sensitive and resistant cells on the intracellular DOX concentration and distribution was shown . In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells . The results of cell survival experiments revealed an enhancement of the cytotoxic effect of DOX induced by voacamine, confirmed by evident morphological changes observed by SEM . These findings suggest promising applications of this natural substance against MDR tumors.

Cancer Res, 2003 Nov 1, 63(21), 7392 - 9
Lamellarin D: a novel potent inhibitor of topoisomerase I; Facompre M et al.; We report the identification and characterization of a novel potent inhibitor of DNA topoisomerase I: lamellarin D (LAM-D), initially isolated from a marine mollusk, Lamellaria sp., and subsequently identified from various ascidians . This alkaloid, which displays potent cytotoxic activities against multidrug-resistant tumor cell lines and is highly cytotoxic to prostate cancer cells, bears a 6H-{1}benzopyrano{4',3':4,5}pyrrolo{2,1-a}isoquinolin-one pentacyclic planar chromophore, whereas its synthetic 5,6-dehydro analogue, LAM-501, has a significantly tilted structure . DNA binding measurements by absorbance, fluorescence, and electric linear dichroism spectroscopy show that LAM-D is a weak DNA binder that intercalates between bp of the double helix . In contrast, the nonplanar analogue LAM-501 did not bind to DNA and failed to inhibit topoisomerase I . DNA intercalation may be required for the stabilization of topoisomerase I-DNA complexes by LAM-D . In the DNA relaxation assay, LAM-D strongly promoted the conversion of supercoiled DNA into nicked DNA in the presence of topoisomerase I . The marine product was approximately 5 times less efficient than camptothecin (CPT) at stabilizing topoisomerase I-DNA complexes, but interestingly, the two drugs exhibited slightly distinct sequence specificity profiles . Topoisomerase I-mediated DNA cleavage in the presence of LAM-D occurred at some sites common to CPT, but a few specific sites identified with CPT but not with LAM-D or conversely unique sites cleaved by LAM-D but not by CPT were detected . The distinct specificity profiles suggest that LAM-D and CPT interact differently with the topoisomerase I-DNA interface . A molecular modeling analysis provided structural information on the orientation of LAM-D within the topoisomerase I-DNA covalent complex . The marine alkaloid did not induce DNA cleavage by topoisomerase II . Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped on DNA by LAM-D in P388 and CEM leukemia cells . P388/CPT5 and CEM/C2 cell lines, both resistant to CPT and expressing a mutated top1 gene, were cross-resistant to LAM-D . Collectively, the results identify LAM-D as a novel lead candidate for the development of topoisomerase I-targeted antitumor agents.

Cancer Res, 2003 Nov 1, 63(21), 7284 - 90
Glucose depletion enhances P-glycoprotein expression in hepatoma cells: role of endoplasmic reticulum stress response; Ledoux S et al.; P-Glycoprotein (P-gp) encoded by the MDR gene is one of the main factors in multidrug resistance . Its expression in cancer cells, which compromises cancer outcome, can be enhanced by some stress signals . Energy depletion, frequently observed in malignant cells, was shown to induce chemoresistance and could be one of these signals . To test this hypothesis, we studied the effect of glucose deprivation on P-gp expression in a rat hepatoma cell line (Fao) . Incubation of Fao cells with a glucose-free medium enhanced P-gp mRNA and protein expression in a time-dependent manner, up to 400% at 40 h . This effect was associated with a stimulation of {(3)H}vinblastine efflux by P-gp despite impaired glycosylation . It was reproduced by inducers of endoplasmic reticulum stress response, such as 2-deoxyglucose (DG), tunicamycin, and thapsigargin . P-gp mRNA induction by DG was preceded by an increase in activator protein binding activity, c-Jun expression, and phosphorylation . In contrast, nuclear factor-kappaB binding activity was unaffected by DG . The antioxidant N-acetylcysteine partially reversed the increase in P-gp mRNA and protein levels induced by DG, as well as the enhancement of c-Jun phosphorylation and activator protein binding activity . Finally, transient transfection of the cells with a deleted mutant of c-Jun, Tam 67, abolished the DG-induced P-gp mRNA expression and mdr1b promoter activation . In conclusion, glucose deprivation enhances P-gp expression and transport function in liver cancer cells . This effect is mediated by endoplasmic reticulum stress response and involves MDR transcriptional induction through c-Jun activation . These results emphasize the importance of glucose metabolism in chemoresistance.

Am J Hum Genet, 2003 Dec, 73(6), 1282 - 92 Epub 2003 Nov 07.
MDR1 Ala893 polymorphism is associated with inflammatory bowel disease; Brant SR et al.; Crohn disease (CD) and ulcerative colitis (UC) are overlapping chronic inflammatory bowel diseases (IBDs) . Suggestive evidence for linkage at chromosome 7q has been reported for both CD and UC . Contained within this region is the gene for MDR1 (multidrug resistance), a membrane transport protein for which human polymorphisms have been reported in Ala893Ser/Thr and C3435T that alter pharmacokinetic profiles for a variety of drugs . Because mdr1 knockout mice spontaneously develop colitis, exonic regions were resequenced and tested for IBD association in a large, multicenter North American cohort . Two missense mutations, Asn21Asp and Ala893Ser/Thr, as well as the expression-associated polymorphism C3435T, described elsewhere, were genotyped in the entire cohort . Significant association of Ala893 with IBD was observed by both case-control analysis (P=.002) and the pedigree disequilibrium test (PDT {P=.00020-.00030}) but not for the Asn21Asp or C3435T polymorphisms . Significant association by PDT was observed within the subset with CD (P=.0014-.00090), with similar, nonsignificant trends in a smaller subset with UC . The Ala893Ser/Thr variant is triallelic, and the associated, common allele is Ala893, with undertransmission of the 893Ser (common) and the 893Thr (rare) variants . The Ala893 variant has decreased activity compared with the 893Ser variant; therefore, the association with human IBD is consistent with the murine model of mdr1 deficiency . Taken together, these data support the association of the common Ala893 polymorphism with IBD specifically and, more broadly, provides additional support for its contribution to interindividual pharmacogenetic variation.

Exp Brain Res, 2003 Dec, 153(3), 356 - 65 Epub 2003 Sep 12.
Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions; Torok M et al.; Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood-brain barrier . The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC . Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3 . It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1) . Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone . Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements . However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose . Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1(MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells . In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased . During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant . These results were unaltered by different cell-culture conditions . In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture conditions.

Tumour Biol, 2003 May-Jun, 24(3), 151 - 5
Technetium-99m tetrofosmin SPECT predicts chemotherapy response in small cell lung cancer; Yeh JJ et al.; The multidrug resistance gene 1 encoding human P-glycoprotein (Pgp) is thought to play an important role in the multidrug resistance of lung cancer . The purpose of this study was to predict chemotherapy response by technetium-99m tetrofosmin (Tc-99m TF) lung single photon emission computed tomography (SPECT) and compare Pgp expression in patients with untreated small cell lung cancer (SCLC) . Forty patients with untreated SCLC received Tc-99m TF lung SPECT prior to chemotherapy . The chemotherapy response was evaluated in the 3rd month after completion of treatment . Immunohistochemical staining of Pgp expression was performed on multiple nonconsecutive sections of biopsy specimens . By quantitative analyses, tumor to background ratios were 1.86 +/- 0.27 and 1.17 +/- 0.26 for patients with a good and poor response, respectively (p < 0.05) . All of the 20 patients with a good chemotherapy response also had a positive Tc-99m TF lung SPECT and negative Pgp expression . In contrast, only 4 of the 20 patients with a poor chemotherapy response had a positive Tc-99m TF lung SPECT . Moreover, 10 of the 20 patients with a poor chemotherapy response also had negative Pgp expression (p < 0.05) . Therefore, we concluded that Tc-99m TF lung SPECT can accurately predict the chemotherapy response, and Tc-99m TF lung SPECT findings can be partially compatible with Pgp expression in patients with untreated SCLC .

Srp Arh Celok Lek, 2003 Mar-Apr, 131(3-4), 168 - 72
{Knowledge level of students at the Kragujevac Medical School about nosocomial infections}; Ilic M et al.; OBJECTIVE: To investigate differences in clinical and pre-clinical medical students' knowledge of nosocomial infections (NI) . DESIGN: Cross-sectional survey . RESULTS: Questionnaires was answered and returned by 352 of 453 student (77.7%) . The results indicated that students knew the definition of NI (70.1% correct answers) and their reservoirs (86%) . The bacteria as etiological agents was the most frequent answer (76.4%), but 30.9% students did not knew at least one multidrug-resistant bacteria . About one half of the students (54.4%) knew that contact was the most frequent mode of NI transmission, but hand washing as preventive measure was cited by only 18.8% of students . Significantly statistical differences about NI in our country, etiology NI and preventive measures, and perception of risk for transmission of hepatitis B for health-care personnel were founded by year of training, by expectation that final-year medical students as more successfully, while pre-clinical students knew more about mode of NI transmission . Pre-clinical students who had previously finished nursing school knew more about multidrug-resistant bacteria than those who had finished some other secondary school, but showed a lower knowledge about definition and most important preventive measures of NI . Clinical students who had previously finished nursing school knew more about frequency NI in our country, reservoirs and preventive measures of NI than those who had finished some other secondary school . CONCLUSION: Data support the need for additional information about nosocomial infections, especially practical work in prevention, in order to get complete knowledge about nosocomial infections.

Int J Lepr Other Mycobact Dis, 2003 Sep, 71(3), 240 - 3
A second case of multidrug-resistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy; Matsuoka M et al.; Emergence of drug resistant strains of Mycobacterium leprae was reported soon after the introduction of dapsone (diamino-diphenyl sulphone, DDS) for leprosy treatment (6, 10, 11) . Three cases of multidrug-resistant strains of M . leprae have been reported recently (2, 8, 9, 13) . In order to prevent multiple drug resistant strains of M . leprae from developing, current leprosy control strategies are based on early detection of cases and treatment with multidrug therapy (MDT) as recommended by the World Health Organization (WHO) . We report here the identification of a multidrug-resistant strain of M . leprae from a patient who received inadequate therapy for leprosy . The drug resistant profile of the isolated strain was confirmed by the mouse footpad method and the identification of mutations in genes previously shown to be associated with resistance to each drug was made.

Am J Infect Control, 2003 Oct, 31(6), 354 - 6
Do physicians examine patients in contact isolation less frequently? A brief report; Saint S et al.; BACKGROUND: Patients who are hospitalized and infected with multidrug-resistant bacteria are usually placed in contact isolation, which requires hospital personnel to gown and glove before patient examination . Contact isolation with active culture surveillance appears beneficial in preventing the spread of drug-resistant infections; however, contact isolation may impede the ability to examine patients as a result of the additional effort required to gown and glove . We assessed whether patients who are hospitalized and placed under contact precautions are examined less often by second- and third-year medical residents (ie, senior medical residents), and attending physicians during morning rounds . METHOD: We conducted a prospective cohort study on the inpatient medical services at 2 university-affiliated medical centers . We directly observed senior medical residents and attending physicians during morning rounds, and recorded the contact precaution status of the patient and whether they were examined by either physician . RESULTS: Of a total of 139 patients, 31 (22%) were in contact isolation . Senior medical residents examined 26 of 31 patients (84%) in contact isolation versus 94 of 108 patients (87%) not in contact isolation (relative risk, 0.96; 95% confidence interval, 0.81-1.14; P =.58) . In comparison, attending physicians examined 11 of 31 patients (35%) in contact isolation versus 79 of 108 patients (73%) not in contact isolation (relative risk, 0.49; 95% confidence interval, 0.30-0.79; P <.001) . DISCUSSION: Attending physicians are about half as likely to examine patients in contact isolation compared with patients not in contact isolation.

Bioorg Med Chem, 2003 Nov 17, 11(23), 5221 - 7
Modified jatrophane diterpenes as modulators of multidrug resistance from Euphorbia dendroides L; Corea G et al.; The new diterpenoids terracinolides J-L (1-3), 13alpha-OH terracinolide F (8), abeodendroidin F (11) and epiabeodendroidin F (12) have been identified from Euphorbia dendroides L . The new compounds and six co-occurring known terracinolides were tested as inhibitors of the drug-efflux activity of P-glycoprotein from cancer cells . The results were used to extend the structure-activity relationships established for this class of compounds highlighting the relevance of substitution at positions 2, 3, 6, and 15 and disclosing a remarkable tolerance toward connectivity changes in the terpenoid core.

J Indian Med Assoc, 2003 Mar, 101(3), 210 - 2
Ofloxacin in multidrug resistant tuberculosis; Walwaikar PP et al.; Tuberculosis (TB) is the leading infectious cause of death today and 3.81 million cases occurred in 1997 and 1998 . Even today in spite of having four drugs like isoniazid, rifampicin, streptomycin/pyrazinamide and ethambutol for the intensive phase, the total duration of treatment remains six months . Because of the global health problems of TB, the increasing rate of multidrug resistant TB (MDR-TB) and the high rate of co-infection with HIV, the need for effective non-toxic antituberculous agents is essential . Fluoroquinolones have been classified as drugs having low bactericidal activity by the WHO . To date, they have been used for preventive therapy, empirical treatment of patients with TB and retreatment of patients with relapsing TB . In-vitro and in-vivo clinical studies have identified ofloxacin as a promising new agent in the treatment of MDR-TB . Ofloxacin has been advocated by the WHO in case of MDR-TB, when susceptibility to test results are not available before starting the new treatment, in the continuation period (18 months) and if resistance is proven to at least isoniazid and rifampicin.

J Indian Med Assoc . 2003 Mar;101(3):190, 192, 194 passim.
Tuberculosis--triumph and tragedy; Singh MM; Tuberculosis has been making havoc worldwide with an 11.9 million cases to be involved by the year 2005 . In India, about 2 million cases are infected every year . Regarding triumphs and tragedies in the control of tuberculosis some points as follows are discussed . (1) Tuberculosis Control Programmes from National Tuberculosis Programme (NTP) to Revised National Tuberculosis Control Programme (RNTCP) and Directly Observed Treatment, Short course (DOTS) . (2) Problem of multidrug resistance (MDR) tuberculosis and (3) HIV and tuberculosis . DOTS being largely based on Indian research . It is now being applied worldwide . MDR is strictly a man made problem . Poor prescriptions, poor case management, lack of coordinated education and haphazard treatment research result in drug resistance . Treatment of MDR is difficult . The drug acceptability, tolerance and toxicity have to be considered . HIV and tuberculosis form a deadly duo . They mean more cases, more costs and more national losses.

J Indian Med Assoc, 2003 Mar, 101(3), 157 - 8, 166
Scientific basis of directly observed treatment, short-course (DOTS); Sharma SK et al.; The introduction of rifampicin, pyrazinamide and ethambutol ushered in the era of "short course chemotherapy" . Multidrug resistance TB (MDR-TB) is threatening to destabilise the best efforts of TB control . Treatment of MDR-TB is difficult, expensive and toxic and is often unsuccessful . DOTS is an interventional strategy designed to effectively diagnose and treat TB . The fundamental principles in the DOTS strategy are: Polititical will, diagnosis by sputum microscopy, directly observed standardised short-course treatment, adequate supply of good quality drugs, systematic monitoring and accountability . Patients with HIV infection and TB disease respond well to antituberculosis treatment if they are given short-course chemotherapy in the programme of DOTS.

J Indian Med Assoc, 2003 Mar, 101(3), 154 - 6
Status of drug resistance in tuberculosis after the introduction of rifampicin in India; Paramasivan CN; The current threat in tuberculosis treatment lies on the fact of emergence of strains resistant to two most antituberculous drugs, isoniazid and rifampicin . Drug resistance to TB may be classified as primary and acquired . Causes of drug resistance are inefficient administration of effective treatment, poor case handling, use of sub-standard drugs, ignorance of healthcare workers, etc . Multidrug resistant TB (MDR-TB) prevalence (median) in new case is highest (14.1%) in Estonia . Studies undertaken in different regions in India by Tuberculosis Research Centre (TRC) during 1997-2000 revealed acquired MDR-TB resistance levels of 25-100% . The key to successful prevention of the emergence of drug resistance remains adequate case finding, prompt and correct diagnosis and effective treatment of infective patients.

J Indian Med Assoc, 2003 Mar, 101(3), 140 - 1, 147
Good progress with DOTS in the South-East Asia Region; Nair N et al.; The South-East Asia Region (SEAR) accounts for 38% of the global tuberculosis (TB) . Encouraging progress has been made since the DOTS strategy was introduced in all SEAR Member States between 1993-94 . Operational guidelines for and joint plans of action for disease control activities in the border districts of Bangladesh, Bhutan, India and Nepal have been drawn up . The key issues involved in the good progress with DOTS are: Resource mobilisation, case detection, case management, drugs and logistics, supervision, monitoring and surveillance, preventing emergence of multidrug resistant TB and lastly health sector reform . Given the current momentum and commitment, it is expected that the region will active the set targets of universal coverage by 2006.

Trop Gastroenterol, 2003 Apr-Jun, 24(2), 70 - 2
The epidemiological and resistogram patterns of enteropathogenic and enterotoxigenic Escherichia coli isolated from diarrhoeal stools in a north Indian hospital; Vaishnavi C et al.; We report our findings on the epidemiological and resistogram patterns of enteropathogenic Escherichia coli (E . coli) (EPEC) and enterotoxigenic E . coli (ETEC) in patients with diarrhoea in a north Indian hospital . A total of 153 diarrhoeic stool samples were cultured for E . coli . Pure or predominant growth obtained was biochemically identified, serogrouped and tested for antibiotic susceptibility . Among the E . coli that could be serogrouped, there were 7 EPEC and 11 ETEC isolates, most of which were multidrug resistant . Apart from this, there were 35 untypable and 4 rough strains of E . coli . Culturing stools and testing the antibiotic susceptibility for most categories of diarrhoeagenic E . coli should be performed in cases of persistent diarrhoea and during outbreaks . This will help to study its epidemiological pattern in a particular locale . Moreover, this information coupled with the clinical picture may be valuable in deciding the need for and type of antibiotic therapy.

J Pharm Sci, 2003 Nov, 92(11), 2152 - 63
Cytokines alter the expression and activity of the multidrug resistance transporters in human hepatoma cell lines; analysis using RT-PCR and cDNA microarrays; Lee G et al.; Pro-inflammatory cytokines suppress the hepatic expression of the multidrug resistance transporters in rodents, indicating potential usefulness in chemotherapy . Our objective was to investigate their impact in human hepatoma cells . HuH 7 and HepG2 cells were treated with IL-1beta, IL-6, or TNF-alpha for 0-72 h . Expression and activity of MDR1 and the MRP (MRP1, 2, 3, and 6) transporters were examined by RT-PCR, efflux assays, and microarrays . Significant reductions in the MDR1-mediated efflux of Rhodamine 123 and MDR1 mRNA levels were observed in HuH 7 cells treated with IL-6, TNF-alpha, or IL-1beta and in TNF-alpha-treated HepG2 cells . However, cytokine-treated HuH7 cells also demonstrated 1.6- to 2.6-fold greater efflux of the MRP substrate, 5-carboxyfluorescein (5-CF) and higher MRP3 mRNA levels (p < 0.05) . IL-1beta and IL-6 treatments increased MRP activity and MRP1 mRNA levels in HepG2 cells (p < 0.05) . Microarrays studies performed in IL-6 and TNF-alpha-treated HepG2 cells detected similar changes in the expression of the MDR1 and MRP transporters, but this did not reach significance . However, the microarrays confirmed cytokine-mediated induction of several acute phase proteins . Our data suggests that although cytokine-mediated suppression of PGP may alter drug resistance in malignant cells, these cytokines may also impose an induction in other multidrug resistance genes .

Hepatobiliary Pancreat Dis Int, 2003 Aug, 2(3), 397 - 403
Clinical significance of mrp gene in primary hepatocellular carcinoma; Wang BL et al.; OBJECTIVE: To study the relations among the expression of the multidrug resistance associated-protein (mrp) gene and clinicopathologic features, the influence of alpha-fetoprotein (AFP), and prognosis of patients who received adjuvant chemotherapy after resection of primary hepatocellular carcinoma (HCC) . METHODS: The expression of the mrp gene encoding MRP and mRNAmrp was determined in tissues from 54 untreated patients with HCC, adjacent tissues from 24 patients with HCC and archival paraffin-embedded tissues from 12 patients with posthepatic cirrhosis . The relationship between the mrp gene expression and the change level of AFP was analyzed in the 24 postoperative HCC patients whose AFP level was measured after 2 weeks . All of the HCC patients were followed up . RESULTS: The percentage of positive expressions of MRP and mRNAmrp in the three kinds of tissues was 57.40%, 25.00%, 16.67%, and 72.22%, 37.50%, 33.33% respectively . Significant difference was noted in the untreated HCC tissue, compared to the other two tissues (P<0.05) . No difference existed between the mrp gene expression and such clinicopathologic findings, as age, sex, and tumor size (P> 0.05), but the expression was related to the degree of differentiation of HCC (P<0.05) . The effective rate of AFP in the mrp gene positive expression group or postoperative chemotherapeutic patients was lower than that in the negative group (P<0.05) . Although no difference was seen in the 1-, 3-, 5-year survival rates of HCC patients (P>0.05), the mean survival time of postoperative HCC patients or the negative mrp gene expression group was longer than that of the positive group (P<0.05) . CONCLUSIONS: Multidrug resistance (MDR) of HCC is related to mrp gene expression and initiates the intrinsic MDR . Detection of mrp gene expression is of great significance in accessing chemotherapeutic resistance of HCC, which provides evidence for reversing MDR in HCC . The mrp gene may be a useful marker in detecting prognosis of HCC patients because its expression is correlated with tumor differention and mean survival time of the patients.

Int J Tuberc Lung Dis, 2003 Nov, 7(11), 1104 - 8
Characterization of clinical isolates of Mycobacterium tuberculosis resistant to drugs and detection of RpoB mutation in multidrug-resistant tuberculosis in the Philippines; Agdamag DM et al.; SETTING: Retrospective study of Mycobacterium tuberculosis isolates at the STD/AIDS Cooperative Central Laboratory, Philippines . OBJECTIVE: To describe patterns of M . tuberculosis resistance against first-line anti-tuberculosis drugs, and to analyze the rpoB gene codon mutation of rifampicin (RMP) resistant isolates and correlate genotypic and phenotypic patterns . DESIGN: One hundred and sixty-four M . tuberculosis complex isolates were retrieved for phenotypic analysis; 89 were resistant to any anti-tuberculosis drug and 50 were RMP-resistant, whereas 48 were multidrug-resistant (MDR) . Of these 48, only 33 were available for genotypic analysis of the rpoB gene . RESULTS: Most drug-resistant isolates were phenotypically resistant to isoniazid (INH) (93%), and the probability of an RMP-resistant isolate becoming MDR was 96% . In 33 MDR isolates, 13 types of mutations in nine independent codons were identified; the most frequently mutated codons were S531L (61%) and G510H (15%), which were present in 76% (25/33) of the isolates . S531L was noted in 85.7% of the RMP + INH + SM resistant isolates, while only 80% of the isolates with INH + RMP, EMB + SM resistance showed this mutation . CONCLUSION: The high probability of RMP isolates being MDR suggests that genetic analysis of RMP resistance is useful in detecting MDR-TB . Worldwide accumulation of findings on circulating MDR-TB strains provides indispensable information about the re-emergence of TB.

Int J Tuberc Lung Dis, 2003 Nov, 7(11), 1097 - 103
Prevalence of Beijing genotype in Latvian multidrug-resistant Mycobacterium tuberculosis isolates; Tracevska T et al.; SETTING: Predominant genotypes of Mycobacterium tuberculosis include the Beijing family, which has caused large tuberculosis outbreaks and has been associated with increased virulence and multidrug resistance (MDR) . OBJECTIVE: To search for the Beijing genotype among Latvian MDR patients to characterise their DNA isolates at the molecular level . DESIGN: MDR isolates were spoligotyped and tested for gene mutations by automatic nucleotide sequencing . RESULTS: Of 109 isolates examined, 95 were located in six clusters of 2 to 63 isolates each . The 63 isolates in the largest cluster had an identical pattern corresponding to the Beijing genotype . The remaining isolates were of a non-Beijing genotype and formed another large group whose similarity ranged from 72% to 100% . Mutations in the rpoB and katG genes were compared in the Beijing and non-Beijing strains . In both groups, the rpoB gene mutations predominated in codons S531L (52.2%) and D516V (14.7%) . Double mutations in the rpoB gene were observed in 8.2% of the isolates, most of them located among Beijing-type isolates . The katG gene mutation S315T (98.4%) was prevalent among all isolates . CONCLUSION: Molecular analysis of MDR isolates of M . tuberculosis demonstrates that the Beijing genotype, most likely due to recent transmission, is prevalent in Latvia among MDR patients and that this genotype can be associated with double mutations.

Int J Tuberc Lung Dis, 2003 Nov, 7(11), 1045 - 51
Encouraging outcomes in the first year of a TB control demonstration program: Orel Oblast, Russia; Kherosheva T et al.; SETTING: Orel, Russia . OBJECTIVE: To evaluate outcomes of tuberculosis (TB) patients treated in the first year of a TB control demonstration project using a revised strategy of directly observed treatment, short-course (DOTS) . Standard methods recommended by World Health Organization (WHO) were adapted to include mycobacterial cultures . DESIGN: Retrospective cohort analysis of TB patients diagnosed between October 1999 and September 2000 . RESULTS: Among 749 TB patients, 65% had bacteriologic confirmation of pulmonary TB, 31% were diagnosed clinically, and 4% had extra-pulmonary TB . Most (92%) had no previous TB treatment, but 8% were identified as retreatment cases . Of all patients, 41% had new sputum smear-positive TB . No patients were HIV-infected . Multidrug-resistant (MDR) TB levels were 3% among new and 17% among retreatment patients . Among new smear-positive patients, treatment success was 79% (72% cure, 7% completion); remaining outcomes were 8% failure, 3% default, 8% death, and 1% transfer . Success rates for new culture-positive and clinically diagnosed patients were 81% and 91%, respectively . CONCLUSION: Despite historical differences, successful implementation of the revised TB strategy in Russia is possible . Treatment success rates were high, suggesting WHO targets of 85% cure for smear-positive patients is attainable . Obstacles include drug resistance and elevated death rates among smear-positive patients.

Probl Tuberk Bolezn Legk, 2003, (9), 29 - 31
{The specific features of a pathogen in acutely progressive pulmonary tuberculosis}; Balasaniants GS et al.; Examination of 318 patients with different forms of acutely progressive pulmonary tuberculosis (APPT) revealed massive bacterial isolation in 181 patients of them . In one fourth of the patients, Mycobacterium tuberculosis (MBT) colonies grew during a month; the mean growth duration was 38.3 +/- 12.7 days, which was indicative of the viability of Mycobacteria and of their high virulence if they were isolated in large amounts . A hundred and thirty-three patients were found to be drug-resistant, which is one of the characteristics of present-day APPT . Such patients more frequently developed multidrug resistance . Sixty-three patients were ascertained to have secondary drug resistance, of them 41 patients were found to have drug resistance that progressed during APPT therapy.

Probl Tuberk Bolezn Legk, 2003, (9), 6 - 9
{Causes and factors of development of drug resistance in pulmonary tuberculosis}; Nechaeva OB et al.; To prevent secondary multidrug resistance and polyresistance of Mycobacterium tuberculosis (MBT), it is necessary to strictly observe the tested chemotherapy regimens during intensive care by using at least four essential antituberculous drugs (ATD) mainly in combination . The rehabilitative phase should be monitored at outpatient units of a day hospital . To prevent cross MBT infection with drug resistance to essential ATD and to efficiently use expensive reserve drugs, it is necessary to set up specialized inpatient units (wards) for treatment and rooms for outpatient receipt of patients who isolate drug-resistant MBT strains.

J Exp Zoolog A Comp Exp Biol, 2003 Nov 1, 300(1), 91 - 7
The xenobiotic transporter, MRP2, in epithelia from insects, sharks, and the human breast: implications for health and disease; Karnaky KJ Jr et al.; A large number of mechanisms, including special excretory transporters, have evolved to help organisms excrete deleterious xenobiotics and endogenous molecules . We have examined the xenobiotic transport function of a putative multidrug resistance associated protein, MRP2, in three different epithelia: the insect renal (Malpighian) tubules, the secretory tubule of the shark rectal gland, and in ductules of the human breast . In the case of the insect and shark, transporter activity occurs in epithelia capable of great fluid transport . In the case of the insect Malpighian tubule, understanding the underlying mechanisms of this transporter may help with efforts to control populations of disease-carrying agriculturally important insects . In striking contrast, ductule architecture in nonlactating human breast ductules is that of an epithelium with a closed lumen . Immunocytochemical studies show that MRP2 is localized in the apical region of the ductule epithelial cells . In this unique case, MRP2 substrates transported into the lumen could possibly be concentrated . Transport substrates of MRP2 include carcinogens as well as antioxidants and other salutary molecules . Thus, in the breast ductule, MRP2 may play a significant role in breast epithelial cell health and cancer carcinogenesis .

FEBS Lett, 2003 Nov 6, 554(1-2), 23 - 9
Differential sensitivity of plant and yeast MRP (ABCC)-mediated organic anion transport processes towards sulfonylureas; Forestier C et al.; The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes . Due to broad substrate spectra, specific modulators of these proteins are still lacking . Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor . Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants . To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated . Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein . Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide . Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist . Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.

Mem Inst Oswaldo Cruz, 2003 Sep, 98(6), 827 - 30 Epub 2003 Oct 29.
Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system; Barreto AM et al.; In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied . Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH . We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system . Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH-95% (81.8-99.1), EMB-94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA-98.3 (89.5-99.9) at 95% confidence interval . The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.

Best Pract Res Clin Haematol, 2003 Dec, 16(4), 629 - 44
Immunophenotyping as a guide for targeted therapy; Sonneveld P et al.; Immunophenotyping of acute and chronic leukaemias has revealed many lineage- and differentiation-specific antigens . It has now become possible to classify leukaemias according to their unique antigenic expression pattern . Among many lineage- and differentiation-specific antigens, disease-specific antigens are increasingly recognized because of their specific prognostic or therapeutic relevance . Expression of the multidrug resistance proteins of the ABC transporter family is associated with a poor response to treatment and a grave clinical prognosis . Recently, attempts to reverse refractory disease by using P-glycoprotein inhibitors have been performed in acute myeloid leukaemia, so far without evidence of clinical benefit.Other new leads to use antigen expression as a way of designing tumour-specific therapy have resulted in imatinib and Flt3 inhibitors which target tyrosine kinases in the leukaemic cell . Clinical trials are underway to investigate the effect of these new agents . The development of an antibody-calicheamycin complex directed against the myeloid-specific antigen CD33 has shown clinical activity in patients with relapsed acute myeloid leukaemia . The further development of these approaches is discussed.

Best Pract Res Clin Haematol, 2003 Dec, 16(4), 613 - 28
The prognostic significance of antigen expression in leukaemia; Schabath R et al.; Numerous immunophenotypic features have been examined for their potential prognostic significance in predicting treatment outcome in leukaemias . These include immunophenotypic subgroups of acute lymphoblastic leukaemia (ALL) and immature acute myeloid leukaemia, expression of individual surface antigens or combined immunophenotypic features, and more recently, molecules mediating the multidrug resistance phenotype or being involved in the regulation of drug-induced apoptosis . Most previous studies investigating the prognostic significance of antigen expression in leukaemia have not used the information provided by multiparameter flow cytometry and have chosen rather arbitrary cut-off points for marker positivity . Moreover, given significant associations between immunophenotypic features and genetic abnormalities in leukaemic cells, immunophenotyping as an independent predictor of treatment outcome has been questioned . Thus, except for lineage assignment of leukaemic blasts and definition of maturational status in ALL, information provided by immunophenotyping is currently not applied to risk-classification systems or used for planning patient treatment in leukaemia . We review some of the recent findings regarding the prognostic impact of distinct immunophenotypic features in acute leukaemias and myelodysplastic syndrome.

Best Pract Res Clin Haematol, 2003 Dec, 16(4), 583 - 97
Flow cytometry in lymphoma diagnosis and prognosis: useful?
Stetler-Stevenson M.
Flow cytometry has become an important tool in the diagnosis of mature lymphoid neoplasms and the determination of prognosis in selected cases . The advantages of flow cytometry are based largely on its ability to analyse, rapidly and simultaneously, multiple cell properties in a quantitative manner . Flow cytometric immunophenotyping is useful in diagnosing lymphoma under the WHO classification system, where lymphoid neoplasms are separated into distinct clinical entities based upon morphology, immunophenotype, genetic abnormalities and clinical features . Flow cytometry can quantify the expression of proteins associated with a good or poor prognosis, detect multidrug resistance, and measure cell proliferation, making it useful in measuring prognostic indicators in lymphoid neoplasia . The unique attributes of flow cytometry therefore make it a valuable technique in the diagnosis and classification of lymphomas as well as the assessment of prognostic markers in lymphoma patients.

Liver Transpl, 2003 Nov, 9(11), 1199 - 210
Bile secretory function after warm hepatic ischemia-reperfusion injury in the rat; Accatino L et al.; Hepatic ischemia-reperfusion (I-R) injury frequently is associated with cholestasis . However, the underlying mechanisms are not fully understood . The aim of the study is to assess bile secretory function in vivo in rats subjected to warm lobar hepatic ischemia at different times during reperfusion . A model of lobar 70% warm hepatic ischemia for 30 minutes was used with studies conducted at 1 and 6 hours and 1, 3, and 7 days after reperfusion . Bile secretory function was assessed after selective cannulation of bile ducts of ischemic (ILs) and nonischemic lobes (NILs) . Serum activity of hepatic alanine and aspartate aminotransferase was slightly increased in rats subjected to I-R, whereas serum bile salt levels increased early during reperfusion, returning to control values after 7 days . ILs showed mild reversible leukocyte infiltration and no significant necrosis . Bile flow and bile salt excretion were significantly decreased in ILs during the first 24-hour reperfusion period compared with sham-operated rats and NILs . A marked reduction in glutathione (GSH) excretion occurred at 1 and 6 hours and 1 and 3 days, which returned to control values after 7 days . Total GSH and both reduced and oxidized GSH levels in liver homogenate and arterial blood GSH levels were unchanged at all times . Protein mass of multidrug resistance protein 2 and its function, assessed by the hepatic maximum secretory rate of ceftriaxone, did not show significant changes in ILs or NILs compared with sham-operated rats . Liver tissue gamma-glutamyl transpeptidase (GGT) and gamma-glutamylcysteine synthetase activities remained unchanged, whereas biliary GGT and cysteine secretory rates were significantly increased in ILs and NILs . Administration of acivicin, a GGT inhibitor, resulted in decreased secretion of this enzyme into bile and a parallel marked increase in biliary GSH secretion compared with untreated ischemic rats . In conclusion, warm hepatic I-R induces reversible cholestatic changes in ILs . GSH secretory rates from both ILs and NILs were markedly decreased during reperfusion . The reversibility of this effect after GGT inhibition, as well as increased release of active GGT into bile and cysteine biliary secretory rates, suggest increased GSH degradation in bile . These findings might be relevant for the I-R-induced clinical cholestasis, as well as cholangiocyte injury, seen after hepatic ischemia.

Int J Clin Oncol, 2003 Oct, 8(5), 326 - 31
C(H)OP refractory chronic lymphocytic leukemia patients in whom salvage chemotherapy chosen by evaluating multiple chemotherapeutic drug-resistant factors was remarkably effective; Matsunaga T et al.; It is well known that the expression of anticancer drug-resistant factors is elevated in patients with primary refractory or relapsed chronic lymphocytic leukemia (CLL) who have been treated with chemotherapy . We report here two C(H)OP refractory patients with CLL in whom salvage chemotherapy chosen by evaluating anticancer drug-resistant factors (glutathione-S-transferase-Pi {GST-Pi}, glycoprotein {GP}-170, multidrug resistance-associated protein {MRP}, and lung resistance protein {LRP}) was remarkably effective . A 71-year-old male patient was refractory to induction therapy with cyclophosphamide, vincristine, and prednisone (COP), and his leukemic cells at diagnosis displayed overexpression of GST-Pi and GP-170 . A 74-year-old female patient's condition had been stable; she had received ten courses of COP over 9 years . However, because systemic lymphadenopathies recurred, she was treated with chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) or dexamethasone, etoposide, ifosphamide, and carboplatin (DeVIC) . However, she did not respond at all, and her leukemic cells at recurrence displayed overexpression of GST-Pi . Therefore, we chose for these patients a salvage therapy consisting of dexamethasone and high-dose cytosine arabinoside (Ara C), to which neither GST-Pi nor GP-170 show any drug resistance . In both patients, this salvage therapy proved effective.

Invest New Drugs, 2003 Nov, 21(4), 413 - 20
Quinolizidinyl derivatives of iminodibenzyl and phenothiazine as multidrug resistance modulators in ovarian cancer cells; Barbieri F et al.; The development of multidrug-resistance (MDR) in neoplastic cells is often responsible for the therapy failure and poor outcome of a number of human cancers . MDR may be associated with the expression of the multidrug transporter glycoprotein p170, encoded by the MDR1 gene, which acts as an ATP-dependent efflux pump by reducing the intracellular accumulation of some cytotoxic agents . A variety of iminodibenzyl and phenothiazine derivatives, characterized by the presence of a bicyclic, strongly basic, and highly lipophilic quinolizidine nucleus, were synthesized to investigate their ability to modulate the MDR phenotype . A set of 10 of them (named 1-10), bearing quinolizidine moiety linked through different connecting chains, were tested as chemoresistance-reversing agents on doxorubicin-resistant ovarian cancer cells (A2780-DX3) . A 51-fold resistance to doxorubicin was reported in the A2780-DX3 compared to the parental sensitive A2780 WT with mean IC(50) values of 0.02 and 1.02 muM, respectively . Moreover, overexpression of the glycoprotein p170 in the resistant cell line was detected by Western blot analysis . By cytotoxicity assays and time-course experiments, different treatment schedules with resistance modulators (including clomipramine as reference drug) and doxorubicin were taken into account . The 16 h exposure of cells to 1 muM of modulator before doxorubicin demonstrated to be superior in sensitizing the resistant cell line . In particular, compounds 8, 7, 10, and 4 increasingly potentiated doxorubicin cytotoxicity, up to 5.6-fold in A2780-DX3 cells . The present results suggest promising indications for further development of these compounds as chemosensitizing drugs.

J Med Chem, 2003 Nov 6, 46(23), 4988 - 5004
Three-dimensional quantitative structure-activity relationship analysis of propafenone-type multidrug resistance modulators: influence of variable selection on test set predictivity; Fleischer R et al.; An extended set of multidrug-resistance modulators of the propafenone type were investigated using CoMFA and CoMSIA . A number of 3D-QSAR models were derived from steric, electrostatic, and hydrophobic fields and their combinations . The hydrophobic fields alone and in combination with the steric and both (steric and electrostatic) fields yielded the models with the highest cross-validated predictivity, in agreement with a previous analysis of a smaller data set of propafenone-type multidrug-resistance (MDR) modulators . Inclusion of lipophilicity did not lead to an improvement of the models . The results point to the importance of hydrophobicity as a space-directed molecular property for MDR-modulating activity . The influence of variable selection applying the GOLPE procedure was investigated with an external test set . Variable-selection procedure was repetitively applied, keeping at each stage variables with uncertain contribution to the models . For the CoMFA-based 3D-QSAR models, an increase in external prediction quality was found . In contrast, the CoMSIA-based 3D-QSAR models were not improved by the GOLPE variable-selection procedure.

Cancer, 2003 Nov 1, 98(9), 1958 - 66
Reduced folate carrier protein expression in osteosarcoma: implications for the prediction of tumor chemosensitivity; Ifergan I et al.; BACKGROUND: High-dose methotrexate (MTX) is an important component of current protocols for the treatment of osteosarcoma . Although MTX uptake proceeds primarily through the reduced folate carrier (RFC) protein and efflux occurs via multidrug resistance protein 1 (MRP1), RFC protein expression in osteosarcoma remains unexamined . METHODS: RFC and MRP1 expression (normalized to beta-actin expression) was examined with Western blot analysis in 11 osteosarcoma specimens obtained at diagnosis and 9 osteosarcoma specimens obtained on recurrence . RESULTS: The average RFC level in specimens obtained on recurrence was significantly higher than the level in specimens obtained at diagnosis (P = 0.0005) . Furthermore, in all three matched pairs of diagnosis and recurrence specimens, RFC levels were higher in recurrence specimens than in the corresponding diagnosis specimens . Potential correlations between RFC and MRP1 expression and histologic response to preoperative chemotherapy were examined . Tumors with poor histologic responses (i.e., </= 90% necrosis) had significantly lower RFC levels than did those with favorable responses to chemotherapy (P = 0.0016) . In contrast, there was no correlation between MRP1 levels at diagnosis and histologic response to chemotherapy (P = 0.8764) . The elevated MRP1 levels in specimens obtained on recurrence relative to MRP1 levels in specimens obtained at diagnosis were not statistically significant (P = 0.2056) . CONCLUSIONS: The significant correlation between low RFC levels at diagnosis and poor histologic response to preoperative chemotherapy suggests that RFC levels at diagnosis may be a useful predictor of chemosensitivity and warrants large-scale studies . In addition, postchemotherapy progression to recurrence is associated with a significant increase in RFC expression . To our knowledge, the current study is the first to examine RFC protein levels in tumor specimens . Cancer 2003 .

Cell Motil Cytoskeleton, 2003 Dec, 56(4), 225 - 36
Vaults bind directly to microtubules via their caps and not their barrels; Eichenmuller B et al.; Vaults are large (13 Mda) ribonucleoprotein particles that are especially abundant in multidrug resistant cancer cells and have been implicated in nucleocytoplasmic drug transport . To understand how these large barrel-shaped complexes are transported through the cytosol, we examined the association of vaults with microtubules both in vitro and in vivo . Within cells, a subpopulation of vaults clearly associates with microtubules, and these vaults remain associated with tubulin dimers/oligomers when microtubules are disassembled by nocodazole treatment . In vitro, a microtubule-pull down assay using highly purified rat vaults and reassembled microtubules reveals that vaults exhibit concentration-dependent binding to microtubules that does not require the carboxyl terminal end of tubulin . Remarkably, negative staining for electron microscopy reveals that vault binding to microtubules is mediated by the vault caps; more than 82% of bound vaults attach to the microtubule lattice with their long axes perpendicular to the long axis of the microtubule . Five to six vault particles were bound per micron of microtubule, with no crosslinking of microtubules observed, suggesting that only one end of the vault can bind microtubules . Taken together, the data support the model of vaults as barrel-shaped containers that transiently interact with microtubules .

Pharmacogenetics, 2003 Nov, 13(11), 675 - 82
ABCB1 C3435T and G2677T/A polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation; Asano T et al.; Advances in transplantation technology have brought about great benefits to patients suffering from organ failure, but the problem still remains of complications induced by steroids used for post-transplant immunosuppression . Among the side-effects caused by steroids, non-traumatic osteonecrosis of the femoral head (ONF) constitutes a serious problem . The same protocol for steroid administration induces ONF in some patients, but not in others, indicating the presence of individual difference in steroid sensitivity . We hypothesized that this difference might be mediated by the drug-transport protein, P-glycoprotein (P-gp), and investigated the relationship between single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1, MDR1) encoding P-gp and ONF . Subjects comprised 136 patients receiving kidney transplantation . Thirty patients developed post-transplant ONF . Genomic DNA was extracted from peripheral blood, and genotypes of ABCB1 C3435T (exon 26) and G2677T/A (exon 21) were determined by direct sequencing . Multivariate analyses based on clinical information were performed to determine the relationship between ABCB1 genotypes and ONF . The dose/concentration (D/C) ratios of tacrolimus were also determined to estimate the activity of P-gp in patients with different genotypes of ABCB1 C3435T (CC, CT, TT), and in those who did and did not develop ONF . The ABCB1 3435TT genotype showed a significantly lower incidence of ONF (adjusted odds ratio = 0.10, P = 0.034) . The D/C ratio in the 3435TT genotype was significantly higher than that in the 3435CC genotype . The D/C ratio in patients developing ONF was significantly higher than in those patients who did not develop ONF . The results suggest increased activity of P-gp in patients with the 3435TT genotype and in those who did not develop ONF . The ABCB1 2677 homozygous variant type also showed a lower incidence of ONF (adjusted odds ratio = 0.26, P = 0.056) . The 3435T and 3435C alleles were in linkage disequilibrium with the 2677T and the 2677G alleles, respectively, in the study population . An assessment of C3435T and G2677T/A polymorphisms preceding steroid treatment could be useful for predicting the resistance to ONF development .

Pharmacogenetics, 2003 Nov, 13(11), 661 - 74
Association between ABCB1 (multidrug resistance transporter) genotype and post-liver transplantation renal dysfunction in patients receiving calcineurin inhibitors; Hebert MF et al.; OBJECTIVE: Renal dysfunction is a common and costly adverse outcome of long-term treatment with calcineurin inhibitors (CNIs) . We conducted a retrospective, case-control study to test whether the risk of renal dysfunction in liver transplantation patients receiving CNIs is associated with the 2677G>T transversion in exon-21 of the gene (ABCB1) encoding P-glycoprotein . A total of 120 non-Hispanic white patients were evaluated . RESULTS: The overall incidence of renal dysfunction by year 3 post-transplantation was 40% . The frequency of renal dysfunction was reduced among patients with an ABCB1 2677TT genotype, as compared to those with a 2677GG genotype . Subjects with a heterozygote genotype behaved phenotypically like the 2677GG group . Comparing those subjects with a 2677TT genotype to the combined group of subjects with a 2677GG, TG, AT, or AG genotype resulted in an odds ratio of 0.26 (0.09-0.77) . When subjects were stratified by gender, the frequency of renal dysfunction was reduced among men with an ABCB1 2677TT genotype, relative to men with different genotypes . A similar odds ratio was obtained for women, but it did not achieve significance . When 18 subjects with an elevated SCr concentration just prior to surgery were excluded from the year 3 analysis, the association between the 2677TT genotype and chronic renal dysfunction in the remaining cohort was strengthened comparing genotype groups . CONCLUSIONS: Based on these results, we conclude that homozygosity for the ABCB1 2677T (S893) allele is associated with reduced risk of chronic renal dysfunction among liver transplantation patients receiving an immunosuppressive regimen containing CNIs .

Pharmacogenetics, 2003 Nov, 13(11), 651 - 60
Effects of ABCB1 (multidrug resistance transporter) gene mutations on disposition and central nervous effects of loperamide in healthy volunteers; Skarke C et al.; OBJECTIVE: Mutations in the ABCB1 gene have been associated with decreased expression and net function of P-glycoprotein (P-gp) . We investigated the modulation of the central nervous effects of loperamide resulting from ABCB1 genetic variants . METHODS: On two occasions, 20 healthy volunteers received 24 mg loperamide suspension orally and, in a double-blind randomized two-way crossover fashion, 800 mg quinidine or placebo orally 1 h before loperamide . Pupil size was measured for 5 h following loperamide administration, and plasma concentrations of loperamide and quinidine were measured for 6 h . Single nucleotide polymorphisms and haplotypes including G2677T(A) (exon 21) and C3435T (exon 26) were analysed for their relation to plasma concentrations of quinidine and loperamide and to the miotic effects of loperamide . RESULTS: Loperamide plasma concentrations with quinidine co-administration were about twice as high as those without quinidine . The ABCB1 haplotype G2677/T3435 was associated with the highest loperamide plasma concentrations, which were about 1.5 times higher than in non-carriers of this haplotype . Plasma concentrations of quinidine did not differ among carriers and non-carriers of genetic variants . When quinidine was co-administered with loperamide, pupil size decreased . Without quinidine it changed only minimally . The ABCB1 TT3435 genotype was associated with the most pronounced increase of the miotic effects of loperamide when quinidine was co-administered . This was accompanied by a tendency toward higher plasma loperamide in TT3435 carriers . CONCLUSIONS: Our data support a functional importance of the ABCB1 mutations for plasma concentrations and central nervous actions of the opioid loperamide .

Cancer Res, 2003 Oct 15, 63(20), 6942 - 7
HMN-176, an active metabolite of the synthetic antitumor agent HMN-214, restores chemosensitivity to multidrug-resistant cells by targeting the transcription factor NF-Y; Tanaka H et al.; HMN-176 ((E)-4-{{2-N-{4-methoxybenzenesulfonyl}amino}stilbazole}1-oxide) is an active metabolite of HMN-214 ((E)-4-{2-{2-(N-acetyl-N-{4-methoxybenzenesulfonyl}amino)stilbazole}}1-oxide), which has a potent antitumor activity in mouse xenograft models . In this study, we show that HMN-176 circumvents multidrug resistance in a K2 human ovarian cancer subline selected for Adriamycin resistance (K2/ARS) . Upon treatment of K2/ARS cells with 3 microM HMN-176, the GI(50) of Adriamycin for the cells decreased by approximately 50% . To explore the molecular mechanism of this effect, we assessed the expression of the multidrug resistance gene (MDR1), which is constitutive in K2/ARS cells, at both the protein and the mRNA level . Western and reverse transcription-PCR analysis revealed that the expression of MDR1 was significantly suppressed by treatment with HMN-176 . Furthermore, when administered p.o., HMN-214 suppressed the expression of MDR1 mRNA in a mouse xenograft model implanted with KB-A.1, an Adriamycin-resistant cell line . Luciferase reporter fusion gene analysis demonstrated that HMN-176 inhibited the Y-box-dependent promoter activity of the MDR1 gene in a dose-dependent manner . Moreover, we show by electrophoretic mobility shift assay that HMN-176 inhibits the binding of NF-Y, which is thought to be an essential factor for the basal expression of MDR1, to its target Y-box consensus sequence in the MDR-1 promoter . Inhibition of MDR-1 expression was achieved with pharmacological concentrations of HMN-176, suggesting that HMN-176 may act by two different mechanisms-cytotoxicity and MDR1 down-regulation-simultaneously . The data presented strongly suggest that the antitumor mechanism of HMN-176 (or its prodrug HMN-214 in vivo) is quite different from those of known antitumor agents.

Eur Respir J, 2003 Oct, 22(4), 637 - 42
Risk factors for recent transmission of Mycobacterium tuberculosis; Heldal E et al.; In recent decades, the decline of tuberculosis has stopped in Western Europe, mainly due to increased immigration from high-prevalence countries . The objective of the current study was to identify risk factors for developing tuberculosis following recent infection, in order to better target interventions . Strains from 861 culture-positive cases, diagnosed in Norway in 1994-1999, were analysed by use of restriction fragment length polymorphism (RFLP) . A cluster was defined as two or more isolates with identical RFLP patterns . Risk factors for being part of a cluster were identified by univariate and multivariate analysis . A total of 134 patients were part of a cluster . These constituted 5% Asian-born, 18% Norwegian-born, 24% European-born and 29% African-born patients . Four independent risk factors for being part of a cluster were identified: being born in Norway, being of young age, being infected with an isoniazid-resistant strain and being infected with a multidrug-resistant strain . Transmission of tuberculosis may be further reduced by improving case management, contact tracing, preventive treatment, screening of immigrants and access to health services for the foreign-born population.

Clin Cancer Res, 2003 Oct 15, 9(13), 5009 - 17
Multidrug-resistant tumor cells remain sensitive to a recombinant interleukin-4-Pseudomonas exotoxin, except when overexpressing the multidrug resistance protein MRP1; de Jong MC et al.; Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5) . In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin) . IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) {IL-4(38-37)-PE38KDEL} . Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin . Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells . In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4 . MRP1-overexpressing cells were not resistant to PE itself . IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine . In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG) . These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1 . Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.

Placenta, 2003 Nov, 24(10), 951 - 8
Basal membrane localization of MRP1 in human placental trophoblast; Nagashige M et al.; The placental trophoblast is considered to act as a barrier between mother and fetus, mediating the exchange of various materials across the placenta . ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug-resistance protein (MRP) are expressed in the placenta and function as efflux transport systems for xenobiotics . In the present study, we aimed to determine the localization of MRP1 in the human placenta in comparison with that of P-gp . Western blotting analysis with human placental membrane vesicles indicated that P-gp and MRP1 are localized on the brush-border membranes and basal membranes, respectively . Immunohistochemical analysis with human normal full-term placenta showed that anti-P-gp monoclonal antibody F4 stained the brush-border side of the trophoblast cells, whereas anti-MRP1 monoclonal antibody MRPr1 stained the basal side . These results confirm that P-gp and MRP1 are located on the brush-border membranes and basal membranes, respectively, of human full-term placental trophoblast . MRP1 was also detected on the abluminal side of blood vessels in the villi . Accordingly, MRP1 may play a role distinct from that of P-gp, which is considered to restrict the influx of xenobiotics into the fetus.

Clin Chem Lab Med, 2003 Oct, 41(10), 1345 - 50
CYP3A4 and MDR1 alleles in a Portuguese population; Cavaco I et al.; Polymorphisms in cytochrome P450 CYP3A4 and multidrug resistance (MDR) 1 genes coding for the important drug-metabolising CYP3A4 and the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) are poorly documented in the Portuguese population . In this study we have determined the frequencies of CYP3A4 and MDR1 alleles in Portuguese Caucasians . Both genes were simultaneously analysed as these genes are known to be frequently co-induced and their products to show a pronounced overlap of substrates . CYP3A4 A-392G (CYP3A4*1B), T673C (CYP3A4*2) and MDR1 T-129C, G2677T and C3435T single nucleotide polymorphisms (SNPs) were analysed in 100 individuals from the southern region of the country . We observed a frequency of 4.0% for CYP3A4*1B, not significantly different from that reported on other Caucasian European populations . CYP3A4*2 was found at an allele frequency of 4.5%, constituting the first report of the presence of this allele outside the Finnish population . Significant differences were found concerning the MDR1 C3435T SNP frequency (64.5%) compared with other European populations, while no differences were found concerning G2677T (47.5%) or T-129C (5%) SNPs . Linkage between the C3435T and G2677T SNPs was observed, although not as evidently as documented in other Caucasian populations . No preferential associations were detected between CYP3A4 and MDR1 alleles.

Am J Pathol, 2003 Nov, 163(5), 1781 - 90
Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells; Sampath J et al.; Our effort to identify novel drug-resistant genes in cyclophosphamide-resistant EMT6 mouse mammary tumors led us to the identification of SPF45 . Simultaneously, other groups identified SPF45 as a component of the spliceosome that is involved in alternative splicing . We isolated the human homologue and examined the normal human tissue expression, tumor expression, and the phenotype caused by overexpression of human SPF45 . Our analyses revealed that SPF45 is expressed in many, but not all, normal tissues tested with predominant expression in normal ductal epithelial cells of the breast, liver, pancreas, and prostate . Our analyses using tissue microarrays and sausages of tumors indicated that SPF45 is highly expressed in numerous carcinomas including bladder, breast, colon, lung, ovarian, pancreatic, and prostate . Interestingly, this study revealed that overexpression of SPF45 in HeLa, a cervical carcinoma cell line, resulted in drug resistance to doxorubicin and vincristine, two chemotherapeutic drugs commonly used in cancer . To our knowledge, this is the first study showing tumor overexpression of an alternate splicing factor resulting in drug resistance.

Mol Cell Biochem, 2003 Oct, 252(1-2), 109 - 16
Resistance to thapsigargin-induced intracellular calcium mobilization in a multidrug resistant tumour cell line; Wagner-Souza K et al.; A multidrug resistant (MDR) cell line, derived from the human leukaemic cell K562 and selected for its resistance to Vincristine, was shown to be resistant to Thapsigargin (TG) . A concentration of 50 nM TG was toxic to K562 cells whereas the MDR cell line, known as Lucena I cells, survived unaffected for up to seven days in culture . Similarly, no intracellular Ca2+ mobilization was observed in the MDR cell line treated with TG . This effect was not a result of TG extrusion by P glycoprotein (Pgp), as no mobilization was observed even in the presence of the Pgp inhibitors Verapamil (5 microM) and Cyclosporin A (0.16 microM) . In the present study, both cell lines expressed comparable levels of Bcl-2 making it unlikely that Bcl-2 was involved in this process . Similarly, no overexpression of the endoplasmic reticulum Ca2+ ATPase (SERCA) could be detected in the MDR cell line and Ca2+ uptake by vesicles of the two cell types were equally sensitive to TG . These results confirm that MDR cells do not mobilize Ca2+ in the presence of TG but go against the possibility that this might be due to TG extrusion or to the overexpression of a resistant SERCA isoform.

Oncogene, 2003 Oct 20, 22(47), 7496 - 511
Transcriptional regulation of ABC drug transporters; Scotto KW; P-glycoprotein, the founding member of the ATP-binding cassette (ABC) family of drug transporters, was first identified almost three decades ago and shown to confer resistance to multiple chemotherapeutic agents when overexpressed in human tumors . Subsequent years have witnessed a tremendous effort to characterize the function and regulation of P-glycoprotein, initially spurred by the hope that its inhibition was the key to overcoming clinical resistance to multiple anticancer agents . However, the identification of MRP1, another member of the ABC drug transporter family, led to the realization that the multidrug resistance (MDR) phenotype is considerably more complex than initially believed . Indeed, at the present time at least 10 members of the ABC transporter family have been implicated in an MDR phenotype, and it is likely that more will be added to this list as studies progress . With this complexity comes the imperative to improve our understanding of the function of individual transporters, as well as to delineate the mechanisms underlying their expression in normal and tumor cells, particularly those that may be amenable to therapeutic intervention . Several articles within this volume address the structure and function of drug transporters . This review will focus on our current understanding of the regulation of ABC drug transporters at the level of transcription.

Oncogene, 2003 Oct 20, 22(47), 7340 - 58
Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2); Doyle LA et al.; Observations of functional adenosine triphosphate (ATP)-dependent drug efflux in certain multidrug-resistant cancer cell lines without overexpression of P-glycoprotein or multidrug resistance protein (MRP) family members suggested the existence of another ATP-binding cassette (ABC) transporter capable of causing cancer drug resistance . In one such cell line (MCF-7/AdrVp), the overexpression of a novel member of the G subfamily of ABC transporters was found . The new transporter was termed the breast cancer resistance protein (BCRP), because of its identification in MCF-7 human breast carcinoma cells . BCRP is a 655 amino-acid polypeptide, formally designated as ABCG2 . Like all members of the ABC G (white) subfamily, BCRP is a half transporter . Transfection and enforced overexpression of BCRP in drug-sensitive MCF-7 or MDA-MB-231 cells recapitulates the drug-resistance phenotype of MCF-7/AdrVp cells, consistent with current evidence suggesting that functional BCRP is a homodimer . BCRP maps to chromosome 4q22, downstream from a TATA-less promoter . The spectrum of anticancer drugs effluxed by BCRP includes mitoxantrone, camptothecin-derived and indolocarbazole topoisomerase I inhibitors, methotrexate, flavopiridol, and quinazoline ErbB1 inhibitors . Transport of anthracyclines is variable and appears to depend on the presence of a BCRP mutation at codon 482 . Potent and specific inhibitors of BCRP are now being developed, opening the door to clinical applications of BCRP inhibition . Owing to tissue localization in the placenta, bile canaliculi, colon, small bowel, and brain microvessel endothelium, BCRP may play a role in protecting the organism from potentially harmful xenobiotics . BCRP expression has also been demonstrated in pluripotential "side population" stem cells, responsible for the characteristic ability of these cells to exclude Hoechst 33342 dye, and possibly for the maintenance of the stem cell phenotype . Studies are emerging on the role of BCRP expression in drug resistance in clinical cancers . More prospective studies are needed, preferably combining BCRP protein or mRNA quantification with functional assays, in order to determine the contribution of BCRP to drug resistance in human cancers.

Antimicrob Agents Chemother, 2003 Nov, 47(11), 3616 - 9
Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method; Martin A et al.; The emergence of multidrug-resistant tuberculosis calls for new, rapid drug susceptibility tests . We have tested 150 Mycobacterium tuberculosis isolates against the second-line drugs ethionamide, kanamycin, capreomycin, ofloxacin, and para-aminosalicylic acid by the colorimetric resazurin microtiter assay and the proportion method . By visual reading, MICs were obtained after 8 days . A very good correlation between results by the colorimetric resazurin microtiter assay and the proportion method was obtained . The colorimetric resazurin microtiter assay is inexpensive, rapid, and simple to perform, and implementation of the assay is feasible for low-resource countries.

Zhonghua Zhong Liu Za Zhi, 2003 Sep, 25(5), 425 - 8
{Mdr-1 ribozyme in the reversal of multidrug resistance in human ovarian cancer}; Yang XK et al.; OBJECTIVE: To study the mechanism of multidrug resistance and its reversal by mdr-1 ribozyme in human ovarian cancer . METHODS: The expression of mdr-1 and p-glycoprotein (p-gp) was studied by confocal laser microscope (Confocal), RT-PCR and Western blot analysis in adriamycin-resistant human ovarian cancer cell line (A2780/ADM) and adriamycin-sensitive one (A2780) . The mdr-1 ribozyme was transfected into the A2780/ADM by Lipofectamine 2000 to overcome the multidrug resistance in ovarian cancer . RESULTS: The expression of mdr-1 mRNA and p-gp in A2780/ADM was significantly higher than that in A2780 . The expression of mdr-1 mRNA and p-gp in A2780/ADM was lowered after being transfected by mdr-1 ribozyme . CONCLUSION: Multidrug resistance of A2780/ADM, possibly being caused by overexpression of mdr-1 gene, can be partially reversed by mdr-1 ribozyme.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Oct, 11(5), 544 - 8
{Multidrug resistance mediated by membrane P-glycoprotein in acute myeloid leukemia}; Su LP; A key issue in the treatment of acute leukemia is the development of resistance to chemotherapeutic drugs . Several mechanisms may account for this phenomenon, including failure of the cell to undergo apoptosis in response to chemotherapy, or failure of the drug to reach and/or affect its intracellular target . This review focuses on the latter mechanisms, and on intracellular drug transport resistance mechanisms in particular . Expression of the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) has generally been reported to correlate with prognosis in acute myeloid leukemia (AML) . Additionally, of more controversial, expression of the ABC transporter multidrug resistance protein (MRP) and the vault-transporter lung resistance protein (LRP) have been correlated with the outcome in AML.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Oct, 11(5), 472 - 5
{Expression of lung resistance protein and multidrug resistance protein genes in bone marrow cells of acute leukemia patients and its clinical significance}; Chi ZH et al.; To study the expression of lung resistance protein (LRP) and multidrug resistance protein (MRP) genes in bone marrow cells in patients with acute leukemia and its clinical significance, expression of LRP and MRP mRNA in bone marrow cells from 47 cases of acute leukemia, including 10 refractory or relapsed cases, and 7 normal individuals were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) . The result s showed that expression of LRP gene was negative in normal individuals . LRP mRNA level in newly treated cases of acute myelocytic leukemia and refractory or relapsed cases was significantly higher than that in normal individuals, increased LRP mRNA level has correlation with lower sensitivity to initial chemotherapy and was associated with reduced overall survival rate . Complete remission (CR) rate in LRP positive patients was lower than that in negative cases . The level of LRP expression was correlated with that of MRP mRNA . In conclusion, the expression of LRP mRNA can predict the treatment outcome and prognosis for acute myelocytic leukemia, prognosis was even worse in LRP and MRP linked expression cases, therefore, LRP was an important resistant factor, determination of LRP and MRP expression can help us to evaluate the prognosis and choose chemotherapy program.

Leukemia, 2004 Jan, 18(1), 78 - 83
Multidrug resistance genes in infant acute lymphoblastic leukemia: Ara-C is not a substrate for the breast cancer resistance protein; Stam RW et al.; Infants with acute lymphoblastic leukemia (ALL) are more resistant to chemotherapeutic drugs than older children with ALL, except for Ara-C . Drug resistance mechanisms in infant ALL, however, remain unknown . Possibly, multidrug resistance (MDR) proteins like P-glycoprotein, MDR-associated protein (MRP1), lung resistance-related protein (LRP/MVP) and the breast cancer resistance protein (BCRP) play a role . Accordingly, we measured the mRNA levels of these proteins in infants (n=13) and non-infants (n=13) with ALL, using quantitative RT-PCR . Infants expressed 2.4-fold less BCRP mRNA (P=0.009) than non-infants with ALL . MDR1, MRP1 and LRP/MVP expression did not differ between both groups . MDR gene expression levels did not correlate to prednisolone, vincristine, daunorubicin or Ara-C cytotoxicity, except for BCRP expression, which correlated with resistance to Ara-C (Rs=0.53, P=0.012), suggesting that Ara-C might be a BCRP substrate . However, culturing patients ALL cells in the presence of the BCRP inhibitor Ko143 had no effect on Ara-C sensitivity . Inhibiting Bcrp1 in the Mdr1a-, Mdr1b- and Mrp1-deficient and Bcrp1-overexpressing mouse cell line Mef3.8/T6400, also did not modulate Ara-C cytotoxicity . Therefore, we conclude that Ara-C is not a substrate for BCRP and that MDR proteins do not play a significant role in drug resistance in infant ALL.

Sex Transm Infect, 2003 Oct, 79(5), 419 - 21
Selective transmission of multidrug resistant HIV to a newborn related to poor maternal adherence; Desai N et al.; OBJECTIVES: To report perinatal transmission of multidrug resistant (MDR) HIV related to variable maternal adherence antenatally . METHODS: Case study including review of clinic records, adherence information, laboratory data, and HIV genotyping results in mother and infant . RESULTS: Poor maternal adherence to clinic visits and antiretroviral therapy contributed to detectable viraemia antenatally . When tested for the first time at age 6 months, the infant was found to have virus with resistance to multiple drugs . In this case, prophylaxis with zidovudine (AZT) failed to prevent the transmission of the MDR strain . CONCLUSIONS: Perinatal transmission of MDR HIV can occur despite standard peripartum prophylaxis with AZT . Perinatal prophylaxis should be tailored to the mother's treatment history and resistance profile . Paediatric HIV specialists should be prepared to deal with a small, but slowly increasing number of babies with a "nightmare" multidrug resistant virus with limited treatment options.

Mol Pharmacol, 2003 Nov, 64(5), 1259 - 69
Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions; Muredda M et al.; The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents . The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression . As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene . All three promoters are GC-rich, TATA-less, and CAAT-less . Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element . The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences . Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity . Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3 . Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes . Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements.

Farmaco, 2003 Nov, 58(11), 1163 - 9
A novel sunscreen agent having antimelanoma activity; Nogueira MA et al.; A novel series of eight dibenzoylmethane derivatives having both sunscreen and cytotoxic activity has been obtained by derivatizing commercial dibenzoyl methanes . Four human cancer cell lines (MCF 7 (breast), NCI ADR (breast expressing the multidrug resistance phenotype), NCI 460 (lung) and UACC 62 (melanoma)) were used for the cytotoxic assay . Eight among the 19 dibenzoylmethane derivatives showed cytotoxicity against these four cell lines . Absorption spectroscopies revealed that these compounds can be used as sunscreens against UV radiation.

Zhonghua Gan Zang Bing Za Zhi, 2003 Oct, 11(10), 609 - 11
{Expression and implication of multidrug resistance associated -protein gene in primary hepatocellular carcinoma}; Wang BL et al.; OBJECTIVES: To investigate the relationship between the expression of mrp and both the responses to chemotherapy and the level of alpha-fetoprotein (AFP) . METHODS: S-P immunohistochemical staining and in situ PCR were adopted to detect MRP and mRNA mrp in 54 cancer tissues taken from untreated HCC patients whose tumor could not be removed during the operation, 24 para-cancer tissues, and 12 posthepatitis cirrhosis paraffin-embedded tissues . The relationship between the expression of mrp and their curative effect to chemotherapy in all the patients was analyzed, so was the relationship between the expression of mrp and the level of AFP in 38 patients whose AFP was positive after operation . RESULTS: The positive rates of expressing MRP and mRNA mrp in the three kinds of tissues were 61.1%, 25.0%, 33.3% and 77.8%, 37.5%, 41.7%, respectively, with higher rates in HCC tissues than those in other tissues (chi2=9.842, P< 0.01; chi2=13.956, P<0.01) . The rates of curative effect to chemotherapy in groups of negative and positive MRP and mRNA mrp expression were 61.9%, 30.3% and 75.0%, 33.3%, respectively, with significant difference between the negative and positive groups (chi2=5.242, P<0.05; chi2=6.627, P< 0.05) . As the same as the percentage of curative effect to chemotherapy, the rates of AFP level decreased evidently were 62.5%, 27.3% and 87.5%, 30.0%, with remarkable difference between the two groups (chi2=4.710, P<0.05; chi2=8.566, P<0.05) . CONCLUSIONS: The multidrug resistance (MDR) of HCC is related to mrp expression, which initiates the intrinsic MDR . There is an important significance by detecting mrp expression in selecting chemotherapeutic method, and the expression of mrp can act as an indicator for chemotherapeutic sensitivity in HCC patients.

Breast Cancer Res Treat, 2003 Sep, 81(2), 149 - 57
Expression of MRP1, LRP and Pgp in breast carcinoma patients treated with preoperative chemotherapy; Rudas M et al.; Our purpose was to determine the expression of the drug resistance factors multidrug resistance protein (MRP1), lung resistance protein (LRP) and P-glycoprotein (Pgp) in breast carcinoma patients treated with preoperative chemotherapy . We have studied the expression of these proteins in breast carcinomas by immunohistochemistry both prior (n = 80) and after (n = 68) preoperative chemotherapy and compared their expression with response to preoperative chemotherapy . In paired samples prior and after chemotherapy expression of drug resistance factors was significantly lower in prechemotherapy samples as compared with postchemotherapy specimens . This was observed for MRP1 (62% vs . 88%, P < 0.001), LRP (65% vs . 97%, P < 0.001) and Pgp (55% vs . 100%, P < 0.001) . Prechemotherapy expression of MRP1 was more frequently observed in patients with distant metastases than in those without (50% vs . 8%, P = 0.02) . No associations were observed between LRP expression and clinical parameters . Pgp expression was more frequently detected in lobular carcinomas than in ductal carcinomas (93% vs . 46%, P = 0.001) and in patients with positive lymph nodes than in patients with negative lymph nodes (65% vs . 31%, P = 0.008) but was independent of other clinical parameters . No significant associations were found between the prechemotherapy or postchemotherapy expression of either of these three proteins and response to preoperative chemotherapy . However, prechemotherapy MRP1 expression was significantly associated with shorter progression-free survival of the patients (P = 0.02), whereas no such associations were observed for either LRP or Pgp . In conclusion, preoperative chemotherapy increases the expression of MRP1, LRP and Pgp . Response to chemotherapy is not associated with pre- or postchemotherapy expression levels of these drug resistance proteins but time to progression may be influenced by prechemotherapy MRP1 expression.

Drug Metab Dispos, 2003 Nov, 31(11), 1337 - 45
Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats; Choudhuri S et al.; The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus . To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum . The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method . Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum . OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum . The remaining transporters {Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8} were expressed at very low levels in choroid plexus . The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain.

Drug Metab Dispos, 2003 Nov, 31(11), 1315 - 9
Induction of multidrug resistance protein 3 (mrp3) in vivo is independent of constitutive androstane receptor; Cherrington NJ et al.; We previously demonstrated that multidrug resistance protein 3 (Mrp3/ABCC3) is induced in rat liver by phenobarbital (PB) and several other microsomal enzyme inducers that induce cytochrome P450 2B (CYP2B) . CYP2B is induced by constitutive androstane receptor (CAR)-retinoid X receptor (RXR) heterodimer binding to a phenobarbital-responsive promoter element in the CYP2B promoter . Hepatic mRNA levels of CYP2B and Mrp3 were measured in three models of altered CAR activity to determine whether CAR is also involved in the induction of Mrp3 . In Wistar Kyoto rats, where males express higher CAR protein levels than females, the induction of CYP2B1/2 was significantly higher in males than in females by PB, diallyl sulfide, and trans-stilbene oxide but not oltipraz . Mrp3 was induced by each of these treatments, but in contrast to CYP2B1/2, to a similar magnitude in males and females . In male hepatocyte-specific RXRalpha-/- mice, CYP2B10 was not induced by diallyl sulfide or oltipraz but remained inducible by PB and trans-stilbene oxide after considering the decrease in basal CYP2B10 expression . Mrp3, however, was induced by PB, diallyl sulfide, trans-stilbene oxide and oltipraz in both wild-type and RXRalpha-/- mice . Additionally, constitutive expression of Mrp3 was significantly reduced in RXRalpha-/- mice . In CAR-/- mice, the robust induction of CYP2B10 by PB was completely absent . However, Mrp3 was equally induced both in wild-type and CAR-/- mice by PB . These data clearly demonstrate that induction of hepatic Mrp3 by PB and other microsomal enzyme inducers is CAR-independent and implies a role for RXRalpha in the constitutive expression of Mrp3.

Drug Metab Dispos, 2003 Nov, 31(11), 1296 - 9
Induction of ABCC3 (MRP3) by pregnane X receptor activators; Teng S et al.; The pregnane X receptor (PXR) mediates the induction of various genes by xenobiotics, including several ATP-binding cassette transporters . PXR is also activated by bile acids likely to prevent their accumulation to toxic levels; however, the role of PXR in the regulation of MRP3, an important bile acid efflux transporter, has not been elucidated . The impact of PXR activators on the hepatic expression of MRP3 was examined in vivo and in vitro . The human hepatoma cell lines HuH7 and HepG2 were treated with PXR activators including clotrimazole, rifampicin, 17beta-hydroxy-11beta-{4-dimethylamino phenyl}-17alpha-{1-propynyl}estra-4,9-dien-3-one (RU486), metyrapone, nifedipine, lithocholic acid, and 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN) . Levels of MRP3 mRNA, as determined by reverse transcription-polymerase chain reaction, were induced 1.6- to 8-fold in a dose-dependent manner (p < 0.05) . Corresponding decreases in the multidrug resistance-associated protein-dependent cellular retention of 5-carboxyfluorescein were also seen in the treated HuH7 cells . In vivo studies demonstrated increased PXR mRNA and induction of MRP3 mRNA in the livers of wild-type mice treated with the PXR activator RU486 . On the other hand, MRP3 induction was not seen in the RU486-treated PXR-null mice . These results suggest that PXR activation may play a role in the regulation of MRP3 expression.

Drug Metab Dispos, 2003 Nov, 31(11), 1288 - 91
The beta-D-glucoside and sodium-dependent glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by both SGLT1 and multidrug resistance-associated proteins 1/2; Walle T et al.; Phloridzin, a glucoside of the flavonoid-like polyphenol phloretin, has long been known to be a specific nontransportable inhibitor of the sodium-dependent glucose transporter SGLT1 . The objective of this study was to determine whether efflux by multidrug resistance-associated protein (MRP) transporters might have masked the absorption by SGLT1 in previous studies . Various cells used as transport models were incubated with phloridzin (50 microM) in the absence and presence of 50 microM 3-{{3-{2-(7-chloroquinolin-2-yl)vinyl}phenyl}-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl} propionic acid (MK-571), a highly selective MRP1/MRP2 inhibitor, and the cellular uptake of phloridzin was measured by high performance liquid chromatography . The uptake of phloridzin by SGLT1-transfected Chinese hamster ovary (CHO) (G6D3) cells was 1.7-fold higher than that by parent CHO cells (p < 0.01) . In the presence of MK-571, the uptake of phloridzin by CHO cells increased 3.7-fold (p < 0.001) . MK-571 caused an 8.0-fold increase in the uptake of phloridzin by G6D3 cells (p < 0.0001) . Thus, in the absence of MRP1 efflux, transport of phloridzin by SGLT1 was clearly demonstrated . Similar results were obtained for the glycosides of the flavonoids quercetin, genistein, and diosmetin . A significantly lower accumulation of phloridzin in MRP2-transfected Madin-Darby canine kidney (MDCK) cells compared with parent MDCK cells demonstrated that phloridzin was a substrate also for MRP2 (p < 0.05) . This conclusion was further strengthened when MK-571 increased the uptake by MRP2-MDCK cells as much as 3.6-fold (p < 0.01) . These results demonstrate that phloridzin, in contrast to previous notions, is transported by SGLT1 . In addition, they demonstrate that this and other flavonoid glycosides unexpectedly are efficiently effluxed by both MRP1 and MRP2.

J Pharmacol Exp Ther, 2004 Jan, 308(1), 260 - 7 Epub 2003 Oct 20.
Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity; Smitherman PK et al.; Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens . The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells . HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control . MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells . Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity . Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance . Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors . Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively . We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function . These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

Neuroscience, 2003, 121(3), 605 - 17
Relationship between expression of multiple drug resistance proteins and p53 tumor suppressor gene proteins in human brain astrocytes; Marroni M et al.; Multiple drug resistance occurs when cells fail to respond to chemotherapy . Although it has been established that the drug efflux protein P-glycoprotein protects the brain from xenobiotics, the mechanisms involved in the regulation of expression of multiple drug resistance genes and proteins are not fully understood . Re-entry into the cell cycle and integrity of the p53 signaling pathway have been proposed as triggers of multiple drug resistance expression in tumor cells . Whether this regulation occurs in non-tumor CNS tissue is not known . Since multiple drug resistance overexpression has been reported in glia and blood vessels from epileptic brain, we investigated the level of expression of multidrug resistance protein, multidrug resistance-associated proteins and lung resistance protein in endothelial cells and astrocytes isolated from epileptic patients or studied in situ in surgical tissue samples by double label immunocytochemistry . Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that multiple drug resistance, multidrug resistance protein, and lung resistance protein are expressed in these cells . Given that lung resistance proteins have been reported to be preferentially expressed by tumors, we investigated expression of tumor suppressor genes in epileptic cortices . The pro-apoptotic proteins p53 and p21 could not be detected in "epileptic" astrocytes, while endothelial cells from the same samples readily expressed these proteins, as did normal brain astroglia and normal endothelial cells . Other apoptotic markers were also absent in epileptic glia.Our results suggest a possible link between loss of p53 function and expression of multiple drug resistance in non-tumor CNS cells.

Exp Cell Res, 2003 Nov 1, 290(2), 177 - 94
The rise of DNA methylation and the importance of chromatin on multidrug resistance in cancer; Baker EK et al.; In recent years, the different classes of drugs and regimens used clinically have provided an improvement in tumour management . However, treatment is often palliative for the majority of cancer patients . Transformed cells respond poorly to chemotherapy mainly due to the development of the multidrug resistance (MDR) phenotype . Response to treatment does not generally result in complete remission and disease cure is uncommon for patients presenting with advanced stage cancer . Successful treatment of cancer requires a clearer understanding of chemotherapeutic resistance . Here, we examine what is known of one of the most extensively studied mechanisms of cellular drug resistance . The human multidrug resistance gene 1 (MDR1) is associated with expression of p-glycoprotein (Pgp) . A transmembrane protein, Pgp acts as an efflux pump and reduces intracellular drug levels and thus its effectiveness as an antitumor agent . The precise mechanism of transcriptional regulation has been unclear due to the complex regulatory nature of the gene . It has become increasingly apparent that trans-activation or genetic amplification is by no means the only mechanism of activation . Consequently, alternative pathways have received more attention in the area of epigenetics to help explain transcriptional competence at a higher level of organization . The goal of this article is to highlight important findings in the field of methylation and explain how they impinge on MDR1 gene regulation . In this review, we cover the current information and postulate that epigenetic modification of MDR1 chromatin influences gene transcription in leukaemia . Finally, we explore transcriptional regulation and highlight recent progress with engineered ZFP's (zinc finger proteins).

Mol Genet Metab, 2003 Sep-Oct, 80(1-2), 216 - 26
Alternative splicing within the ligand binding domain of the human constitutive androstane receptor; Savkur RS et al.; The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily . The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites . hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2) . Thus, hCAR is believed to be a mediator of drug-drug interactions . We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion . Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions . Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver . Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized . Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

Biochemistry, 2003 Oct 28, 42(42), 12163 - 73
A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity; Karwatsky J et al.; Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression . These cells are cross-resistant to several structurally and functionally dissimilar drugs . Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics . In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines . Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells . This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene . The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM) . We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations . To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin . These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells . In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells . Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.

Pharm Res, 2003 Sep, 20(9), 1394 - 400
Involvement of multidrug resistance associated protein 1 (Mrp1) in the efflux transport of 17beta estradiol-D-17beta-glucuronide (E217betaG) across the blood-brain barrier; Sugiyama D et al.; PURPOSE: The purpose of present study is to investigate the involve ment of multidrug resistance-associated protein 1 (Mrp1), Mrp2, an P-glycoprotein (Mdr1a) in the efflux transport of 17beta-estradiol-D-17beta-glucuronide (E217betaG) across the blood-brain barrier (BBB) . METHOD: The expression of Mrp1 and Mrp2 at the BBB was investigated by RT-PCR and Western blot analyses . The time profiles of the remaining radioactivity of {3H}E217pG in the brain were compared in wild-type, Mdr1a/Mdr1b and Mrp1 knockout mice and normal and Mrp2-deficient mutant rats {Sprague-Dawley and Eisai hyperbilirubinemic rats (EHBR), respectively} after intracerebral microinjection . RESULTS: RT-PCR and Western blot analyses revealed the expression of Mrp1 in isolated rat brain capillary; however, RT-PCR was unable to detect any expression of Mrp2 . Significant elimination of E217betaG was observed in wild-type mice at a rate constant of 0.007 min(-1) which was significantly decreased (0.004 min(-1)) in Mrp1 knockout mice . In contrast, there was no difference in the efflux of E217betaG from the brain in wild-type and Mdr1a/Mdr1b knockout mice and in normal and EHBR . No significant difference was observed in the accumulation of E217betaG by brain slices prepared from wild-type and Mrp1 knockout mice . CONCLUSION: Mrp1, but not Mrp2, is involved in the excretion of E217betaG at the BBB and provides a barrier function by extruding conjugated metabolites into the blood.

Aktuelle Urol, 2003 Jul, 34(4), 250 - 2
Taxol resistance and its reversal by synthetic isoprenoids in human bladder cancer cell line; Nakagawa M et al.; PURPOSE: We investigated the mechanism of action of reversal agents for taxol-resistance in bladder cancers . MATERIALS AND METHODS: We isolated a taxol-resistant cell line (KK47/TX30) from a human KK47 bladder cancer cell line (KK47/WT) . We characterized KK47/TX30 cells and screened reversal agents for taxol-resistance . RESULTS: KK47/TX30 cells exhibited approximately 700-fold resistance to taxol and cross-resistance to Vinca alkaloids and topoisomerase II inhibitors . Western blot analysis demonstrated P-glycoprotein (P-gp) overexpression in taxol-resistant cells . Drug accumulation and efflux studies showed that the decreased taxol accumulation in the resistant cell line was due to enhanced taxol efflux . We synthesized 31 isoprenoids based on the structure of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB), which could completely reverse multidrug resistance (MDR) as shown previously . Among those examined, trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane (N-5228) could completely reverse taxol-resistance in KK47/TX30 cells . Results of a structure-activity relationship study of isoprenoids suggested that the following structural features were important for overcoming taxol-resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3,4-dimethoxy functionalities with moderate electron-donation . CONCLUSIONS: Taxol-resistance was primarily mediated by P-gp overexpression in KK47/TX30 cells . One of the synthetic isoprenoids, N-5228 could completely reverse taxol-resistance in KK47/TX30 cells.

Leukemia, 2003 Dec, 17(12), 2358 - 82
Treatment by design in leukemia, a meeting report, Philadelphia, Pennsylvania, December 2002; Larson RA et al.; Novel approaches have been designed to treat leukemia based on our understanding of the genetic and biochemical lesions present in different malignancies . This meeting report summarizes some of the recent advances in leukemia treatment . Based on the discoveries of cellular oncogenes, chromosomal translocations, monoclonal antibodies, multidrug resistance pumps, signal transduction pathways, genomics/proteonomic approaches to clinical diagnosis and mutations in biochemical pathways, clinicians and basic scientists have been able to identify the particular genetic mutations and signal transduction pathways involved as well as design more appropriate treatments for the leukemia patient . This meeting report discusses these exciting new therapies and the results obtained from ongoing clinical trials . Furthermore, rational approaches to treat complications of tumor lysis syndrome by administration of the recombinant urate oxidase protein, also known as rasburicase, which corrects the biochemical defect present in humans, were discussed . Clearly, over the past 25 years, molecular biology and biotechnology has provided the hematologist/oncologist novel bullets in their arsenal that will allow treatment by design in leukemia.

J Biol Chem, 2004 Jan 2, 279(1), 463 - 8 Epub 2003 Oct 15.
Molecular modeling correctly predicts the functional importance of Phe594 in transmembrane helix 11 of the multidrug resistance protein, MRP1 (ABCC1); Campbell JD et al.; The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions . At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport . Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains . Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C . J., Haimeur, A., Olsen, S . L., Deeley, R . G., and Cole, S . P . C . (2002) J . Biol . Chem . 277, 49495-49503), were found to line the "pore" in our model proximal to the membrane cytosol interface . A fifth aromatic residue (Phe594) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance . To test this idea, MRP1-Phe594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles . Substitution of Phe594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C4 . On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate- and substitution-specific changes were observed . These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.

Antibiot Khimioter, 2003, 48(6), 33 - 41
{A novel marker of multiple drug resistance BCRP}; Bogush TA et al.; Data on a new marker of multidrug resistance from the group of ABC-transporters--BCRP (breast cancer resistance protein) and in particular the data on the general characteristics of BCRP, spectrum of substrates for this transporter, data on the role of BCRP in human organism, pathways of inhibiting BCRP-dependent transport by pharmacological agents are presented . The data on BCRP expression in human normal tissues and tumor cells are considered in detail . Possible reasons of results inconsistency in evaluation of BCRP expression in different tumors and prognostic significance of BCRP in prediction of sensitivity to chemotherapy and aggressiveness of disease process are discussed.

Gene, 2003 Oct 2, 315, 153 - 64
Isolation and characterisation of two multidrug resistance associated protein genes from maize; Swarbreck D et al.; Two genes encoding ATP-binding cassette (ABC) transporters were isolated from the crop plant Zea mays (maize) . The clones, designated ZmMRP1 and ZmMRP2, were highly homologous to members of the multidrug resistance associated protein (MRP) subfamily . Genomic Southern analysis and characterisation of bacterial artificial chromosome (BAC) clones demonstrated that both genes are present in two copies in maize, which are located in proximity to each other, suggesting the occurrence of duplication events . The full-length genomic and cDNA sequences of ZmMRP1 and 2 were obtained, permitting analysis of the intron/exon structures and protein domains . Intron positions and phasing were conserved between ZmMRP1 and 2 and their closest Arabidopsis homologues . Both clones contained two copies each of the membrane spanning domains and nucleotide-binding folds diagnostic of the ABC superfamily, and ZmMRP1 contained an additional N-terminal membrane-spanning domain (MSD0) that is typical of MRP transporters but which is lacking in the most closely related Arabidopsis and rice MRPs . In contrast, ZmMRP2 and its closest rice but not Arabidopsis homologues lacked MSD0, suggesting the repeated loss of this domain in MRP family evolution . ZmMRP1 and 2 were expressed in all tissues examined but displayed distinct expression profiles in response to herbicide safeners and pro-oxidants . ZmMRP1 was induced by aminotriazole and to a lesser extent by menadione, whereas ZmMRP2 was expressed at a lower constitutive level and did not exhibit strong induction by any of the compounds tested . The characterisation of these clones represents an important step in the experimental analysis of the MRP subfamily in a monocotyledonous crop plant.

Mol Cancer Ther, 2003 Sep, 2(9), 911 - 7
Transduction of green fluorescent protein increased oxidative stress and enhanced sensitivity to cytotoxic drugs in neuroblastoma cell lines; Goto H et al.; Green fluorescent protein (GFP) is employed as a selection marker for gene transduction and to track tumor cells . Transduction of enhanced GFP (eGFP) into human neuroblastoma cell lines via a lentiviral vector significantly sensitized CHLA-20 (wild-type and functional TP53), and to a lesser extent CHLA-90 cells (multidrug-resistant, mutant, and nonfunctional TP53) to carboplatin, doxorubicin, etoposide, or melphalan, relative to cells transduced using the cell surface antigen CD80 as a selection marker . Total glutathione (GSH) was significantly up-regulated (1.8- to 2.8-fold) after eGFP (but not CD80) transduction in cell lines with, but not in those lacking, functional p53 . Cytotoxicity of GSH depletion by buthionine sulfoximine in CHLA-20 (but not in CHLA-20-eGFP) was diminished by hypoxia (2% O(2)) . Thus, oxidative stress produced by GFP selects for cells with up-regulated GSH in a p53-dependent manner, and also enhanced the cytotoxicity of anticancer drugs in neuroblastoma cell lines . Our data suggest caution when employing GFP-transduced cells to assess drug sensitivity and that using a cell surface antigen as a selection marker for gene transduction may perturb cells less than GFP.

Mol Cancer Ther, 2003 Sep, 2(9), 863 - 72
Kahalalide F, a new marine-derived compound, induces oncosis in human prostate and breast cancer cells; Suarez Y et al.; Kahalalide F (KF) is a novel antitumor drug of marine origin under clinical investigation . KF showed a potent cytotoxic activity against a panel of human prostate and breast cancer cell lines, with IC(50) ranging from 0.07 micro M (PC3) to 0.28 micro M (DU145, LNCaP, SKBR-3, BT474, MCF7) . Importantly, nontumor human cells (MCF10A, HUVEC, HMEC-1, IMR90) were 5-40 times less sensitive to the drug (IC(50) = 1.6-3.1 micro M) . KF cytotoxicity did not correlate with the expression level of the multidrug resistance MDR1 and of the tyrosine kinase HER2/NEU, and only slightly by the anti-apoptotic BCL-2 protein . KF action was triggered rapidly by short pulse treatments (15 min caused 50% maximum cytotoxicity) . Neither a general caspase inhibitor (Z-VAD-fmk) nor transcription or translation inhibitors (actinomycin D, cycloheximide) blocked KF action . Flow cytometry analysis revealed that KF induced neither cell-cycle arrest nor apoptotic hypodiploid peak . Using mitochondrial (JC-1)- and lysosomal (LysoTracker Green, Acridine Orange)-specific fluorophores, we detected loss of mitochondrial membrane potential and of lysosomal integrity following KF treatment . Confocal laser and electron microscopy revealed that KF-treated cells underwent a series of profound alterations including severe cytoplasmic swelling and vacuolization, dilation and vesiculation of the endoplasmic reticulum, mitochondrial damage, and plasma membrane rupture . In contrast, the cell nucleus showed irregular clumping of chromatin into small, condensed masses, while chromatin disappeared from other nuclear domains, but the nuclear envelope was preserved and no DNA degradation was detected . Together, these data indicate that KF induces cell death via oncosis preferentially in tumor cells.

Chest, 2003 Oct, 124(4), 1476 - 81
Comparative roles of levofloxacin and ofloxacin in the treatment of multidrug-resistant tuberculosis: preliminary results of a retrospective study from Hong Kong; Yew WW et al.; OBJECTIVE: To compare levofloxacin and ofloxacin in the treatment of multidrug-resistant tuberculosis (MDR-TB) . PATIENTS AND METHODS: A retrospective analysis of 106 patients with MDR-TB (February 1990 through December 2000) receiving directly observed therapy with fluoroquinolone and accompanying drugs, which mainly included aminoglycosides, cycloserine, ethionamide/prothionamide, and pyrazinamide, was performed . Clinical data from 99 suitable patients were subjected to univariate analysis, stratification, and multiple logistic regression to compare the roles of levofloxacin and ofloxacin in multidrug regimens . RESULTS: Forty patients received 612.5 +/- 79.0 mg qd levofloxacin (mean +/- SD), and 59 patients received 628.8 +/- 101.8 mg qd ofloxacin together with similar active second-line drugs for similar durations . The times to sputum smear (both 1.8 months) and culture conversion (both 2.1 months) were equivalent . Adverse reactions occurred at similar rates (10.0% vs 11.9%) . The combined treatment success rate was 83.8%, being higher among ofloxacin-susceptible than ofloxacin-resistant cases (90.5% vs 64.0%, p < 0.01) . The success rates for the levofloxacin group were 90.0% (overall), 96.2% (ofloxacin-susceptible cases), and 78.6% (ofloxacin-resistant cases) in comparison with 79.7%, 87.5%, and 45.5%, respectively, for the ofloxacin group (Mantel-Haenszel common odds ratio estimate, 4.0; p < 0.05) . Bacillary susceptibility to ofloxacin, good adherence, radiographic extent of one lung or less, and use of levofloxacin were independent predictors of favorable outcome (odds ratios, 7.6 to 21.3) . One patient each from both groups relapsed . CONCLUSION: Levofloxacin was found to be more efficacious than ofloxacin when incorporated into multidrug regimens used for treatment of MDR-TB.

Oncol Res, 2003, 14(1), 49 - 60
Development of a syngeneic in vivo tumor model and its use in evaluating a novel P-glycoprotein modulator, PGP-4008; Lee BD et al.; Multidrug resistance (MDR) is a phenomenon by which tumor cells develop reduced sensitivity to anticancer drugs, which often leads to the failure of cancer chemotherapy . A prominent mechanism of MDR is the overexpression of the multidrug efflux pump, P-glycoprotein (P-gp), that decreases the intracellular accumulation of many anticancer drugs, leading to increased tumor growth . Intensive efforts are under way to develop clinically useful MDR modulators that inhibit the function of P-gp for use in combination with established anticancer drugs . Our goal was to develop an improved in vivo solid tumor model utilizing immunocompetent animals to examine the efficacy of P-gp-specific MDR modulators . Using in vitro cytotoxicity and drug accumulation assays, two transformed murine cell lines, JC and TIB-75, were found to demonstrate the P-gp-mediated MDR phenotype . In contrast, two similar lines did not express functional P-gp . Western blot analyses confirmed the expression of P-gp and the lack of expression of the closely related drug efflux protein MRP1 in the JC and TIB-75 cell lines . The JC cell line displayed excellent tumorigenicity and consistent growth kinetics when implanted into immune-competent Balb/c mice . Animals treated with a combination of a known MDR modulator, cyclosporin A, and a cytotoxic drug, doxorubicin, exhibited significantly reduced tumor growth compared with untreated controls or animals treated with either cyclosporin A or doxorubicin alone . Similarly, a novel P-gp-specific MDR modulator, PGP-4008, in combination with doxorubicin showed inhibition of tumor growth . However, in contrast with the significant loss of body weight observed in the animals treated with the combination of cyclosporin A and doxorubicin, those treated with PGP-4008 plus doxorubicin did not experience weight loss . Therefore, this syngeneic solid tumor model provides a new in vivo system that can be used to evaluate the efficacy of P-gp inhibitors in an immune-competent host . This should allow improved prediction of the clinical utility of these compounds.

Oncol Res, 2003, 14(1), 39 - 48
Binding site(s) on P-glycoprotein for a newly synthesized photoaffinity analog of agosterol A; Mitsuo M et al.; Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR) . We have synthesized {125I}11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells . The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp . {125I}AzidoAG-A is transported by P-gp and the intracellular accumulation of both {125I}azidoAG-A and {3H}AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells . {125I}AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents . The binding of {125I}azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of {125I}azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding . Thus, {125I}azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.

Int J Tuberc Lung Dis, 2003 Oct, 7(10), 912 - 9
The treatment of tuberculosis in South Korea; Seung KJ et al.; South Korea's complex system of tuberculosis control has never been fully described . The prevalence of tuberculosis has dropped dramatically since 1965, partly because of farsighted governmental policy that provided low-cost, accessible tuberculosis treatment to the entire population . Within the tuberculosis control system, public and private sector entities provide a wide variety of treatment options . The National Tuberculosis Program focuses on improving cure rates for new cases, while the private sector has taken more of a role in the treatment of drug-resistant tuberculosis and other types of complicated cases . There has been a decrease in drug-resistant tuberculosis since 1980 for multiple reasons, including increased cure rates from the introduction of rifampin-based regimens, improved nutrition and living standards, and the treatment of drug-resistant cases in the private sector . Multidrug-resistant tuberculosis, however, still poses a significant threat to public health . The limited outcomes data that exist in South Korea for multidrug-resistant tuberculosis treatment suggest that cure rates are low and failure and abandonment rates are high . New public health measures are needed to improve the control of multidrug-resistant tuberculosis.

J Heart Lung Transplant, 2003 Oct, 22(10), 1168 - 73
Multidrug-resistant tuberculosis in a lung transplant recipient; Lee J et al.; Tuberculosis infection has been a relatively rare complication after lung transplantation . However, as more countries in which Mycobacterium tuberculosis infection remains endemic embark on lung transplant programs, the occurrence of multidrug-resistant tuberculosis after transplantation is a genuine threat . We report the first case of multidrug-resistant tuberculosis in a double-lung transplant recipient who probably acquired the disease from the donor . We discuss the problems in clinical management of post-transplant tuberculosis infection and of drug-resistance.

Biochem Biophys Res Commun, 2003 Oct 24, 310(3), 824 - 9
Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts; Hayashi A et al.; The nuclear factor-E2 p45-related factor (Nrf) 2 is a transcription factor for the antioxidant responsive element-mediated induction of enzymes responsible for conjugation . Since multidrug resistance-associated protein1 (Mrp1/Abcc1) is an ATP-binding cassette transporter which plays an important role in the cellular extrusion of conjugated metabolites and, therefore, acts synergistically with conjugating enzymes for the cellular detoxification of xenobiotics, we examined the possibility that Nrf2 is also involved in the expression of Mrp1 in mouse embryo fibroblasts . The constitutive expression levels of Mrp1 mRNA and protein were significantly lower in Nrf2 (-/-) cells compared with those in wild type cells . In addition, significant induction by diethyl maleate was observed in wild type, but not in Nrf2 (-/-), cells, suggesting the involvement of Nrf2 in both the constitutive and inducible mRNA and protein expression of Mrp1 . In addition, the uptake of {3H}2,4-dinitrophenyl-S-glutathione, a typical substrate of Mrp1, into isolated membrane vesicles also demonstrated that Nrf2 regulates the transport activity of glutathione conjugates in mouse fibroblasts . This is the first demonstration that Nrf2 is required for the constitutive and inducible expression of Mrp family proteins.

J Gastroenterol Hepatol, 2003 Nov, 18(11), 1283 - 6
Obstetric cholestasis with elevated gamma glutamyl transpeptidase: incidence, presentation and treatment; Milkiewicz P et al.; BACKGROUND: Obstetric cholestasis (OC) may cause severe pruritus in the mother and lead to fetal distress and stillbirth . The etiology of OC is multifactorial, but includes inherited dysfunction of bile canalicular transporters . One of these, multidrug resistant protein 3 (MDR3), a phospholipid transporter, when dysfunctional is associated with elevated levels of gamma glutamyl transpeptidase (GGT) . The aim of the present study was to assess the incidence of OC associated with elevated GGT . We compared the natural history of a cholestatic pregnancy and the efficacy of ursodeoxycholic acid (URSO) in OC patients grouped according to a normal or raised GGT level . METHODS: Eighty-one patients with OC were analyzed . OC was diagnosed in patients with pruritus and elevated serum bile acids (SBA) . Fifty-seven consenting volunteer patients (70%) were treated with URSO . RESULTS: Elevated GGT at presentation was found in 21 patients (30%) and was associated with significantly higher serum levels of aspartate transaminase (AST), bilirubin (BIL) and SBA . OC presented at approximately the same gestation week in both groups of patients . In patients not treated with URSO, liver function tests (LFT) showed no significant change from the time of diagnosis to delivery . Patients from both groups responded to URSO with significant improvement in their AST and alanine aminotransferase (ALT) levels, but SBA fell significantly only in the normal GGT group . CONCLUSIONS: An elevated GGT occurs in less than one-third of patients with OC in the UK and, when present, is associated with greater impairment of LFT, but no difference in gestational age at onset . Treatment with URSO appears to be safe and significantly improves LFT in patients with OC, with the exception of SBA in the high GGT group.

Folia Microbiol (Praha), 2003, 48(4), 496 - 500
Cross-resistance to strobilurin fungicides in mitochondrial and nuclear mutants of Saccharomyces cerevisiae; Hnatova M et al.; In yeast the resistance to kresoxim-methyl and azoxystrobin, like the resistance to strobilurin A (mucidin) is under the control of both mitochondrial cob gene and the PDR network of nuclear genes involved in multidrug resistance . The mucidin-resistant mucl (G137R) and muc2 (L275S) mutants of Saccharomyces cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoxim-methyl and azoxystrobin . Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclear PDR3 gene . On the other hand, nuclear mutants containing disrupted chromosomal copies of the PDR1 and PDR3 genes or the PDR5 gene alone were hypersensitive to kresoxim-methyl, azoxystrobin and strobilurin A . The frequencies of spontaneous mutants selected for resistance either to kresoxim-methyl, azoxystrobin or strobilurin A were similar and resulted from mutations both in mitochondrial and nuclear genes . The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochrome b and/or increased efflux of strobilurins from fungal cells.

J Clin Microbiol, 2003 Oct, 41(10), 4865 - 9
Photographic and luminometric detection of luciferase reporter phages for drug susceptibility testing of clinical Mycobacterium tuberculosis isolates; Hazbon MH et al.; Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis . Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a "low-tech" inexpensive alternative that is called the Bronx box . In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M . tuberculosis, with the agar proportion method (PM) serving as reference . The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods . The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively . These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis.

Cancer Chemother Pharmacol, 2004 Feb, 53(2), 179 - 85 Epub 2003 Oct 03.
P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells; Bielak-Mijewska A et al.; PURPOSE: One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene . The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3 . MATERIALS AND METHODS: Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 micro M or with UVC to induce apoptosis . Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting . RESULTS: Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation . No active caspase-8 was detected . Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells . CONCLUSIONS: Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells . However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.

Oncologist, 2003, 8(5), 411 - 24
The role of ABC transporters in clinical practice; Leonard GD et al.; Drug resistance remains one of the primary causes of suboptimal outcomes in cancer therapy . ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via ATP-dependent drug efflux pumps . P-glycoprotein (P-gp), encoded by the MDR1 gene, is an ABC transporter normally involved in the excretion of toxins from cells . It also confers resistance to certain chemotherapeutic agents . P-gp is overexpressed at baseline in chemotherapy-resistant tumors, such as colon and kidney cancers, and is upregulated after disease progression following chemotherapy in malignancies such as leukemia and breast cancer . Other transporter proteins mediating drug resistance include those in the multidrug-resistance-associated protein (MRP) family, notably MRP1, and ABCG2 . These transporters are also involved in normal physiologic functions . The expressions of MRP family members and ABCG2 have not been well worked out in cancer . Increased drug accumulation and drug resistance reversal with P-gp inhibitors have been well documented in vitro, but only suggested in clinical trials . Limitations in the design of early resistance reversal trials contributed to disappointing results . Despite this, three randomized trials have shown statistically significant benefits with the use of a P-gp inhibitor in combination with chemotherapy . Improved diagnostic techniques aimed at the selection of patients with tumors that express P-gp should result in more successful outcomes . Further optimism is warranted with the advent of potent, nontoxic inhibitors and new treatment strategies, including the combination of new targeted therapies with therapies aimed at the prevention of drug resistance.

J Endocrinol, 2003 Oct, 179(1), 41 - 53
Oestrogen receptor alpha increases p21(WAF1/CIP1) gene expression and the antiproliferative activity of histone deacetylase inhibitors in human breast cancer cells; Margueron R et al.; We analysed the antiproliferative activity of various histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) on human breast cancer cells . We observed a lower sensitivity to HDAC inhibition for oestrogen receptor negative (ER-) versus positive (ER+) cell lines . This differential response was associated neither with a modification of drug efflux via the multidrug resistance system nor with a global modification of histone acetyltransferase (HAT)/HDAC activities . In contrast, we demonstrated that in ER+ breast cancer cells the p21(WAF1/CIP1) gene was more sensitive to TSA regulation and was expressed at higher levels . These differences were observed both in transient transfection experiments and on the endogenous p21(WAF1/CIP1) gene . The Sp1 transcription factor, which was shown to interact in vitro with both class I and class II HDACs, is sufficient to confer the differential sensitivity to TSA and participated in the control of p21(WAF1/CIP1) basal expression . Finally, re-expression of ERalpha following adenoviral infection of ER- breast cancer cells increased both p21(WAF1/CIP1) protein accumulation and the growth inhibitory activity of TSA . Altogether, our results highlight the key role of ERalpha and p21(WAF1/CIP1) gene expression in the sensitivity of breast cancer cells to hyperacetylating agents.

J Biol Chem, 2003 Dec 19, 278(51), 51085 - 90 Epub 2003 Oct 02.
Farnesoid X receptor activates transcription of the phospholipid pump MDR3; Huang L et al.; The human multidrug resistance gene MDR3 encodes a P-glycoprotein that belongs to the ATP-binding cassette transporter family (ABCB4) . MDR3 is a critical trans-locator for phospholipids across canalicular membranes of hepatocytes, evidenced by the fact that human MDR3 deficiencies result in progressive familial intrahepatic cholestasis type III . It has been reported previously that MDR3 expression is modulated by hormones, cellular stress, and xenobiotics . Here we show that the MDR3 gene is trans-activated by the farnesoid X receptor (FXR) via a direct binding of FXR/retinoid X receptor alpha heterodimers to a highly conserved inverted repeat element (a FXR response element) at the distal promoter (-1970 to -1958) . In FXR trans-activation assays, both the endogenous FXR agonist chenodeoxycholate and the synthetic agonist GW4064 activated the MDR3 promoter . Deletion or mutation of this inverted repeat element abolished FXR-mediated MDR3 promoter activation . Consistent with these data, MDR3 mRNA was significantly induced by both chenodeoxycholate and GW4064 in primary human hepatocytes in time- and dose-dependent fashions . In conclusion, we demonstrate that MDR3 expression is directly up-regulated by FXR . These results, together with the previous report that the bile salt export pump is a direct FXR target, suggest that FXR coordinately controls secretion of bile salts and phospholipids . Results of this study further support the notion that FXR is a master regulator of lipid metabolism.

FEBS Lett, 2003 Sep 25, 552(2-3), 141 - 4
Artemisinin inhibits inducible nitric oxide synthase and nuclear factor NF-kB activation; Aldieri E et al.; Artemisinin is a natural product used as an alternative drug in the treatment of severe and multidrug-resistant malaria . In the present work we show that artemisinin shares with other sesquiterpene lactones the ability to inhibit the activation of the nuclear factor NF-kB: by this mechanism, artemisinin, as well as parthenolide, inhibits nitric oxide synthesis in cytokine-stimulated human astrocytoma T67 cells . These results suggest that artemisinin, in addition to its antiparasitic properties, could also exert a therapeutic effect on neurological complications of malaria.

HIV Med, 2003 Oct, 4(4), 338 - 45
Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells; Dupuis ML et al.; BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) is expressed in HIV-1 target cells, in a range of pharmacological barriers and in AIDS-associated tumours . P-gp substrates include HIV-1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug-resistant (MDR) cells . OBJECTIVES: The aim of this study was to investigate the effect on human CD4 T-lymphoblastoid CEMrev cells of saquinavir and other PIs in terms of P-gp expression and to characterize the functional and biochemical patterns of PI-induced P-gp molecules . METHODS: CEMrev cells no longer expressing detectable amounts of P-gp were cultured for a prolonged period in the presence of 10 microg/mL saquinavir (CEMsaq10) and tested for P-gp expression and function . Subsequently, CEMsaq10 cells were transferred into medium containing 15 microg/mL saquinavir (CEMsaq15) and cultured for several months . These cell lines were continuously monitored for P-gp expression, function and immunochemical patterns . A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P-gp expression in CEMrev cells . RESULTS: Compared with the drug-diluent control, the exposure of CEMrev cells to 10 microg/mL saquinavir induced, in a consistent fraction of cells (45-50%), de novo expression of functioning P-gp molecules . The transfer of CEMsaq10 cells to 15 microg/mL saquinavir was associated with a dramatic increase in P-gp expression and function (85-90% of CEMsaq15 cells expressed P-gp and effluxed P-gp dye substrates) . These saquinavir-induced P-gp molecules included 75-kDa molecules as well as the classical 170-kDa form of P-gp, suggesting induction of a particular isoform of P-gp termed mini-P-glycoprotein . Conversely, ritonavir and indinavir induced transient P-gp expression in a small percentage of the CEMrev cells . CONCLUSIONS: Treatment of human CD4 T-lymphoblastoid CEMrev cells with saquinavir caused over-expression of functioning P-gp molecules . This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P-gp were observed in CEMsaq10 and CEMsaq15 cells during prolonged in vitro culturing, even in drug-free conditions.

Probl Tuberk Bolezn Legk, 2003, (8), 3 - 5
{Some specific features of drug resistance of Mycobacteria tuberculosis in patients with acutely progressive destructive forms of pulmonary tuberculosis}; Kibrik BS et al.; A clear correlation has been found between the drug resistance of Mycobacterium tuberculosis (MBT) and the degree of immunodeficiency in 127 patients with acute destructive tuberculosis . The findings indicate that it is possible to use significant immunodeficiency as an early marker of the multidrug resistance of MBT and as a predictor of cheesy pneumonia . The fact that there was X-ray involution in one lung and concomitantly progression in the other suggests the presence of MBT strains showing different drug resistance . The efficiency of treatment for acute tuberculosis has been ascertained to be determined by MBT drug resistance.

J Biol Chem, 2003 Dec 12, 278(50), 50234 - 9 Epub 2003 Sep 30.
Separate roles for the Golgi apparatus and lysosomes in the sequestration of drugs in the multidrug-resistant human leukemic cell line HL-60; Gong Y et al.; The sequestration of drugs away from cellular target sites into cytoplasmic organelles of multidrug-resistant (MDR) cancer cells has been recently shown to be a cause for ineffective drug therapy . This process is poorly understood despite the fact that it has been observed in a large number of MDR cancer cell lines . Analysis of drug sequestration in these cells has traditionally been done using fluorescent anthracycline antibiotics (i.e . daunorubicin, doxorubicin) . This narrow selection of substrates has resulted in a limited understanding of sequestration mechanisms and the intracellular compartments that are involved . To better characterize this phenotype, we chose to examine the sequestration of molecules having different acid/base properties in the MDR HL-60 human leukemic cell line . Here we show that weakly basic drug daunorubicin is sequestered into lysosomes according to a pH partitioning type mechanism, whereas sulforhodamime 101, a zwitterionic molecule, is sequestered into the Golgi apparatus through a drug transporter-mediated process . Quantitative intracellular pH measurements reveal that the lysosome-tocytosol pH gradient is expanded in the MDR line . Moreover, the MDR cells overexpress the multidrug resistance-related protein (MRP1), which is localized to the Golgi apparatus . These results demonstrate, for the first time, that two distinct mechanisms for intracellular compartmentalization are operational in a single MDR cell line.

J Biol Chem, 2003 Dec 12, 278(50), 50136 - 41 Epub 2003 Oct 01.
Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites; Loo TW et al.; The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell . Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux . We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity . The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions . Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine . One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine . Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site . The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM . By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C . These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.

Cancer Res, 2003 Sep 15, 63(18), 5962 - 9
Discovery and evaluation of inhibitors of human sphingosine kinase; French KJ et al.; Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of bioactive lipids, including the proapoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate . Accumulating evidence indicates that sphingosine kinase (SK) plays a pivotal role in regulating tumor growth and that SK can act as an oncogene . Despite the importance of SK for cell proliferation, pharmacological inhibition of SK is an untested means of treating cancer because of the current lack of nonlipid inhibitors of this enzyme . To further assess the involvement of SK in human tumors, levels of RNA for SK in paired samples of cDNA prepared from tumors and normal adjacent tissue were analyzed . Expression of SK RNA was significantly elevated in a variety of solid tumors, compared with normal tissue from the same patient . To identify and evaluate inhibitors of SK, a medium throughput assay for recombinant human SK fused to glutathione S-transferase was developed, validated, and used to screen a library of synthetic compounds . A number of novel inhibitors of human SK were identified, and several representative compounds were characterized in detail . These compounds demonstrated activity at sub- to micromolar concentrations, making them more potent than any other reported SK inhibitor, and were selective toward SK compared with a panel of human lipid and protein kinases . Kinetic studies revealed that the compounds were not competitive inhibitors of the ATP-binding site of SK . The SK inhibitors were antiproliferative toward a panel of tumor cell lines, including lines with the multidrug resistance phenotype because of overexpression of either P-glycoprotein or multidrug resistance phenotype 1, and were shown to inhibit endogenous human SK activity in intact cells . Furthermore, each inhibitor induced apoptosis concomitant with tumor cell cytotoxicity . Methods for the synthesis of a series of aurone inhibitors of SK were established, and a prototypical dihydroxyaurone was found to have moderate antitumor activity in vivo in the absence of overt toxicity to the mice . These compounds are the first examples of nonlipid inhibitors of SK with in vivo antitumor activity and so provide leads for additional development of inhibitors of this important molecular target.

Cancer Res, 2003 Sep 15, 63(18), 5909 - 16
Mislocalization of membrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines; Liang XJ et al.; The accumulation of {(14)C}carboplatin and {(3)H}methotrexate is reduced in single-step KB epidermoid adenocarcinoma (KB-CP) cells, which are cross-resistant to carboplatin, methotrexate, and sodium arsenite . In these KB-CP cells, multidrug resistance is accompanied by mislocalization of multidrug resistance associated protein (MRP) 1 and other membrane proteins such as folate-binding protein . MRP1 was not decreased in amount in single-step variants but accumulates in a cytoplasmic fraction, and its apparent molecular weight was altered probably because of reduced glycosylation in resistant cells . This low-density compartment was partially labeled with antibodies to lectin-GSII (a Golgi marker) and Bip/GRP78 (an endoplasmic reticulum marker) . Pulse-chase labeling of MRP1 with (35)S-methionine and (35)S-cysteine and pulse-chase biotinylation of cell surface MRP1 suggests that membrane protein mislocalization is caused mainly by a defect of plasma membrane protein recycling, manifested also as a defect in acidification of lysosomes . The reduced accumulation of cytotoxic compounds in the KB-CP cells is presumed to result from the failure of carrier proteins and/or transporters to localize to the plasma membrane.

Lancet Infect Dis, 2003 Oct, 3(10), 624 - 32
Diagnosis and treatment of tuberculosis in children; Shingadia D et al.; There has been a recent global resurgence of tuberculosis in both resource-limited and some resource-rich countries . Several factors have contributed to this resurgence, including HIV infection, overcrowding, and immigration . Childhood tuberculosis represents a sentinel event in the community suggesting recent transmission from an infectious adult . The diagnosis of tuberculosis in children is traditionally based on chest radiography, tuberculin skin testing, and mycobacterial staining/culture although these investigations may not always be positive in children with tuberculosis . Newer diagnostic methods, such as PCR and immune-based methods, are increasingly being used although they are not widely available and have a limited role in routine clinical practice . Diagnostic approaches have been developed for use in resource-limited settings; however, these diagnostic methods have not been standardised and few have been validated . Short-course, multidrug treatment has been adopted as standard therapy for adults and children with tuberculosis, with or without directly observed therapy . Compliance is a major determinant of the success of drug treatment . Although uncommon in children, multidrug-resistant tuberculosis is also increasing and treatment will often involve longer courses of therapy with second-line antituberculosis drugs . Treatment of latent infection and chemoprophylaxis of young household contacts is also recommended for tuberculosis prevention, although this may not always be carried out, particularly in high incidence areas.

Int J Antimicrob Agents, 2003 Oct, 22(4), 380 - 7
Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine); Seifert K et al.; Leishmania donovani promastigote lines resistant to hexadecylphosphocholine (HePC, miltefosine) at 2.5, 5.0, 10.0, 20.0 and 40.0 microM were developed in vitro by continuous step-wise drug pressure . The 40 microM line was 15 times more resistant to HePC than the wild-type clone and showed cross-resistance to the ether lipid ET-18-OCH3 (edelfosine) but not to the standard anti-leishmanial drugs . Resistance was stable up to 12 weeks in drug-free culture medium . No amplification of specific genes, including the multidrug resistance P-glycoprotein gene, could be detected in the resistant parasites.

Mol Biol Cell, 2003 Oct, 14(10), 4238 - 49 Epub 2003 Aug 07.
TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19; Geisler M et al.; Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs . Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction . Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1 . This interaction was verified in vitro . Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction . Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane . TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1 . The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

Acta Biochim Pol, 2003, 50(3), 883 - 90
YOR129c, a new element interacting with Cnm67p, a component of the spindle pole body of Saccharomyces cerevisiae; Wysocka M et al.; The Saccharomyces cerevisiae spindle pole body (SPB) consists of numerous proteins forming the outer, inner and central plaques . The protein Cnm67 is an important component of the outer plaque . The C-terminus of this protein contains a determinant important for its SPB localization . We identified a protein encoded by YOR129c which interacts with this C-terminus in the two-hybrid system . YOR129c and CNM67 exhibit weak genetic interaction . The double deletion strain yor129cdelta cnm67delta exhibits moderately increased resistance to 0.1M LiCl and hygromycin B compared with the cnm67delta single mutant . We propose that the YOR129c protein is an accessory factor associated with the cytoplasmic face of SPB and plays a role in cation homeostasis and/or multidrug resistance.

Yakugaku Zasshi, 2003 Sep, 123(9), 773 - 9
{MDR1 genotypes related to pharmacokinetics and MDR1 expression}; Nakamura T; The multidrug-resistant transporter encoded by the MDR1 gene belongs to the ATP-binding cassette superfamily of membrane transporters . It is involved not only in the acquisition of multidrug-resistance phenotypes in cancer cells but also in normal tissues such as the brain, kidneys, liver, and intestines . This transporter has the potential to export unnecessary or toxic exogenous substances or metabolites, and in the intestine it is thought to play a role in limiting the oral absorption of a number of structurally unrelated drugs . In 2000, Hoffmeyer et al . performed a systemic screening for MDR1 polymorphisms and suggested that a single-nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene (C3435T) was associated with a lower level of intestinal MDR1 expression, and thereby with lower plasma concentrations of digoxin after oral administration . At present, over 20 SNPs have been found in the MDR1 gene . Clinical studies on the effects of C3435T on MDR1 expression and function in the tissues, and consequently on the pharmacokinetics, have been performed worldwide . In this review, the latest reports concerning the relationship of MDR1 genotypes with pharmacokinetics and MDR1 expression are summarized . Our experimental results demonstrate the importance of genetic polymorphisms at positions 3435 and 2677 in the MDR1 gene on pharmacokinetics and intestinal MDR1 expression . In the future, haplotype analysis of the MDR1 gene and subsequent classification of subjects are needed for individualized pharmacotherapy based on MDR1 genotyping.

Leukemia, 2003 Oct, 17(10), 1948 - 60
Current trends in large cell lymphoma; Fisher RI et al.; For the last decade, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) has been the best available standard of care for aggressive non-Hodgkin's lymphoma (NHL), based on equivalent therapeutic results with other multiagent chemotherapy accompanied by lower costs and lesser toxicity . However, only 40-45% of these patients are cured with CHOP . New treatment strategies have been employed, including the addition of Rituximab to CHOP in elderly patients; dose escalation using granulocyte-colony-stimulating factor; overcoming the multidrug resistance phenotype with infusional chemotherapeutic regimens and use of some newer agents . Furthermore, the International Prognostic Factor index (IPI) has permitted identification of subsets of patients with large variations in prognosis, allowing prognosis specific therapy to be tested . There is now accumulating evidence that the clinical behavior of certain NHL can be profiled by the expression of certain molecular markers, which will undoubtedly play a role in the development of new prognostic models that may refine our ability to identify poor-risk patients . Regardless, there is still significant opportunity for improving survival in large cell lymphomas.

J Virol, 2003 Oct, 77(20), 11193 - 200
High rates of human immunodeficiency virus type 1 recombination: near-random segregation of markers one kilobase apart in one round of viral replication; Rhodes T et al.; One of the genetic consequences of packaging two copies of full-length viral RNA into a single retroviral virion is frequent recombination during reverse transcription . Many of the currently circulating strains of human immunodeficiency virus type 1 (HIV-1) are recombinants . Recombination can also accelerate the generation of multidrug-resistant HIV-1 and therefore presents challenges to effective antiviral therapy . In this study, we determined that HIV-1 recombination rates with markers 1.0, 1.3, and 1.9 kb apart were 42.4, 50.4, and 47.4% in one round of viral replication . Because the predicted recombination rate of two unlinked markers is 50%, we conclude that markers 1 kb apart segregated in a manner similar to that for two unlinked markers in one round of retroviral replication . These recombination rates are exceedingly high even among retroviruses . Recombination rates of markers separated by 1 kb are 4 and 4.7% in one round of spleen necrosis virus and murine leukemia virus replication, respectively . Therefore, HIV-1 recombination can be 10-fold higher than that of other retroviruses . Recombination can be observed only in the proviruses derived from heterozygous virions that contain two genotypically different RNAs . The high rates of HIV-1 recombination observed in our studies also indicate that heterozygous virions are formed efficiently during HIV-1 replication and most HIV-1 virions are capable of undergoing recombination . Our results demonstrate that recombination is an effective mechanism to break the genetic linkage between neighboring sequences, thereby reassorting the HIV-1 genome and increasing the diversity in the viral population.

J Biol Chem, 2003 Dec 26, 278(52), 52641 - 50 Epub 2003 Sep 25.
Competitive promoter occupancy by two yeast paralogous transcription factors controlling the multidrug resistance phenomenon; Lucau-Danila A et al.; Highly flexible gene expression programs are required to allow cell growth in the presence of a wide variety of chemicals . We used genome-wide expression analyses coupled with chromatin immunoprecipitation experiments to study the regulatory relationships between two very similar yeast transcription factors involved in the control of the multidrug resistance phenomenon . Yrm1 (Yor172w) is a new zinc finger transcription factor, the overproduction of which decreases the level of transcription of the target genes of Yrr1, a zinc finger transcription factor controlling the expression of several membrane transporter-encoding genes . Surprisingly, the absence of YRR1 releases the transcriptional activity of Yrm1, which then up-regulates 23 genes, 14 of which are also direct target genes of Yrr1 . Chromatin immunoprecipitation experiments confirmed that Yrm1 binds to the promoters of the up-regulated genes only in yeast strains from which YRR1 has been deleted . This sophisticated regulatory program can be associated with drug resistance phenotypes of the cell . The program-specific distribution of paired transcription factors throughout the genome may be a general mechanism by which similar transcription factors regulate overlapping gene expression programs in response to chemical stress.

Life Sci, 2003 Oct 17, 73(22), 2841 - 54
Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2); Jager W et al.; Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients . Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea . To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system . Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats . While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold . This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2 . Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination . FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2 . FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates . In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin . This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.

Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 222 - 7
Involvement of multidrug resistance-associated proteins in regulating cellular levels of (-)-epigallocatechin-3-gallate and its methyl metabolites; Hong J et al.; (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, has many interesting biological activities . The uptake of EGCG and involvement of specific efflux pumps were studied in MDCKII cells transfected with hPgp, hMRP1, and hMRP2 genes . Total cell associated {3H}EGCG increased 7-fold in the presence of the MRP inhibitors, indomethacin and probenecid, in MDCKII/MRP1 cells, compared to a 2-fold increase in wild-type cells . Intracellular levels of EGCG, 4(")-O-methyl EGCG, and 4('),4(")-di-O-methyl EGCG were increased by 13-, 11-, and 3-fold, respectively, by indomethacin in MDCKII/MRP1 cells . Accumulation of EGCG and its methyl metabolites was also increased approximately 10-fold in the presence of MK-571 in MDCKII/MRP2 cells . Co-treatment with isoflavones, curcumin and tetrahydrocurcumin, increased {3H}EGCG accumulation significantly in MDCKII/MRP1 and HT-29 cells . The results indicate that EGCG and its methyl metabolites are substrates for MRP1 and MRP2, but not for Pgp . MRP type efflux pumps may limit the bioavailability of EGCG.

Ann Trop Med Parasitol, 2003 Sep, 97(6), 575 - 80
Atovaquone-proguanil for recrudescent Plasmodium falciparum in Vietnam; Giao PT et al.; Malarone, a fixed combination of atovaquone with proguanil (AP), has recently been recognized as a promising treatment against multidrug-resistant Plasmodium falciparum . In Vietnam, the first-line treatment for P . falciparum malaria is currently a combination of mefloquine and an artemisinin derivative, and the use of AP has not been explored . The aim of the present study, based in Vietnam, was to assess the efficacy of AP when used to treat P . falciparum recrudescences that had occurred after primary treatment with mefloquine-artesunate . All but two of the 39 patients investigated completed follow-up . The mean parasite- and fever-clearance times {and 95% confidence intervals (CI)} after AP treatment were 36 (30-42) and 21 (18-24) h, respectively . Most (32) of the 37 infections that were followed adequately appeared to be eradicated by the AP, the other five recrudescing once more . The overall cure 'rate' and (CI) was 86% (76%-98%) . All of the patients tolerated the AP well . Atovaquone-proguanil appears to be a safe and promising alternative treatment for P . falciparum infections in South-east Asia, although the combination is relatively expensive and may not clear some infections with multidrug-resistant parasites.

J Nat Prod, 2003 Sep, 66(9), 1181 - 5
New polyhydroxy sterols: proteasome inhibitors from a marine sponge Acanthodendrilla sp; Tsukamoto S et al.; Seven new polyhydroxylated sterols, as well as the known agosterols A, C, and D2 (8-10), were isolated from a marine sponge Acanthodendrilla sp . as proteasome inhibitors . Their structures were elucidated on the basis of their spectral data, and they were characterized as agosterol congeners, which had been initially isolated from a marine sponge Spongia sp . and reported to reverse multidrug resistance in tumor cells . Among them, agosterol C (9) most strongly inhibited chymotrypsin-like activity of the proteasome with an IC50 value of 10 microg/mL.

Strahlenther Onkol, 2003 Aug, 179(8), 564 - 70
UV or X-irradiation increases the cytoplasmic accumulation of rhodamine 123 in various cancer cell lines; Dumitriu IE et al.; PURPOSE: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis . We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines . MATERIAL AND METHODS: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL) . Cells were irradiated with up to 960 mJ/cm2 and up to 50 Gy of UVB and X-ray, respectively . RESULTS: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion . For PBL, this effect did not correlate with an apoptotic phenotype . In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period . Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis . CONCLUSION: Irradiation inhibits the export function of ABC transporters . Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death . The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells.

Am J Pathol, 2003 Oct, 163(4), 1567 - 77
Expression and localization of the multidrug resistance protein 5 (MRP5/ABCC5), a cellular export pump for cyclic nucleotides, in human heart; Dazert P et al.; The multidrug resistance protein 5 (MRP5/ABCC5) has been recently identified as cellular export pump for cyclic nucleotides with 3',5'-cyclic GMP (cGMP) as a high-affinity substrate . In view of the important role of cGMP for cardiovascular function, expression of this transport protein in human heart is of relevance . We analyzed the expression and localization of MRP5 in human heart {21 auricular (AS) and 15 left ventricular samples (LV) including 5 samples of dilated and ischemic cardiomyopathy} . Quantitative real-time polymerase chain reaction normalized to beta-actin revealed expression of the MRP5 gene in all samples (LV, 38.5 +/- 12.9; AS, 12.7 +/- 5.6; P < 0.001) . An MRP5-specific polyclonal antibody detected a glycoprotein of approximately 190 kd in crude cell membrane fractions from these samples . Immunohistochemistry with the affinity-purified antibody revealed localization of MRP5 in cardiomyocytes as well as in cardiovascular endothelial and smooth muscle cells . Furthermore, we could detect MRP5 and ATP-dependent transport of {(3)H}cGMP in sarcolemma vesicles of human heart . Quantitative analysis of the immunoblots indicated an interindividual variability with a higher expression of MRP5 in the ischemic (104 +/- 38% of recombinant MRP5 standard) compared to normal ventricular samples (53 +/- 36%, P < 0.05) . In addition, we screened genomic DNA from our samples for 20 single-nucleotide polymorphisms in the MRP5 gene . These results indicate that MRP5 is localized in cardiac and cardiovascular myocytes as well as endothelial cells with increased expression in ischemic cardiomyopathy . Therefore, MRP5-mediated cellular export may represent a novel, disease-dependent pathway for cGMP removal from cardiac cells.

Am J Trop Med Hyg, 2003 Aug, 69(2), 179 - 83
Molecular epidemiology of malaria in Cameroon . XVII . Baseline monitoring of atovaquone-resistant Plasmodium falciparum by in vitro drug assays and cytochrome b gene sequence analysis; Basco LK; Atovaquone is a new broad-spectrum antiprotozoal drug with high in vitro activity against multidrug-resistant Plasmodium falciparum . Its specific action against protozoans is based on the inhibition of the parasite cytochrome bc1 complex of the mitochondrial electron transport system . Protozoans may develop atovaquone resistance by the selection of a mutant cytochrome b gene . With the increasing availability of atovaquone-proguanil combination for prophylaxis and treatment of malarial infections, it is necessary to establish baseline data on atovaquone sensitivity before the drug is introduced massively in an endemic region . For this purpose, the activity of atovaquone was assessed indirectly by in vitro drug sensitivity assays with several serum substitutes and DNA sequencing of the cytochrome b gene . Using the standard in vitro assay procedures with 10% human serum, the geometric mean 50% inhibitory concentration (IC50) for atovaquone was calculated to be 1.15 nM (range = 0.460-4.17 nM), while the use of 10% fetal calf serum resulted in lower IC50s (geometric mean = 0.575, range = 0.266-2.20 nM) . The use of Albumax, a lipid-enriched bovine albumin, over the same concentration range (0.25-16 nM) showed poor results . None of the 37 isolates with an atovaquone IC50 < 4.17 nM displayed any mutation . Further monitoring of atovaquone-resistant P . falciparum is warranted for the rational use of this new antimalarial drug.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3208 - 13
A rifampin-hypersensitive mutant reveals differences between strains of Mycobacterium smegmatis and presence of a novel transposon, IS1623; Alexander DC et al.; Rifampin is a front-line antibiotic for the treatment of tuberculosis . Infections caused by rifampin- and multidrug-resistant Mycobacterium tuberculosis strains are difficult to treat and contribute to a poor clinical outcome . Rifampin resistance most often results from mutations in rpoB . However, some drug-resistant strains have rpoB alleles that encode the phenotype for susceptibility . Similarly, non-M . tuberculosis mycobacteria exhibit higher levels of baseline resistance to rifampin, despite the presence of rpoB alleles that encode the phenotype for susceptibility . To identify other genes involved in rifampin resistance, we generated a library of Mycobacterium smegmatis mc(2)155 transposon insertion mutants . Upon screening this library, we identified one mutant that was hypersensitive to rifampin . The transposon insertion was localized to the arr gene, which encodes rifampin ADP ribosyltransferase, an enzyme able to inactivate rifampin . Sequence analysis revealed differences in the arr alleles of M . smegmatis strain mc(2)155 and previously described strain DSM 43756 . The arr region of strain mc(2)155 contains a second, partial copy of the arr gene plus a novel insertion sequence, IS1623.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3117 - 22
Fluoroquinolone-containing third-line regimen against Mycobacterium tuberculosis in vivo; Veziris N et al.; The objective of the present study was to compare the activities of a third-line regimen recommended by the World Health Organization (WHO) and two derivatives of that regimen with the activity of the standard combination of isoniazid, rifampin, and pyrazinamide as a positive control against Mycobacterium tuberculosis in a murine model . The WHO regimen combines ofloxacin (OFX), ethionamide, amikacin, and pyrazinamide; in the two derivatives of this regimen, OFX was replaced by levofloxacin (LVX) or moxifloxacin (MXF) . The four drugs, a fluoroquinolone (either OFX, LVX, or MXF), ethionamide, pyrazinamide, and amikacin, were administered for the first 2 months (initial phase); and two drugs, a fluoroquinolone (either OFX, LVX, or MXF) and ethionamide, were administered for the following 10 months (continuation phase) . After 6 months of treatment, only the spleens and lungs of mice treated with the standard regimen became culture negative . From 9 months onward, all of the organs of mice treated with the MXF-containing third-line regimen were culture negative . The majority of organs from mice treated with the OFX-containing regimen continued to be culture positive, and the mean CFU counts remained unchanged for as long as 12 months . The results for mice treated with the LVX-containing regimen fell between those for the groups receiving the MXF- and OFX-containing regimens . In conclusion, the activity of the OFX-containing third-line regimen against M . tuberculosis was rather weak in vivo, whereas when OFX was replaced by MXF, 9 months of treatment with a modified third-line regimen displayed bactericidal activity comparable to that of 6 months of treatment with the standard regimen in mice . The MXF-containing third-line regimen seems to be a powerful alternative for the treatment of tuberculosis (TB) when isoniazid and rifampin cannot be used, which is the main feature of multidrug-resistant TB.

Endocr Relat Cancer, 2003 Sep, 10(3), 409 - 18
Validation of real-time RT-PCR for analysis of human breast cancer cell lines resistant or sensitive to treatment with antiestrogens; de Cremoux P et al.; Using a quantitative real-time RT-PCR technique we have compared the expression of a number of genes in two different human breast cancer model systems for development of acquired resistance to antiestrogens . The model system developed at the Danish Cancer Society comprises the cell lines MCF-7, MCF-7/TAMR-1, MCF-7/182R-6 and MCF-7/182R-7, and the model system developed at the Lombardi Cancer Research Center consists of the cell lines MCF-7/LCC1, MCF-7/LCC2 and MCF-7/LCC9 . The findings on the well-known parameters estrogen receptor (ER)alpha, progesterone receptor (PR) and epidermal growth factor receptor (EGFR) are in good agreement with previous reports, thus documenting the usefulness of the real-time RT-PCR technique for multiparametric RNA analysis . The gene expression levels in the two model systems were found to be quite similar in relation to ERalpha, AIB1 (amplified in breast cancer-1), breast cancer antiestrogen resistance gene 1 (BCAR1) and ErbB-2 mRNA expression, whereas significant differences were observed on the expression of ERbeta, multidrug resistance gene 1 (MDR1), PR and EGFR . Furthermore, the presented data suggest that ERbeta, AIB1, BCAR1, CYP19 and MDR1 are unlikely to be causally involved in development of antiestrogen resistance in these breast cancer cell lines.

Breast Cancer Res Treat, 2003 Aug, 80(3), 321 - 30
Novel drug amooranin induces apoptosis through caspase activity in human breast carcinoma cell lines; Rabi T et al.; Amooranin (AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India . A . rohituka stem bark is one of the components of a medicinal preparation used in the Indian Ayurvedic system of medicine for the treatment of human malignancies . We investigated the mechanism of cell death associated with AMR cytotoxicity in human mammary carcinoma MCF-7, multidrug resistant breast carcinoma MCF-7/TH and breast epithelial MCF-10A cell lines . AMR IC50 values ranged between 3.8-6.9 microg/ml among MCF-7, MCF-7/TH and MCF-10A cells . AMR induced oligonucleosome-sized DNA ladder formation characteristic of apoptosis when tumor cells were treated with 1-8 microg/ml AMR for 48 h . In situ cell death detection assay indicated that AMR caused 37.3-72.1% apoptotic cells in MCF-7, 32-48.7% in MCF-7/TH and 0-37.1% in MCF- 10A cells at 1-8 microg/ml concentrations . The induction of apoptosis in AMR treated cells was accompanied by the elevation of total caspase and caspase-8 activities . Flow cytometric analysis showed that AMR induced caspase-8 activation in 40.8-71% MCF-7, 28.5-43.2% MCF-7/TH and 4-32.8% MCF-10A cells at 1-8 microg/ml concentrations . Our results suggest that AMR is a novel drug having potential for clinical development against human malignancies.

J Pharm Sci, 2003 Oct, 92(10), 1968 - 80
Characterization of dexloxiglumide in vitro biopharmaceutical properties and active transport; Tolle-Sander S et al.; The objective of this work was to characterize dexloxiglumide biopharmaceutical properties in vitro and relate these characteristics to its in vivo absorption performance, and to assess dexloxiglumide interaction with P-glycoprotein (P-gp) and MRP1 to anticipate its drug interaction potential . Dexloxiglumide aqueous solubility was moderate and pH dependent . Dexloxiglumide exhibited moderate Caco-2 permeability that was polarized, concentration dependent, and pH dependent . The apical-to-basolateral (AP-BL) permeability at pH 5 {14.5 (+/-1.8) x 10(-6) cm/s} was 2-fold higher than at pH 7.5 {7.24 (+/-0.27) x 10(-6) cm/s} . Neutral and ionized dexloxiglumide species displayed permeabilities of 30.8 (+/-8.4) x 10(-6) cm/s and 9.03 (+/-1.31) x 10(-6) cm/s, respectively . The transport of dexloxiglumide across MDR1-MDCK (P-gp overexpressing Madine Darby canine kidney cells) monolayers was polarized, with a BL-AP/AP-BL permeability ratio of 9.35 (+/-0.73), which was reduced to 1.03 (+/-0.03) by P-gp inhibition . Rhodamine 123 efflux was reduced by dexloxiglumide from 4.06 (+/-0.34) to 2.84 (+/-0.15) across Caco-2 monolayers, and from 17.3 (+/-0.9) to 8.26 (+/-1.38) across MDR1-MDCK monolayers, further indicating dexloxiglumide interaction with P-gp . Additionally, P-gp ATPase activity increased with dexloxiglumide concentration . Dexloxiglumide was effluxed from MRP1-NIH3T3 cells (NIH-3T3 cells expressing the multidrug resistance-associated protein 1) . Dexloxiglumide increased MRP1-substrate fluorescein uptake 4-fold, and fluorescein increased dexloxiglumide uptake 1.8-fold . Overall, in vitro transport studies indicate dexloxiglumide to be moderately soluble and moderately permeable, which is in agreement with the incomplete oral absorption of dexloxiglumide . In vitro, dexloxiglumide was moderately modulated by P-gp and MRP1, which provides a rationale for the design of drug interaction studies .

J Urol, 2003 Oct, 170(4 Pt 1), 1383 - 7
Expression of multidrug resistance proteins P-glycoprotein, multidrug resistance protein 1, breast cancer resistance protein and lung resistance related protein in locally advanced bladder cancer treated with neoadjuvant chemotherapy: biological and clinical implications; Diestra JE et al.; PURPOSE: Resistance to chemotherapy is a major obstacle to overcome in the conservative treatment of patients with locally advanced bladder cancer (LABC) . We investigated the predictive value of the response to neoadjuvant chemotherapy (NACT) and prognosis of the expression of multidrug resistance (MDR) related proteins, P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP) and lung resistance related protein/major vault protein (LRP/MVP) in LABC . MATERIALS AND METHODS: Using immunohistochemistry we studied the expression of MDR proteins in tumors from 83 patients with LABC treated with NACT using a bladder sparing approach . Expression was related to the response to NACT, bladder preservation and prognosis . RESULTS: P-gp, MRP1, BCRP and LRP/MVP were expressed at high levels in 53%, 59%, 28% and 70% of cases, respectively . P-gp expression correlated with shorter progression-free survival (p = 0.04) but not with overall survival . Surprisingly MRP1 expression correlated with a higher response (p = 0.005) and a higher probability of bladder preservation following NACT (p = 0.001) . BCRP did not show any prognostic impact . High LRP/MVP expression was significantly associated with a worse response to NACT and a decreased probability of bladder preservation (p = 0.035) . CONCLUSIONS: Our data suggest that MRP1 and LRP/MVP may be useful in combination with other clinicopathological prognostic factors for selecting patients with LABC to be candidates for bladder preservation after NACT . A large prospective study is warranted to confirm the prognostic value of these MDR proteins.

J Mol Neurosci, 2003, 20(3), 339 - 43
Overcoming the blood-brain barrier to taxane delivery for neurodegenerative diseases and brain tumors; Rice A et al.; The blood-brain barrier (BBB) effectively prevents microtubule (MT)-stabilizing drugs from readily entering the central nervous system (CNS) . A major limiting factor for microtubule-stabilizing drug permeation across the BBB is the active efflux back into the circulation by the overexpression of the multidrug-resistant gene product 1 (MDR1) or P-glycoprotein (P-gp) . This study has focused on strategies to overcome P-gp-mediated efflux of Taxol analogs, MT-stabilizing agents that could be used to treat brain tumors and, potentially, neurodegenerative diseases such as Alzheimer's disease . However, taxol is a strong P-gp substrate that limits its distribution across the BBB and therapeutic potential in the CNS . We have found that addition of a succinate group to the C-10 position of paclitaxel (Taxol) results in an agent, Tx-67, with reduced interactions with P-gp and enhanced permeation across the BBB in both in vitro and in situ models . Our studies demonstrate the feasibility of making small chemical modifications to Taxol to generate analogs with reduced affinity for the P-gp but retention of MT-stabilizing properties, i.e., a taxane that may reach and treat therapeutic targets in the CNS.

Cancer Res, 2003 Sep 1, 63(17), 5538 - 43
Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter; Volk EL et al.; The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown . We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002) . Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918 . Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system . Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP . In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C . In contrast, no transport was observed in vesicles containing the mutant form of BCRP . Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min . MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport . Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX . Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP . Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.

Biomed Pharmacother, 2003 Sep, 57(7), 301 - 8
N-methylation of anthracyclines modulates their cytotoxicity and pharmacokinetic in wild type and multidrug resistant cells; Gate L et al.; Anthracyclines are the most commonly used classes of anticancer agents in chemotherapy . Development of resistance to these molecules is one of the major reasons for treatment failure . The overexpression of the membrane transporter P-glycoprotein (P-gp) is among the principal mechanisms involved in this phenomenon . This pump, which is responsible for the multidrug resistance (MDR) phenotype, decreases the toxicity of a wide range of unrelated anticancer drugs by increasing their cellular efflux . Structure-activity relationship experiments have shown that the positively charged amino group of the anthracyclines could be responsible for their transport by P-gp . Here, we used three new anthracyclines that shared the same chromophore but differed by the degree of N-methylation of their sugar moiety . Oxaunomycin (OXN) possessed a non-methylated amino group, while LB-1 was monomethylated and beta-clamycin T (BCT) was dimethylated . In sensitive cells (FLC), reduced cytotoxicity was related to the level of N-methylation; whereas in resistant cells (DOX-RFLC(1) and DOX-RFLC(2)) overexpressing different levels of P-gp, increased N-methylation enhanced anthracycline cytotoxicity . Decreased resistance in DOX-RFLCs was associated with an increased drug accumulation due to a reduced cellular efflux . As expected, the MDR modulator verapamil decreased resistance to these anthracyclines by increasing the cellular accumulation . These results suggest that N-methylation of anthracyclines circumvents resistance by diminishing drug transport by P-gp in MDR-positive cells . These observations could be the consequence of the steric hindrance created by the methyl group(s) which may impair the interaction between the positively charged amino group and the active site of P-gp.

Phytother Res, 2003 Sep, 17(8), 903 - 8
Activity against multidrug-resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases; Jimenez-Arellanes A et al.; The increase of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) demands the search for alternative antimycobacterial drugs . The aim of this study was to evaluate plants used in Mexican traditional medicine to treat respiratory diseases for activity against MDR-TB . A group of 22 plants was screened for activity against Mycobacterium tuberculosis H37Rv and Mycobacterium avium at concentrations from 50 to 200 microg/mL . The antimycobacterial effect was determined by a microcolorimetric assay with Alamar blue dye . None of the aqueous extracts had antimycobacterial activity . Hexane extracts from Artemisia ludoviciana, Chamaedora tepejilote, Lantana hispida, Juniperus communis and Malva parviflora, and methanol extracts from Artemisia ludoviciana and Juniperus communis inhibited the growth of Mycobacterium tuberculosis . Mycobacterium avium was inhibited by Juniperus communis hexane extract and by Malva parviflora methanol extract . The active extracts were tested against monoresistant variants of Mycobacterium tuberculosis H37Rv (isoniazid, rifampin, streptomycin and ethambutol resistant) and the hexane extract of Lantana hispida showed the best activity . Lantana hispida hexane extract was also active against a group of MDR-TB clinical isolates . In contrast, it did not inhibit the growth of non-tuberculous mycobacteria . The hexane extract of Lantana hispida was fractionated by column chromatography and one of its fractions (FVI) inhibited the growth of all the MDR-TB clinical isolates at concentrations up to 25 microg/mL . This study supports the fact that selecting plants by ethnobotanical criteria enhances the probability of finding species with activity against mycobacteria, and our results point to Lantana hispida as an important source of potential compounds against MDR-TB .

Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 937 - 45
Functional androgen receptor confers sensitization of androgen-independent prostate cancer cells to anticancer therapy via caspase activation; Davis R et al.; Therapeutic resistance remains an unresolved problem in the clinical management of human prostate cancer (PC) . Despite initial positive response to androgen ablation therapy (AAT), virtually all PC patients will relapse due to acquisition of hormone refractory disease and selective outgrowth of tumor cells with multidrug resistance phenotype . We here provide the first experimental evidence that restoring a functional androgen receptor (AR) in the androgen-independent prostate cancer PC3 cells enhances their sensitivity to growth arrest and suppresses their colony-forming ability in response to paclitaxel and gamma-irradiation . Furthermore, functional AR increases the susceptibility of these cells to the apoptotic potentials of therapeutic agents, as evidenced by an increase in caspase activity, annexin V binding, and internucleosomal DNA fragmentation, by inducing caspase activation . The abrogation of the cytotoxic effects by 4-hydroxyflutamide suggests a crucial role for AR activation in enhancing the therapeutic sensitivity of these cells in a ligand-independent fashion . Our data thus demonstrate that a functional AR is a prerequisite for effective therapeutic response and that aberrant expression or blockade by AAT may trigger pathways leading to emergence of PC cells with therapeutic resistance phenotype . Since the mainstay of primary therapy for PC has been AAT by pharmaco-therapeutic or surgical means, this study thus provides a new frontier for revising the AAT therapeutic strategy in conjunction with radiation and/or chemotherapeutic agents.

Int J Antimicrob Agents, 2003 Sep, 22(3), 284 - 90
Screening for effectors that modify multidrug resistance in yeast; Kozovska Z et al.; The yeast transcription factors Pdr1p and Pdr3p regulate the expression of several genes that encode energy-dependent efflux pumps involved in multidrug resistance . They recognize specific pleiotropic drug resistance elements in the promoters of the target gene such as PDR5 coding for a major multidrug transporter . Gain-of-function mutations in Pdr1p/Pdr3p result in over-expression of transporter genes and establishment of multidrug resistance . We developed a novel yeast-based screening procedure designed to detect compounds that specifically modify multidrug resistance due to an interference with the expression of drug efflux transporter genes . The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose induced Pdr3p driven over-expression of a dominant-lethal allele of the PMA1 gene placed under the control of the PDR5 promoter . Validation of the assay was achieved by showing that growth inhibition was relieved by mutant Pdr3p devoid of activation domain . This screening system may also be used to select the loss-of-function pdr3 (or pdr1) mutants and to identify specific gene(s) whose over-expression or deletion will suppress the expression of multidrug transporters and increase the susceptibility of yeast cells to antifungals.

Int J Antimicrob Agents, 2003 Sep, 22(3), 242 - 9
Cloning, expression, purification and properties of a putative multidrug resistance efflux protein from Helicobacter pylori; Morrison S et al.; Helicobacter pylori is a common pathogen of humans, which predisposes individuals to gastric inflammation and a variety of diseases including gastric cancer . Sequencing of the organism's genome reveals an extensive portfolio of predicted membrane transport proteins, although few of the proteins encoded by these genes have yet been isolated . We describe here the cloning and expression in Escherichia coli of the H . pylori gene hp1181 encoding a putative multidrug resistance membrane transport protein . Substantial overexpression was accomplished, and the protein was tagged with RGS(His)(6) at the C-terminus, which enabled its purification in mg quantities . Identification of the full-length protein was achieved by N-terminal amino acid sequencing and Western blotting using an antibody to the RGS(His)(6) epitope . The retention of structural integrity and occurrence of predicted alpha-helix in the protein were verified by circular dichroism spectroscopy.

J Med Invest, 2003 Aug, 50(3-4), 126 - 35
Multidrug resistance in hematological malignancy; Hirose M et al.; The recent treatment of hematological malignancies appears to be unsatisfactory in child and adult patients with acute myeloid leukemia and adult patients with acute lymphocytic leukemia . A major problem in the treatment of leukemia is caused by the development of drug resistance to chemotherapeutic agents, which is already present at diagnosis or after chemotherapy as a minimal residual disease, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemia clone . It was suggested that the mechanisms of drug resistance consist of drug resistance proteins, which work as a drug efflux pump . These are the permeability-related glycoprotein (P-Gp), the multidrug-resistance associated protein (MRP), the lung resistance protein (LRP), and other MDR proteins such as the transporter associated with antigen processing (TAP), anthracyclin resistance associated protein (ARA), MRP 2-7, and breast cancer resistance protein (BCRP) . In addition, anti-apoptosis mechanisms, alterations of tumor suppressor genes, altered immunogenicity, drug resistance mechanisms for individual drugs, and clinical risk factors such as white blood cell count, age, and other factors have been reported to act in drug resistance singly or in combinations . Here we describe the update of research on the biology of MDR in the hematological malignancies and also discuss how to overcome MDR and adapt the updated treatment methods in the clinical medical field.

Drug Metab Dispos, 2003 Oct, 31(10), 1235 - 9
Expression profiles of drug-metabolizing enzyme CYP3A and drug efflux transporter multidrug resistance 1 subfamily mRNAS in small intestine; Takara K et al.; The purpose of this study is to examine the expression profiles of CYP3A1, CYP3A2, CYP3A9, and CYP3A18 mRNAs as well as multidrug resistance (mdr)1a and mdr1b mRNAs in the liver and small intestine of normal male Wistar rats using a reverse transcription-polymerase chain reaction (PCR) . In the rat liver, the PCR products for CYP3A1, CYP3A2, and CYP3A18 were readily detectable, whereas CYP3A9 was slightly and mdr1a and mdr1b barely detected . Surprisingly, no PCR products for CYP3A1 and CYP3A2 were detected in the small intestine, but those for CYP3A9, CYP3A18, and mdr1a were readily detectable, and a faint band for mdr1b was also observed . Both CYP3A9 and CYP3A18 levels were found to be high in the duodenum and decreased from the top to bottom of the gut, indicating regional differences in both CYP3A9 and CYP3A18 expression in the small intestine . In contrast, mdr1a expression increased gradually from the upper to lower intestine . Consequently, it was suggested that drug metabolism in the small intestine of normal rats was mediated by CYP3A9 and CYP3A18 rather than CYP3A1 and CYP3A2 . Also, regional differences of CYP3A9, CYP3A18, and mdr1a expression were found in the small intestine . The distributions of CYP3A9 and CYP3A18 were different from the distribution of mdr1a, suggesting the cooperative action of drug clearance pathways . This information is important to drug metabolism research based on ex vivo and in vivo studies using rats.

Arch Bronconeumol, 2003 Sep, 39(9), 382 - 6
{Prognosis of mono- and polydrug resistant pulmonary tuberculosis in the city of Santa Cruz, Bolivia}; Olle Goig JE et al.; OBJECTIVE: To evaluate the results of treatment in patients with pulmonary tuberculosis (TB) resistant to one antituberculosis drug (single drug resistance {SDR}) or to more than one drug (polyresistance {PDR}), excluding patients with resistance to the combination of isoniazid and rifampicin (multidrug resistance) . PATIENTS AND METHOD: Retrospective review of all the records of patients with pulmonary TB diagnosed in an outpatient clinic in Santa Cruz, Bolivia, from 1983 through 1993 whose first cultures were SDR or PDR and who were started on treatment in the clinic . RESULTS: We identified 368 patients: 276 (75%) with SDR TB and 92 (25%) with PDR TB . There were 164 new patients among the SDR cases (59%) and 41 (45%) among the PDR cases (P<.05) . The mean (SD) age of PDR patients was 31.5 (14.2) years and there were 165 (68%) males . The mean age of SDR patients was 29.4 (13.4) years and there were 50 (54%) males . Eleven patients (3%) experienced a change in the type of resistance (SDR: 7 {3%}; PDR: 4 {4%}) and in 119 cases (32%) the initial treatment regimen was changed (SDR: 84 {30%}; PDR: 35 {38%}) . One hundred ninety-six SDR patients (71%) and 56 PDR patients (61%) were cured (P>.05) . There were no significant differences in the cure rates in either of the 2 groups between new patients and patients with a history of previous treatment . Seventy-three SDR (26%) and 35 PDR patients (38%) failed to complete treatment (P<.05) . CONCLUSIONS: The cure rates among patients who presented with SDR TB and PDR TB were similar to those of drug-sensitive patients; there were no significant differences between new and previously treated patients . The low cure rates were due to the high number of patients who abandoned treatment . Unless a system of therapy administered under supervision is set up it is not likely that these figures can be improved in Santa Cruz, Bolivia.

Int J Tuberc Lung Dis, 2003 Sep, 7(9), 903 - 6
Impact of the growing HIV-1 epidemic on multidrug-resistant tuberculosis control in Latvia; Morozova I et al.; Latvia, a country with levels of multidrug-resistant (MDR) TB among the highest in the world, experienced a 58-fold increase in HIV seroprevalence among all persons tested in the country from 1996 through 2001 . In addition, HIV seroprevalence among TB cases increased from 0.4% to 1.4%, and among MDR-TB cases from 0% to 5.6% from 1998 through 2001, potentially compromising gains made to date in controlling the country's MDR-TB epidemic . The following will be critical to the future of MDR-TB control in Latvia: containing HIV transmission in the country, particularly among injection drug users who comprised 72% of all HIV cases reported in the country by the end of 2001, as well as 81% of all MDR-TB cases co-infected with HIV; expanding capabilities to more rapidly detect and successfully treat patients with MDR-TB; developing mutual TB control strategies between the National TB and AIDS programs; and continuing to improve institutional infection control measures, particularly in hospitals and prisons where an increasing number of persons infected with HIV come into contact with persons with active MDR-TB.

Int J Tuberc Lung Dis, 2003 Sep, 7(9), 873 - 8
Standardisation and evaluation of DNA-lanthanide fluorescence spectroscopy for determining rifampicin resistance in Mycobacterium tuberculosis clinical isolates; Mani C et al.; SETTING: Tuberculosis Research Centre, Chennai . OBJECTIVE: To rapidly identify multidrug-resistant Mycobacterium tuberculosis using a novel method . DESIGN: A new assay, based on DNA-lanthanide fluorescence, was standardised and evaluated using 93 each of coded rifampicin-resistant and rifampicin-sensitive M . tuberculosis clinical isolates for the correct identification of rifampicin resistance . The results obtained by the new assay were compared with the conventional results . RESULTS AND CONCLUSION: The new assay gave a sensitivity and specificity of 88% and 85%, respectively . It is simple, easy to perform and requires 48 hours for the drug susceptibility results to be available after obtaining the primary culture.

Southeast Asian J Trop Med Public Health, 2003 Jun, 34(2), 316 - 21
Comparative clinical trial of two-fixed combinations dihydroartemisinin-napthoquine-trimethoprim (DNP) and artemether-lumefantrine (Coartem/Riamet) in the treatment of acute uncomplicated falciparum malaria in Thailand; Krudsood S et al.; An open randomized comparison of two-fixed dose artemisinin derivative-containing combination regimens was conducted in adults with acute uncomplicated multidrug resistant falciparum malaria in Thailand . DNP, a combination of dihydroartemisinin with napthoquine and trimethoprim developed recently in China, has been evaluated in China, Vietnam, Cambodia and Thailand . This study was performed to compare the safety, tolerability and efficacy of DNP and artemether-lumefantrine/Coartem . One hundred and thirty eligible uncomplicated falciparum malaria patients were enrolled into the study . Patients were randomly assigned in a 2:1 ratio into group A, which received DNP one tablet twice a day for one day; and group B, which received Coartem/Riamet four tablets twice a day for 3 days . The cure rates at 28-day were 99% and 97% in group A and group B, respectively . No serious adverse events occurred . We concluded that both DNP and Coartem/ Riamet were safe, well tolerated and highly efficacious in the treatment of acute uncomplicated falciparum malaria in Thailand.

Chest, 2003 Sep, 124(3), 915 - 21
Tuberculosis among Tibetan refugee claimants in Toronto: 1998 to 2000; Marras TK et al.; BACKGROUND AND OBJECTIVES: Between 1998 and 2000, approximately 525 Tibetan people previously living in the United States claimed refugee status in Canada, many of whom were referred to our centers for completion of tuberculosis (TB) screening . We reviewed TB-related outcomes in this cohort, to compare our experience with previously published work, and to assess follow-up after a stay in a low-incidence region . METHODS: We performed a retrospective study of all patients of Tibetan origin assessed at our centers (St . Michael's Hospital and West Park Healthcare Centre, both in Toronto) for completion of TB screening, referred because of abnormal chest radiographic findings or positive tuberculin skin test (TST) result . We compared rates of active and drug-resistant TB in our cohort with local and national rates, as well as those previously published in similar groups . RESULTS: One hundred eighty-nine individuals were referred to us for assessment, and 181 records were available for review . The mean duration of stay in Canada prior to presentation was 2.6 months, after having spent a mean of 11 months in the United States . Thirty-two percent of patients gave a history of previous TB, and 97% were TST positive . Culture-positive TB was diagnosed in 24 patients (13%, 4,571 per 100,000), 12 patients had at least one drug resistance (50% of cases), and 4 patients were resistant to at least isoniazid and rifampin (multidrug resistant, 17% of cases) . INTERPRETATION: People from highly TB endemic areas retain a very high risk of active TB and drug resistance, despite an intervening period in a low-prevalence country . It is important to maintain a high degree of suspicion for TB in all people from high-incidence areas . Treatment of all cases of latent TB infection or ongoing medical surveillance is likely justified in this population.

J Biol Chem, 2003 Nov 21, 278(47), 46576 - 82 Epub 2003 Sep 10.
Identification of two different states of P-glycoprotein in its catalytic cycle: role of the linker region in the transition between these two states; Rao US et al.; P-glycoprotein (Pgp) is a drug-translocating ATPase responsible for multidrug resistance in cancer . Although it is well-established that Pgp exhibits drug-dependent ATPase and ATP-dependent drug transport functions, the mechanism by which these two reactions are coupled remains unclear . We have shown recently that proteolytic cleavage of the linker region, which joins the NH2 and COOH halves of the Pgp molecule, results in a Pgp form that exhibits drug-independent and -dependent ATPase activities (Nuti et al., (2000) Biochemistry 39, 3424-3432; Nuti, S . L., and Rao, U . S . (2002) J . Biol . Chem . 277, 29417-29423) . To understand the mechanism underlying this phenomenon, we used the procedure of vanadate-mediated trapping of the Pgp transport cycle intermediates to determine the steps in the catalytic cycle that are being regulated by the linker region . We show that vanadate stably traps Pgp under two different conditions, one in the presence of ATP alone and the other in the presence of ATP and drug, suggesting the existence of two Pgp conformations . These two conformations, one mediating basal and the other drug-stimulated ATPase reactions, represent different transport cycle intermediates of Pgp, because arresting Pgp in either conformation prevents the catalytic cycle from proceeding to completion . The results also show that these two conformations are uncoupled and appear simultaneously in Pgp that was cleaved in the linker region . These results together suggest that Pgp assumes at least two distinct conformational states, which catalyze two ATP hydrolysis events in the drug transport cycle, and the linker region mediates the transition between these two states of Pgp.

Diagn Microbiol Infect Dis, 2003 Sep, 47(1), 331 - 9
Colony morphology switching of Candida lusitaniae and acquisition of multidrug resistance during treatment of a renal infection in a newborn: case report and review of the literature; Favel A et al.; Candida lusitaniae is an emerging opportunistic pathogen which exhibits an unusual antifungal susceptibility pattern . We describe a case of fatal renal infection due to C . lusitaniae in a very low birth weight neonate who was treated with short courses of fluconazole given alternately with amphotericin B . A colony morphology switching was detected on the standard primary culture medium by changes in colony size . Switching was shown to affect deeply the susceptibility to amphotericin B . Afterwards, the switched phenotype developed a cross resistance to fluconazole and itraconazole . Several issues raised by this case are discussed in the light of an extensive review of the literature . Our observations point out the importance of both the detection of colony morphology switching and the close monitoring of antifungal susceptibility in the management of infections due to C . lusitaniae . A judicious therapeutic strategy should prevent the acquisition of multidrug resistance during antifungal therapy.

Pharmacol Res, 2003 Nov, 48(5), 467 - 72
Effect of mdr2 mutation with combined tandem disruption of canalicular glycoprotein transporters by cyclosporine A on bile formation in mice; Elamiri A et al.; The inhibition of canalicular glycoprotein transporters has been suggested as the cause of familial intrahepatic cholestasis . Mutations in multidrug resistance 3-glycoprotein (MDR3) gene induce progressive familial intrahepatic cholestasis type 3 (PFIC3) . Phenotypically, mutation in mdr2 in mice resembles the disruption of MDR3 in human . Secondly, mutation in the bile salt exporting pump (BSEP)/sister of P-glycoprotein (spgp) gene causes progressive familial intrahepatic cholestasis type 2 in human . However, in spgp knock-out mice only a mild persistent cholestasis occurs . The aim of this study is to evaluate the effects of various P-glycoprotein (Pgp) transporters on bile formation and the canalicular transport of taurocholic acid (TCA) in an attempt to understand the combined role of these transporters in the pathogenesis of familial intrahepatic cholestasis . Total bile acid (TBA) and cholic acid secretion rate were decreased in the mdr2 knock-out mice . However, bile flow (BF) and the secretion of muricholic acids were increased . Secretion of cholesterol was negligible and no phospholipids were detected in bile of mdr2 knock-out mice . Treatment with cyclosporine A (CsA) decreased the BF, and the biliary secretion of bile salts (BS) and phospholipids as compared to wild type mice, but after the injection of TCA+CsA, the BF, and the biliary secretion of BS and lipids were increased as compared to the wild type mice treated with CsA alone . In the mdr2 knock-out mice, CsA treatment decreased the BF and the secretion of BS but after the injection of TCA+CsA, the BF and the biliary secretion of BS were increased and the phospholipid secretion was slightly stimulated as compared to the mdr2 knock-out mice treated with CsA alone . CONCLUSION: Disruption of the mdr2 gene and the inhibition of glycoprotein transporters by CsA induce cholestasis in mice which is characterized by reduced BF, BS and biliary lipid secretion . However, CsA treatment did not significantly increase the cholestatic effect in the mdr2 knock-out mice . The injection of TCA decreased the cholestatic effect in the mdr2 knock-out mice as well as the inhibition of glycoproteins transporters by CsA . These data suggest that mutation in the canalicular mdr2 is an important factor during the development of progressive familial cholestasis.

Emerg Infect Dis, 2003 Aug, 9(8), 965 - 9
Multidrug-resistant tuberculosis in HIV-negative patients, Buenos Aires, Argentina; Palmero D et al.; Initial multidrug-resistant (MDR) tuberculosis (TB) in HIV-negative patients treated at a Buenos Aires referral hospital from 1991 to 2000 was examined by using molecular clustering of available isolates . Of 291 HIV-negative MDRTB patients, 79 were initially MDR . We observed an ascending trend of initial MDRTB during this decade (p=0.0033) . The M strain, which was responsible for an institutional AIDS-associated outbreak that peaked in 1995 to 1997, caused 24 of the 49 initial MDRTB cases available for restriction fragment length polymorphism . Of those, 21 were diagnosed in 1997 or later . Hospital exposure increased the risk of acquiring M strain-associated MDRTB by approximately two and a half times . The emergence of initial MDRTB among HIV-negative patients after 1997 was apparently a sequel of the AIDS-related outbreak . Because the prevalence of M strain-associated disease in the study population did not level out by the end of the decade, further expansion of this disease is possible.

J Endocrinol, 2003 Sep, 178(3), 339 - 46
Glucocorticoid resistance in inflammatory bowel disease; Farrell RJ et al.; Glucocorticoids are potent inhibitors of T cell activation and proinflammatory cytokines and are highly effective treatment for active inflammatory bowel disease (IBD) . However, failure to respond, acutely or chronically, to glucocorticoid therapy is a common indication for surgery in IBD, with as many as 50% of patients with Crohn's disease (CD) and approximately 20% of patients with ulcerative colitis (UC) requiring surgery in their lifetime as a result of poor response to glucocorticoids . Studies report that approximately one-third of patients with CD are steroid dependent and one-fifth are steroid resistant while approximately one-quarter of patients with UC are steroid dependent and one-sixth are steroid resistant . While the molecular basis of glucocorticoid resistance has been widely assessed in other inflammatory conditions, the pathophysiology of the glucocorticoid resistance in IBD is poorly understood . Research in IBD suggests that the phenomenon of glucocorticoid resistance is compartmentalised to T-lymphocytes and possibly other target inflammatory cells . This review focuses on three key molecular mechanisms of glucocorticoid resistance in IBD: (i) decreased cytoplasmic glucocorticoid concentration secondary to increased P-glycoprotein-mediated efflux of glucocorticoid from target cells due to overexpression of the multidrug resistance gene (MDR1); (ii) impaired glucocorticoid signaling because of dysfunction at the level of the glucocorticoid receptor; and (iii) constitutive epithelial activation of proinflammatory mediators, including nuclear factor kappa B, resulting in inhibition of glucocorticoid receptor transcriptional activity . In addition, the impact of disease heterogeneity on glucocorticoid responsiveness and recent advances in IBD pharmacogenetics are discussed.

J Pharmacol Exp Ther, 2003 Nov, 307(2), 589 - 96 Epub 2003 Sep 09.
A subset of highly effective propafenone-type multidrug resistance modulators lacks effects on cardiac action potential and mechanical twitch parameters of rat papillary muscles; Schmid D et al.; In this study, we tested a series of 12 previously identified, highly effective propafenone-type multidrug resistance (MDR) modulators for their possible undesirable effects on cardiac tissue . We used rat papillary muscle preparations and quantitatively determined the potency of these substances to block action potential (AP) upstroke velocity (Vmax) and to prolong APD50 . Simultaneously, the effects on isometric twitch parameters were evaluated . Concentration-response curves were obtained for all parameters . Within a subset of the compounds, we found a significant rank correlation (r' = 0.87; p < 0.05) between potencies to block Vmax (kiVmax) and to inhibit daunomycin efflux in MDR cells (IC50) . Surprisingly, the most lipophilic compounds with additional aromatic side chains completely lacked effects on AP and mechanical twitch parameters, although they are the most effective MDR modulators . Additional structural modifications such as fluoride substitution of the aromatic ring, introduction of arylpiperazine or piperidine side chains, as well as modifying the hydrogen bond acceptor strength of the carbonyl group did not reestablish cardiac side effects . In contrast, when these substances were truncated at the phenylpropiophenone moiety of the propafenone core structure, cardiac effects reoccurred . We conclude that aromatic substituents in the vicinity of the nitrogen atom prevent interaction with ion channels, likely due to steric hindrance, and are thus a prerequisite for eliminating unwanted cardiac effects.

Int J Oncol, 2003 Oct, 23(4), 1225 - 30
Nuclear texture and chromatin structure in OV1/VCR human multidrug-resistant cell line; Yatouji S et al.; Previous studies have demonstrated that multidrug-resistant leukemic cells displayed nuclear texture changes . In this work, the human ovarian carcinoma cell line IGROV1 and its multidrug-resistant variant OV1/VCR were studied . Cell smears of these cell populations were analysed by image cytometry . As compared to sensitive cells, OV1/VCR display a chromatin global decondensation as assessed by textural features analysis . In order to correlate this decondensation with alterations in chromatin structure, DNase I was used . OV1/VCR DNA displayed an increased DNase I sensitivity, suggesting an increased chromatin accessibility . Furthermore, OV1/VCR cells displayed an increased level in acetylated histone H4, a mechanism known to be associated with transcriptionally active chromatin and relaxed chromatin conformation.

Int J Oncol, 2003 Oct, 23(4), 1195 - 201
Enhanced cytotoxicity and nuclear accumulation of doxorubicin-loaded nanospheres in human breast cancer MCF7 cells expressing MRP1; Aouali N et al.; Previous studies have demonstrated that doxorubicin (DOX) encapsulated in polyisohexylcyano-acrylate nanospheres (NS-DOX) circumvented the resistance of breast cancer cells overexpressing P-glycoprotein (Pgp) . Another protein is involved in multidrug resistance phenotype, the multidrug resistance associated protein (MRP1) . We report that NS-DOX overcomes multidrug resistance in breast cancer cells overexpressing MRP1 . Taking into account that anthracyclines are conjugated to or co-transported with glutathione by MRP1, these data suggest that probably due to ion pair formation (NS-DOX), MRP1 could not transport the anthracycline . Pgp is probably able to transport the ion pair drug complex and the mechanisms of drug resistance reversion in Pgp expressing cells need to be further elucidated.

Cancer Lett, 2003 Sep 10, 199(1), 61 - 8
Anti-proliferative effect of the abl tyrosine kinase inhibitor STI571 on the P-glycoprotein positive K562/ADM cell line; Kotaki M et al.; STI571, an abl tyrosine kinase inhibitor, is less effective in chronic myelogenous leukemia (CML) patients in the accelerated phase and in blastic crisis . We addressed whether STI571 is effective for the CML blastic crisis cell line K562 and the P-glycoprotein (P-gp) positive, multidrug resistance cell line K562/ADM . The present results demonstrate that P-gp positive K562/ADM cells were more resistant than K562 cells to the anti-proliferative and apoptotic effect of STI571, but the co-addition of a P-gp modulator augmented the sensitivity of K562/ADM cells to STI571 . For patients in CML blastic crisis, simultaneous use of a P-gp modulator may increase the efficacy of STI571.

Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 612 - 8
Constitutive rat multidrug-resistance protein 2 gene transcription is down-regulated by Y-box protein 1; Geier A et al.; BACKGROUND/AIMS: Molecular mechanisms underlying transcriptional rat multidrug-resistance protein 2 (Mrp2, Abcc2) gene regulation are mostly unclear . Given the presence of putative binding sites for the Y-box binding protein YB-1 in the regulatory sequence, its trans-regulatory influence was analyzed . METHODS: Reporter assays in HepG2 cells with various Mrp2 deletion constructs in the absence and presence of co-transfected YB-1 were performed . DNA binding studies with recombinant YB-1 protein and nuclear extracts obtained from HepG2 cells and rat liver tissue were carried out . RESULTS: The minimal promoter sequence was confined to the proximal 186 bp . A YB-1 responsive element, Mrp2 YRE-1, was mapped at -186/-157, which exhibits specific YB-1 binding . YB-1 acts as a potent repressor of Mrp2 promoter activity in vitro . CONCLUSIONS: Constitutive Mrp2 gene expression is conferred through the proximal -186 bp . YB-1 acts as a repressor in vitro by specific binding to a defined element in the proximal promoter sequence.

J Chemother, 2003 Aug, 15(4), 374 - 9
The clinical relevance of the expression of several multidrug-resistant-related genes in patients with primary acute myeloid leukemia; Galimberti S et al.; Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms . The co-expression of several genes could be at the basis of the resistant phenotype in vivo . In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIalpha, topoisomerase IIbeta, MRP, GSTpi, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays . In our series, topoisomerase IIbeta was significantly co-expressed with MRP (p = 0.05), GSTpi (p = 0.017) and LRP (p = 0.005) . GSTpi was co-expressed with LRP (p = 0.03) and MRP (p = 0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p = 0.02) . The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival . Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.

Anticancer Drugs, 2003 Aug, 14(7), 503 - 14
Antitumor triptycene bisquinones: a novel synthetic class of dual inhibitors of DNA topoisomerase I and II activities; Wang B et al.; Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin in the nanomolar range in vitro, but have the advantage of blocking nucleoside transport and retaining their efficacy in multidrug-resistant (MDR) tumor cells . Since TT bisquinones induce poly(ADP-ribose) polymerase-1 cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these lead antitumor drugs were tested for their ability to trigger the DNA topoisomerase (Topo) inhibitions responsible for the initial and massive high-molecular-weight cleavage of DNA required for tumor cells to commit apoptosis . Interestingly, antitumor TTs have the unusual ability to inhibit, in a concentration-dependent manner, the relaxation of supercoiled plasmid DNA catalyzed by both purified human Topo I and II enzymes . However, if there is a relationship between the ability of TT analogs to inhibit Topo activities and their quinone functionality and cytotoxicity, it is far from perfect, suggesting that other molecular targets may be involved in the mechanism of action of these antitumor drugs . Moreover, one of the most cytotoxic TT bisquinone, 6-bromo-7-methoxy- or 7-bromo-6-methoxy-2-N-methylamino-1 H,4 H,5 H,8H-9,10-dihydro-9,10-{1',2'}benzenoanthracene-1,4,5,8-tetraone (TT24), inhibits Topo II activity more effectively than amsacrine (m-AMSA) and matches the Topo I inhibitory effect of camptothecin (CPT) . The dual inhibitory activity of TT24 is substantiated by the findings that TT24 mimics the action of m-AMSA in the Topo II assay, where the Topo I inhibitor CPT is ineffective, and also mimics the action of CPT in the Topo I assay, where the Topo II inhibitor etoposide is ineffective . Because of their ability to target nucleoside transport and topoisomerase activities, synthetic TT bisquinones might represent a novel class of bifunctional drugs valuable to develop new means of polychemotherapy and circumvent MDR.

J Nucl Med, 2003 Sep, 44(9), 1394 - 401
99mTc-MIBI imaging as a predictor of therapy response in osteosarcoma compared with multidrug resistance-associated protein and P-glycoprotein expression; Burak Z et al.; In vitro studies have demonstrated that (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) is a transport substrate of multidrug resistance (MDR)-related proteins . The aim of this clinical study was to evaluate whether (99m)Tc-MIBI scintigraphy was a functional imaging tool for in vivo detection of multidrug resistance-associated protein (MRP) expression in osteosarcoma and to investigate the role of MRP and (99m)Tc-MIBI imaging to predict the clinical outcome . We also examined whether the scintigraphic parameters would help to distinguish the functional capacity of P-glycoprotein (Pgp) and MRP . METHODS: Twenty-four patients with a diagnosis of osteosarcoma were studied before neoadjuvant chemotherapy . Tumor-to-background ratios of both early (10 min) and delayed (1 h) images and the percentage washout rate (WR%) of (99m)Tc-MIBI were calculated . Immunohistochemical analysis of MRP and Pgp was performed on biopsy specimens, and the response to preoperative chemotherapy was assessed by histopathologic examination . RESULTS: Fifteen of 24 osteosarcoma samples in our series (62.5%) showed significant expression of MRP . The level of MRP expression was significantly correlated with the WR% of (99m)Tc-MIBI (r = 0.58, P = 0.003), and the WR% of (99m)Tc-MIBI was significantly faster in patients with high MRP expression than in those with a low MRP score (P = 0.007) . The clearance rate of (99m)Tc-MIBI was significantly slower in tumor samples with negative or low expression of both Pgp and MRP (16% +/- 6.2%) when compared with osteosarcomas with high expression of both proteins (31.7% +/- 8.7%) (P = 0.001) . There was not a significant difference between the WR% of (99m)Tc-MIBI in tumors with coexpression of both proteins and in tumors with high expression of either Pgp or MRP . Both the rate of MRP expression and the WR% of (99m)Tc-MIBI were significantly correlated with response rate . CONCLUSION: Our results suggest that the WR% of (99m)Tc-MIBI is correlated with MRP expression . Both the WR% of (99m)Tc-MIBI and MRP expression are correlated with therapy response . (99m)Tc-MIBI can be used as a general probe for functional imaging of both Pgp and MRP; however, it is not capable of differentiating the functional status of either MDR-related glycoprotein.

Clin Cancer Res, 2003 Aug 15, 9(9), 3492 - 502
Pyrazoloacridine is active in multidrug-resistant neuroblastoma cell lines with nonfunctional p53; Keshelava N et al.; PURPOSE: The purpose of this study was to determine the activity of pyrazoloacridine (PZA) in neuroblastomas that have acquired high-level resistance to multiple drugs (not associated with multidrug resistance-associated protein or P-glycoprotein) during therapy, including those with loss of p53 function . EXPERIMENTAL DESIGN: We determined the activity of PZA in 12 drug-sensitive and 10 multidrug-resistant (MDR) neuroblastoma cell lines . Six of the MDR cell lines lacked functional p53 . Drug cytotoxicity was measured using the DIMSCAN fluorescence/digital imaging microscopy assay with a 4-log dynamic range . RESULTS: LC(90) (i.e., the drug concentration that was lethal for 90% of the cell population) values ranged from 0.01 to 1.1 microM for the drug-sensitive cell lines, from 0.8 to 2.4 microM for the MDR cell lines with functional p53, and from 0.9 to 2.1 microM for the MDR cell lines that lacked functional p53 . To confirm that PZA cytotoxicity is independent of p53 function, we compared two neuroblastoma cell lines in which p53 function was abrogated via human papilloma virus-16 E6 transduction (which mediates increased degradation of p53) to the mock-transduced (LXSN) controls . LC(90) values for human papilloma virus-16 E6 clones (abrogated p53) ranged from 0.2 to 2.04 micro M, whereas LC(90) values for LXSN controls (functional p53) were 0.1 and 0.5 microM . PZA was active with 72-h in vitro exposures against p53-nonfunctional MDR cells at drug levels (2-3 microM) obtained for shorter periods (1-3 h) in Phase I and II clinical trials . Washout experiments showed that 3 micro M PZA achieved 0.5-1 log of cell kill with 1-3-h exposures versus 3 logs at 24 h . CONCLUSIONS: These data suggest that expanding the time of PZA systemic exposure, which may be clinically tolerable with hematopoietic stem cell support, should be tested in clinical trials . Prolonged systemic exposure to PZA with hematopoietic stem cell support may be effective against recurrent neuroblastomas that have failed conventional chemotherapeutic regimens, including those neuroblastomas with loss of p53 function.

Clin Cancer Res, 2003 Aug 15, 9(9), 3246 - 53
Irinotecan pathway genotype analysis to predict pharmacokinetics; Mathijssen RH et al.; PURPOSE: The purpose was to explore the relationships between irinotecan disposition and allelic variants of genes coding for adenosine triphosphate binding cassette transporters and enzymes of putative relevance for irinotecan . EXPERIMENTAL DESIGN: Irinotecan was administered to 65 cancer patients as a 90-min infusion (dose, 200-350 mg/m(2)), and pharmacokinetic data were obtained during the first cycle . All patients were genotyped for variants in genes encoding MDR1 P-glycoprotein (ABCB1), multidrug resistance-associated proteins MRP-1 (ABCC1) and MRP-2 (canalicular multispecific organic anion transporter; ABCC2), breast cancer resistance protein (ABCG2), carboxylesterases (CES1, CES2), cytochrome p450 isozymes (CYP3A4, CYP3A5), UDP glucuronosyltransferase (UGT1A1), and a DNA-repair enzyme (XRCC1), which was included as a nonmechanistic control . RESULTS: Eighteen genetic variants were found in nine genes of putative importance for irinotecan disposition . The homozygous T allele of the ABCB1 1236C>T polymorphism was associated with significantly increased exposure to irinotecan (P = 0.038) and its active metabolite SN-38 (P = 0.031) . Pharmacokinetic parameters were not related to any of the other multiple variant genotypes, possibly because of the low allele frequency . The extent of SN-38 glucuronidation was slightly impaired in homozygous variants of UGT1A1*28, although differences were not statistically significant (P = 0.22) . CONCLUSIONS: It is concluded that genotyping for ABCB1 1236C>T may be one of the factors assisting with dose optimization of irinotecan chemotherapy in cancer patients . Additional investigation is required to confirm these findings in a larger population and to assess relationships between irinotecan disposition and the rare variant genotypes, especially in other ethnic groups.

Pathology, 2003 Aug, 35(4), 315 - 8
Multidrug resistance proteins and topoisomerase IIalpha expression in colon cancer: association with metastatic potential; Zorzos HS et al.; AIMS: To investigate the role of multidrug resistance proteins and topoisomerase IIalpha in colon cancer . METHODS: Tissue sections from 89 Dukes' stage B-D colon cancer patients were selected . The expression of multidrug resistance proteins and topoisomerase IIalpha in primary tumour cells was assessed by standard immunohistochemistry . The extent of their expression was measured by image analysis and was correlated with clinicopathological features of the patients . RESULTS: P-glycoprotein was associated with the presence of lymph node metastasis (P=0.005), vessel invasion (P=0.0001) and perineural invasion (P=0.020) . CONCLUSIONS: P-glycoprotein is probably involved in the processes of local invasion and metastatic dissemination.

Biochem J, 2003 Dec 15, 376(Pt 3), 801 - 5
Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates; Mallea M et al.; Over the last decade, MDR (multidrug resistance) has increased worldwide in microbial pathogens by efflux mechanisms, leading to treatment failures in human infections . Several Gram-negative bacteria efflux pumps have been described . These proteinaceous channels are capable of expelling structurally different drugs across the envelope and conferring antibiotic resistance in various bacterial pathogens . Combating antibiotic resistance is an urgency and the blocking of efflux pumps is an attractive response to the emergence of MDR phenotypes in infectious bacteria . In the present study, various alkylaminoquinolines were tested as potential inhibitors of drug transporters . We showed that alkylaminoquinolines are capable of restoring susceptibilities to structurally unrelated antibiotics in clinical isolates of MDR Gram-negative bacteria . Antibiotic efflux studies indicated that 7-nitro-8-methyl-4-{2'-(piperidino)ethyl}aminoquinoline acts as an inhibitor of the AcrAB-TolC efflux pump and restores a high level of intracellular drug concentration . Inhibitory activity of this alkylaminoquinoline is observed on clinical isolates showing different resistance phenotypes.

J Clin Microbiol, 2003 Sep, 41(9), 4454 - 6
Direct detection of multidrug-resistant Mycobacterium tuberculosis in clinical specimens in low- and high-incidence countries by line probe assay; Johansen IS et al.; The INNO-LiPA Rif.TB assay is designed for the detection of rpoB gene mutations causing rifampin resistance in isolates . We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly . Results were obtained in 78.3% of clinical specimens, and all were concordant with those obtained by BACTEC 460 . The assay could have major impact on the management of multidrug-resistant tuberculosis.

J Clin Microbiol, 2003 Sep, 41(9), 4372 - 7
Molecular epidemiology of pulmonary tuberculosis in belgrade, central serbia; Vukovic D et al.; In order to gain precise data on the actual epidemiology of tuberculosis (TB) in Belgrade, central Serbia, we conducted the molecular epidemiological investigation described herein . IS6110 restriction fragment length polymorphism (RFLP) typing of 176 Mycobacterium tuberculosis isolates was performed . These strains were obtained from 48.4% of all patients diagnosed with culture-proven pulmonary TB from April through September 1998 and from May through October 1999 . Clusters containing strains with identical RFLP IS6110 patterns were assumed to have arisen from recent transmission . Of the 176 cases, 55 (31.2%) were grouped into 23 clusters ranging in size from two to six patients . Nearly 80% of clustered patients were directly interviewed, and transmission between family-unrelated contacts was found to be predominant in the study population . Classical contact investigation identified only 2 (3.6%) of the 55 clustered patients . The clustering of TB patients was not associated with any demographic or clinical characteristic other than infection with multidrug-resistant (MDR) M . tuberculosis strains . Nearly 70% of MDR strains were clustered, which indicates active transmission of MDR TB in Belgrade . However, this was not observed by conventional epidemiologic surveillance . In conclusion, the first molecular epidemiologic analysis of TB in the region revealed frequent recent transmission of TB and pointed out significant shortcomings of the current concept for conventional contact tracing . The results presented also demonstrate that transmission of MDR TB in Belgrade is not optimally controlled, and they provide support for the development of improved control strategies, including application of molecular methods.

J Pharm Pharmacol, 2003 Aug, 55(8), 1063 - 73
Delivery of an anticancer drug and a chemosensitizer to murine breast sarcoma by intratumoral injection of sulfopropyl dextran microspheres; Liu Z et al.; Intratumoral injection of controlled-release microsphere formulations of anticancer compounds has the potential to selectively increase tumour exposure to drugs . This work aimed to evaluate the therapeutic effect and toxicity of microsphere formulations containing the anticancer drug, doxorubicin, in a murine tumour model . The effect of co-administration of verapamil, a P-glycoprotein modulator or chemosensitizer, was investigated . Initial in-vitro studies confirmed the ability of verapamil to enhance the accumulation of both doxorubicin and {(99mTc)}sestamibi, also a P-glycoprotein substrate, in EMT6 murine breast sarcoma cells and a doxorubicin-selected multidrug-resistant variant, EMT6/AR1.0 . Ex-vivo studies using confocal microscopy demonstrated release of doxorubicin from microspheres and diffusion of the drug through tissue . For in-vivo studies, EMT6 and EMT6/AR1.0 cells were grown in BALB/c mice . Following intratumoral injection of doxorubicin-loaded microspheres, alone or in combination with verapamil-loaded microspheres, the tumour diameter was measured serially as an indication of therapeutic effect, while the weight, appearance, and behaviour of the mice were monitored as an indication of general toxicity . Intratumoral injections of doxorubicin-loaded microspheres were tolerated much better than systemic administration of equivalent drug concentrations . There was a modest (up to 34%) delay of tumour growth compared with groups receiving no treatment or blank microspheres . Co-injection of verapamil microspheres with doxorubicin microspheres produced a moderate increase in toxicity but no further delay in tumour growth . Controlled-release microsphere formulations of anticancer agents administered intratumorally were an efficient way to deliver high drug doses to the tumour with little systemic toxicity.

Cancer Chemother Pharmacol, 2004 Jan, 53(1), 61 - 7 Epub 2003 Sep 03.
Increased myelotoxicity of idarubicin: is there a pharmacological basis? Results of a pharmacokinetic and an in vitro cytotoxicity study; Kroschinsky F et al.; BACKGROUND: Clinical trials evaluating idarubicin (IDA) in acute myeloid leukemia, multiple myeloma and non-Hodgkin's lymphoma (NHL) have provided some evidence for an increased myelotoxicity of IDA compared to other anthracyclines . IDA is known to be less sensitive towards multidrug resistance mediated by P-glycoprotein (P-gp) . This phenotype is a major impediment to successful antineoplastic treatment, but P-gp is also expressed on hematopoietic stem cells (HSC) . METHODS: We investigated the pharmacokinetics of IDA and etoposide (ETO) in seven previously untreated patients with aggressive NHL . The patients received a CHOP-derived protocol (CIVEP) in which doxorubicin (DOX) was substituted by IDA 11-16 mg/m(2) and ETO 3 x 100 mg/m(2) was added . Furthermore, we evaluated in vitro the impact of P-gp expression on the cytotoxicity of DOX and IDA in cells from three parental chemosensitive leukemia and lymphoma cell lines (HL60, U937, CCRF) and their resistant sublines, as well as in CD34-positive HSC . RESULTS: The peak plasma levels (C(max)), terminal elimination half-life (t(1/2)) and area under the concentration curve (AUC) both for IDA and for ETO did not differ from published data . In cell line models the numbers of viable cells in a P-gp-expressing resistant CCRF-VCR100 subline were significantly more reduced by IDA ( P<0.001), but there was no difference in the cytotoxicities of IDA and DOX in chemosensitive CCRF cells and in the (non-P-gp-expressing) resistant U937 and HL60 sublines . Cytotoxicity against HSC was more pronounced after incubation with IDA than after treatment with DOX ( P=0.014), even when a tenfold higher concentration of DOX than of IDA was used . The addition of cyclosporin A increased the cytotoxic effect of DOX but not that of IDA in HSC . CONCLUSIONS: The pharmacokinetics of IDA and its main metabolite idarubicinol in CHOP-derived protocols were not different from data obtained with other combinations or monotherapy . The increased myelotoxicity of IDA may be a consequence of P-gp expression in CD34-positive HSC.

Biochem J, 2003 Dec 15, 376(Pt 3), 749 - 56
Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653; Ramaen O et al.; Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs . Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function . To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli . NBD1 was highly purified under native conditions and was characterized as a soluble monomer . (15)N-labelling allowed recording of the first two-dimensional NMR spectra of this domain . The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction . Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence . The affinity for ATP and ADP were in the same range (K (d(ATP))=118 microM and K (d(ADP))=139 microM) . Binding of nucleotides did not influence the monomeric state of NBD1 . The ATPase activity of NBD1 was magnesium-dependent and very low {V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively} . The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation.

Drugs Exp Clin Res, 2003, 29(2), 53 - 67
Activation of p53 protein in normal and in tumor cells by a novel anticancer agent CHS 828; Wojciechowski J et al.; CHS 828, a novel cyanoguanidine, represents a new class of drugs for cancer therapy, with an unknown primary mechanism of action . It is generally known that anticancer drugs induce p53 response thereby triggering cell cycle arrest or apoptosis . We investigated the effect of CHS 828 on p53 response in normal and tumor cells and compared this effect with that exerted by conventional anticancer drugs . After 24 h of treatment with CHS 828, we observed a dose-dependent up-regulation of wild type (WT) p53 protein in human breast carcinoma MCF-7 cells as well as in normal human and mouse fibroblasts . The highest p53 increase was observed at 300 nM to 1 microM CHS 828 . CHS 828 induced phosphorylation of p53 protein at Ser-15 in normal cells . However, the drug failed to induce p53 protein in mouse cells in which the poly(ADP-ribose)-1 gene (PARP-1) was disrupted even at a 30-fold higher dose and after prolonged treatment . Combined treatment of PARP-1 -/- cells by multidrug resistance modulators did not alter p53 expression . CHS 828 inhibited cell proliferation and DNA replication in the tested cells . Interestingly, DNA synthesis as well as proliferation of PARP-1 deficient cells was inhibited by drug concentrations that were approximately 3-fold lower than their conventional counterparts . Treatment of cells with CHS 828 for 48 h did not induce apoptosis.

Biochem Biophys Res Commun, 2003 Sep 19, 309(2), 324 - 30
Expressions of hepatobiliary organic anion transporters and bilirubin-conjugating enzyme in differentiating embryonic stem cells; Tanaka Y et al.; Mouse embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability . We previously reported that ES cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (HNF-3beta) . In the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugt1a1) in undifferentiated and differentiating HNF-3beta-transfected ES (HNF-3beta-ES) cells . The expression of organic anion transporting polypeptide 1 (oatp1), multidrug resistance-associated protein 1 (mrp1), mrp2, mrp3, and ugt1a1 was not seen in the undifferentiated HNF-3beta-ES cells by RT-PCR, whereas all were expressed in differentiating HNF-3beta-ES cells . Protein expression for oatp1, mrp1, mrp2, mrp3, and ugt1a1 was also observed in the differentiating HNF-3beta-ES cells by Western blotting . An immunofluorescence examination revealed that oatp1 was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with CD26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes.

J Neurochem, 2003 Sep, 86(6), 1564 - 7
Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier; Bourasset F et al.; Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability . The aim of the study was to elucidate its transport mechanism across the BBB . We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others . Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1 . The brain uptake of {14C}M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice {mdr1a(-/-) and mrp1(-/-)}, with and without probenecid, digoxin, PSC833 or d-glucose . No difference was found between P-gp and mrp1 competent and deficient mice . The brain uptake of {14C}M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with {14C}M6G alone . The co-perfusion of {14C}M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved . The co-perfusion of {14C}M6G with d-glucose revealed a threefold decrease in M6G uptake . In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.

Dev Growth Differ, 2003 Aug, 45(4), 377 - 87
Physiological regulation of P-glycoprotein, MRP1, MRP2 and cytochrome P450 3A2 during rat ontogeny; Rosati A et al.; P-glycoprotein and the multidrug resistance-related proteins MRP1 and MRP2 belong to the ATP binding cassette family of proteins and transport a wide range of substrates . These proteins are also involved in metabolic and excretory processes of xenobiotics . The rat genes mdr1a and mdr1b code for P-glycoproteins, while mrp1 and mrp2 genes code for MRP1 and MRP2 proteins, respectively . In this study, the physiological modulation of the level of transcript for these genes during rat ontogeny in the liver, kidney, lung, brain and heart was analyzed by reverse transcription-polymerase chain reaction . An increasing level of transcript during ontogeny was demonstrated for mdr1a and mdr1b in all tissues considered, as well as for mrp2, which was detected only in the liver and kidney . In contrast, mrp1 transcript, present in all tissues, did not show any modulation . The maximum level of expression was reached in adult animals and a significant decrease was demonstrated in aging rats . Western blot analysis with C219 and M2III-6 monoclonal antibodies confirmed this different pattern of expression during ontogeny in the liver . The physiological regulation of cytochrome P450 3A2 was also considered: in the rat liver, an increase in the level of transcript during ontogeny, with a maximum in 60-day-old rats and a decrease in 8-month-old rats, was evident.

Eur J Biochem, 2003 Sep, 270(18), 3696 - 708
cGMP and glutathione-conjugate transport in human erythrocytes; Klokouzas A et al.; The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated . MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes . MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells . MRP4 is detected in MOR/R but not COR-L23/R cells . Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m . This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1 . It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4 . Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.

Gastroenterology, 2003 Sep, 125(3), 825 - 38
Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice; Wagner M et al.; BACKGROUND & AIMS: Cholestasis induces changes in hepatic adenosine triphosphate-binding cassette (ABC) transporter expression . We aimed to investigate the role of the nuclear bile acid receptor (farnesoid X receptor {FXR}) in mediating changes in ABC transporter expression and in determining liver injury . METHODS: Hepatic ABC transporter (multidrug resistance-associated proteins {Mrp} 2-4 and bile salt export pump {Bsep}) expression and localization were studied in common bile duct-ligated (CBDL) FXR knockout (FXR(-/-)), wild-type (FXR(+/+)), and sham-operated mice . Serum alanine aminotransferase, alkaline phosphatase, bilirubin and bile acid levels, hepatic bile acid composition, and liver histology were investigated . Cholangiomanometry and bile duct morphometry were performed . RESULTS: CBDL induced expression of Mrp 3 and Mrp 4 in FXR(+/+) and even more in FXR(-/-), whereas Mrp 2 expression remained unchanged . Bsep expression was maintained in CBDL FXR(+/+) but remained undetectable in CBDL FXR(-/-) . Alanine aminotransferase levels and mortality rates did not differ between CBDL FXR(+/+) and FXR(-/-) . CBDL increased biliary pressure and induced bile ductular proliferation and bile infarcts in FXR(+/+), whereas FXR(-/-) had lower biliary pressures, less ductular proliferation, and developed disseminated liver cell necroses . CONCLUSIONS: Overexpression of Mrp 3 and Mrp 4 in CBDL mice is FXR independent and could play an important role in the adaptive hepatic ABC transporter response to cholestasis . Maintenance of Bsep expression strictly depends on FXR and is a critical determinant of the cholestatic phenotype . Lack of bile infarcts in CBDL FXR(-/-) suggests that development of bile infarcts is related to bile acid-dependent bile flow and biliary pressure . This information is relevant for the potential use of FXR modulators in the treatment of cholestatic liver diseases.

Biochem Pharmacol, 2003 Sep 1, 66(5), 739 - 48
Pyrazolotriazolopyrimidine derivatives sensitize melanoma cells to the chemotherapic drugs: taxol and vindesine; Merighi S et al.; In this study, we have evaluated the "in vitro" modulatory activity of a series of pyrazolotriazolopyrimidine derivatives (PTP-d) in sensitizing malignant melanoma cells to the chemotherapic drugs: taxol and vindesine . To that end, we have described the impact of chemotherapeutic agents on the cell cycle and on the induction of apoptosis when used alone or in combination with PTP-d . We have demonstrated that four PTP-d reduced chemotherapic drugs EC(50) doses of the G(2)/M accumulation with an average of 1.7-fold for taxol and 9.5-fold for vindesine when challenged on A375 human melanoma cell line . This sensitization activity was also confirmed by analyzing the apoptosis degree induced by the chemotherapic drugs . Interestingly, PTP-d had no effects on the response to cytotoxic agents by skin-derived human keratinocyte cells, NCTC 2544 . Therefore, we have investigated the signaling pathway sustaining the sensitizing effect of PTP-d, providing functional evidence that active compounds are able to inhibit multidrug resistance-associated ATP-binding cassette drug transporter . These results suggested that PTP-d hold great promise for the treatment of multidrug resistance in cancers, leading to potential new therapies for melanoma.

Biochim Biophys Acta, 2003 Sep 2, 1615(1-2), 103 - 14
Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein, MRP1 (ABCC1): effects on protein expression, membrane localization, and transport function; Ito K et al.; The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG) . Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function . We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity . All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A . The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1 . Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions . MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted . In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1 . However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3 . We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.

J Mol Biol, 2003 Sep 5, 332(1), 229 - 42
Conformational changes in the multidrug transporter EmrE associated with substrate binding; Tate CG et al.; EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism . Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer . Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals . Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution . An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound . The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.

N Engl J Med, 2003 Aug 28, 349(9), 837 - 46
Structured treatment interruption in patients with multidrug-resistant human immunodeficiency virus; Lawrence J et al.; BACKGROUND: We compared two strategies for treating patients infected with multidrug-resistant human immunodeficiency virus (HIV) . METHODS: Patients with multidrug-resistant HIV and HIV RNA levels of more than 5000 copies per milliliter were randomly assigned to a four-month structured interruption of treatment followed by a change in antiretroviral regimen (treatment-interruption group) or to an immediate change in regimen (control group) . Genotypic and phenotypic resistance testing was performed . Disease progression, death, and changes in genotypic resistance, CD4 cell counts, HIV RNA levels, and quality of life were assessed . RESULTS: After a median follow-up of 11.6 months, disease progression or death occurred in 22 of the 138 patients in the treatment-interruption group and in 12 of the 132 patients in the control group (P=0.01), with a hazard ratio of 2.57 (95 percent confidence interval, 1.2 to 5.5) for the treatment-interruption group . There were eight deaths in each group . In the treatment-interruption group, the mutant HIV populations completely or partially reverted to wild type by four months in 64.0 percent of patients . As compared with the control group, the treatment-interruption group had a mean CD4 cell count that was 85 cells per cubic millimeter lower from months 0 through 4 (P<0.001), 47 cells per cubic millimeter lower from months 5 through 8 (P<0.001), and 31 cells per cubic millimeter lower after eight months (P=0.11) . The mean HIV RNA levels were 1.2 log copies per milliliter higher (on a base-10 scale) in the treatment-interruption group during months 0 through 4 (P<0.001), but they were not significantly different from those in the control group after month 4 . The overall quality of life was similar in the two groups . CONCLUSIONS: In patients infected with multidrug-resistant HIV, structured interruption of treatment was associated with greater progression of disease and did not confer immunologic or virologic benefits or improve the overall quality of life .

Biophys J, 2003 Sep, 85(3), 2028 - 34
Assessment of multidrug resistance reversal using dielectrophoresis and flow cytometry; Labeed FH et al.; In cancer, multidrug resistance (MDR) is the simultaneous resistance of tumor cells to different natural product anticancer drugs that have no common structure . This is an impediment to the successful treatment of many human cancers . A common correlate of MDR is the overexpression of a membrane protein, P-glycoprotein . Many studies have shown that MDR can be reversed after the use of substrate analogs, called MDR modulators . However, our understanding of MDR modulation is incomplete . In this article, we examine the electrical properties of the human leukemic cells (K562) and its MDR counterpart (K562AR) using dielectrophoresis and flow cytometry (with a membrane potential sensitive dye, DIOC5), both before and after treatment with XR9576 (a P-glycoprotein-specific MDR-reversal agent) . The results show significant differences in the cytoplasmic conductivity between the cell lines themselves, but indicate no significant changes after modulation therapy . We conclude that the process of MDR modulation is not associated with changes in the electrical properties of cancer cells . Moreover, the results demonstrate that using the flow cytometry method alone, with MDR cells, may produce artifactual results--whereas in combination with dielectrophoresis, the results show the role of MDR modulators in preventing drug efflux in MDR cells.

Biophys J, 2003 Sep, 85(3), 2006 - 14
Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1); Saengkhae C et al.; Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR . Several studies have suggested that Rh123 is also a substrate for MRP1 . However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed . Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism . In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells . The formation of a substrate concentration gradient was observed . MRP1-mediated transport of rhodamine was glutathione-dependent . The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline . The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.

Plant J, 2003 Jan, 33(1), 119 - 29
The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use; Klein M et al.; Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance . Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels . Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1) . Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark . Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements . Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark . In contrast, opening of stomata of mrp5-1 plants was reduced in the light . In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress . In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness . All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct . Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light . Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level . Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use . Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency . Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics . These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning.

Clin Infect Dis, 2003 Sep 1, 37(5), 665 - 72 Epub 2003 Aug 14.
Molecular epidemiology and drug resistance of Mycobacterium tuberculosis isolates in the Archangel prison in Russia: predominance of the W-Beijing clone family; Toungoussova OS et al.; Prisons play a significant role in the epidemiology of drug-resistant tuberculosis . A total of 114 Mycobacterium tuberculosis isolates recovered from patients in the Archangel prison (Archangel, Russia) in 2001 were studied using restriction fragment-length polymorphism analysis and spoligotyping . Drug susceptibility was analyzed by the radiometric broth method (BACTEC; Becton Dickinson Diagnostic Systems) . According to genotyping studies, 87 (76.3%) of the isolates belonged to the W-Beijing family . Nearly all (96.6%) W-Beijing isolates were part of a cluster, whereas only 25.9% of the other isolates were clustered (P<.001) . The largest cluster comprised 43 patients . Multidrug resistance was high among new (34.0%) and previously treated (55.0%) cases . Resistance to ethambutol (OR, 3.4; 95% CI, 1.0-12.7; P=.03) and streptomycin (OR, 4.2; 95% CI, 1.5-11.6; P=.001) was significantly associated with infection with W-Beijing isolates . Tuberculosis due to drug-resistant W-Beijing isolates is a major problem in the Archangel prison.

Eur Radiol, 2003 Aug, 13(8), 1771 - 85 Epub 2002 Aug 10.
Imaging of pulmonary tuberculosis; Van Dyck P et al.; Tuberculosis, more than any other infectious disease, has always been a challenge, since it has been responsible for a great amount of morbidity and mortality in humans . After a steady decline in the number of new cases during the twentieth century, due to improved social and environmental conditions, early diagnosis, and the development of antituberculous medication, a stagnation and even an increase in the number of new cases was noted in the mid-1980s . The epidemiological alteration is multifactorial: global increase in developing countries; minority groups (HIV and other immunocompromised patients); and elderly patients due to an altered immune status . Other factors that may be responsible are a delayed diagnosis, especially in elderly patients, incomplete or inadequate therapy, and the emergence of multidrug-resistant tuberculosis . The course of the disease and its corresponding clinicoradiological pattern depends on the interaction between the organism and the host response . Classically, pulmonary tuberculosis has been classified in primary tuberculosis, which occurred previously in children, and postprimary tuberculosis, occurring in adult patients . In industrialized countries, however, there seems to be a shift of primary tuberculosis towards adults . Furthermore, due to an altered immunological response in certain groups, such as immunocompromised and elderly patients, an atypical radioclinical pattern may occur . The changing landscape, in which tuberculosis occurs, as well as the global resurgence, and the changed spectrum of the clinical and radiological presentation, justify a renewed interest of radiologists for the imaging features of pulmonary tuberculosis . This article deals with the usual imaging features of pulmonary tuberculosis as well as the atypical patterns encountered in immunodepressed and elderly patients.

Exp Toxicol Pathol, 2003 Jul, 55(1), 39 - 44
Repetitive doxorubicin treatment of glioblastoma enhances the PGP expression--a special role for endothelial cells; Rittierodt M et al.; The glioblastoma is the highest dedifferentiated form of astrocytic brain tumors, which is refractory to chemotherapy in most cases . The lack of chemotherapeutic success is correlated with overexpression of the product P-glycoprotein (PGP) coded by the multidrug resistance 1 (MDR1) gene and a subsequent release of drugs from the tumor cells . For the chemotherapeutical treatment of glioblastomas, the endothel cell is of special importance since due to its manifold metabolic and protective tasks within the blood-brain barrier, it already has a relatively high PGP expression under physiological conditions . The aim of the present study was to analyze the uptake of the antimitotic drug Doxorubicin (DOX) and the expression of PGP in human and rat glioblastoma cell lines and in a human endothelial cell line at different time points . In the following in vivo approach DOX enriched glioblastoma cells were transplanted into rats and the developed tumor was investigated histologically . The results showed an increased uptake and an enhanced expression of PGP at certain time points in every cell line . In the tissue a DOX release was mainly observed in perivascular surroundings . It was concluded that DOX enhanced the constitutive PGP expression which led to a subsequent exclusion of DOX in tumor cells but also in the endothelial cells of the tumor vasculature . Since the vascularization is a prerequisite for tumor growth, the inhibition of the PGP expression in tumor endothelial cells might be a clinical approach to make the DOX treatment more effective.

Probl Tuberk Bolezn Legk, 2003, (7), 29 - 31
{Complex treatment of destructive pulmonary tuberculosis}; Shul'ga IA et al.; Fifty-five patients with destructive pulmonary tuberculosis were examined . Of them 35 isolated drug-resistant Mycobacterium tuberculosis . Of the 35 patients, 12 were first diagnosed as having pulmonary tuberculosis and 23 had its chronic forms . Due to the complex inpatient treatment involving pneumoperitoneum, in patients with first diagnosed pulmonary tuberculosis, bacterial isolation ceased and decay cavities closed in 100 and 87.5% of cases, respectively . In patients with chronic pulmonary tuberculosis and multidrug-resistant Mycobacterium tuberculosis, bacterial isolation ceased in 10 of the 23 cases and fresh decay cavities closed in 7 patients . The results of the complex treatment for destructive tuberculosis with mycobacterial drug resistance may recommend this treatment modality since the latter is most effective.

Semin Oncol, 2003 Aug, 30(4), 545 - 57
Immunotoxin therapy of hematologic malignancies; Frankel AE et al.; Patients with chemotherapy relapsed or refractory hematologic malignancies may be effectively treated with allogeneic or autologous stem cell transplants . However, many patients cannot be transplanted due to age, comorbidities, or lack of suitable donors . Further, a fraction of patients relapse post-transplant . Novel therapeutic agents that can kill multidrug-resistant malignant stem cells and are not myelosuppressive are needed . One class of such agents is immunotoxins . Immunotoxins consist of cell-selective ligands covalently linked to peptide toxins . The ligand delivers the molecule to specific cell surface receptors on malignant cells . The toxin triggers cell death either by reaching the cytosol and catalytically inactivating vital cell processes or by modifying the tumor cell surface membrane . We have synthesized immunotoxins for therapy of chemoresistant hematologic diseases . In this review, we will detail the synthesis of a number of these drugs and describe their preclinical and clinical activity . Several of these agents have shown dramatic antitumor effects in patients with hematologic neoplasms, and one immunotoxin has been approved for use by the US Food and Drug Administration (FDA) . Over the next several decades, a growing number of these agents should reach the clinic.

J Tongji Med Univ, 1999, 19(4), 260 - 3
Multidrug resistance P-glycoprotein function of bone marrow hematopoietic cells and the reversal agent effect; Chen Z et al.; The multidrug resistance P-glycoprotein (P-gp) expression and function in hematopoietic stem/progenitor cells were studied to investigate whether the inhibition of hematopoietic cell P-gp function by multidrug resistance reversal agent increases the cytotoxicity of chemotherapy drugs on the hematopoietic cells . The expression of P-gp on the surface of CD34+ cells from healthy human marrow was examined by flow cytometry . The multidrug resistance reversal agent MS-209 was used to measure the effects of MS-209 on the Rhodamin-123 uptaking of CD34+ hematopoietic cells . By using methylcellulose semi-solid culture, normal human granulocyte-macrophage clonal formation unit (CFU-GM) was cultured . The changes in CFU-GM inhibitory rate caused by daunorubicin were determined in the presence or absence of MS-209 . The results showed that the P-gp expression rate of bone marrow CD34+ cells was 13.3% . MS-209 obviously increased the Rhodamin-123 uptake of CD34+ positive cells . The mean inhibitory rate of daunorubicin for CFU-GM was 29.6%, but it was increased to 43.3% in the presence of MS-209 with the difference being significant (P < 0.05) . It was concluded that hematopoietic cells expressed P-gp protein and possessed active function . MS-209 could inhibit the membrane efflux pump and increase the cytotoxicity of chemotherapy drugs to the clonal growth of hematopoetic stem cells, suggesting the side effects of these drugs on the hematopoietic system should be taken into consideration in the clinical use.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2971 - 3
Discordant resistance to kanamycin and amikacin in drug-resistant Mycobacterium tuberculosis; Kruuner A et al.; It is generally thought that there is full cross-resistance in Mycobacterium tuberculosis between the aminoglycoside drugs kanamycin and amikacin . However, kanamycin resistance and amikacin susceptibility were seen in 43 of 79 (54%) multidrug-resistant Estonian isolates, indicating that there might be a need to test the resistance of M . tuberculosis isolates to both drugs.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2810 - 6
Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1; Chen X et al.; Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1) . G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells . In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication . The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 {monocyte chemoattractant protein 1}, CCL3 {macrophage inflammatory protein 1alpha}, and CCL5 {regulated upon activation, normal T-cell expressed and secreted protein}), the CXC chemokine (CXCL12 {stromal cell-derived factor 1alpha}), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a) . Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did . CCR5 mRNA expression was also down-regulated by the compound . Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM . Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM . Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function . Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

Endocrinology, 2003 Sep, 144(9), 4008 - 17
Growth hormone modulation of the rat hepatic bile transporter system in endotoxin-induced cholestasis; Mesotten D et al.; Treatment with high dose human GH, although an effective anabolic agent, has been associated with increased incidence of sepsis, inflammation, multiple organ failure, and death in critically ill patients . We hypothesized that GH might increase mortality by exacerbating cholestasis through modulation of bile acid transporter expression . High dose GH was continuously infused over 4 d into rats, and on the final day lipopolysaccharides were injected . Hepatic bile acid transporter expression was measured by Northern analysis and immunoblotting and compared with serum markers of cholestasis and endotoxinemia . Compared with non-GH-treated controls, GH increased endotoxin-induced markers of cholestasis and liver damage as well as augmented IL-6 induction . In endotoxinemia, GH treatment significantly induced multidrug resistance-associated protein 1 mRNA and protein and suppressed organic anion transporting polypeptides, Oatp1 and Oatp4, mRNA, suggesting impaired uptake of bilirubin and bile acids at the basolateral surface of the hepatocyte, which could contribute to the observed worsening of cholestasis by GH . This study of endotoxinemia may thus provide a mechanistic link between GH treatment and exacerbation of cholestasis through modulation of basolateral bile acid transporter expression in the rat hepatocyte.

J Control Release, 2003 Aug 28, 91(1-2), 75 - 83
An essential relationship between ATP depletion and chemosensitizing activity of Pluronic block copolymers; Kabanov AV et al.; Pluronic block copolymers are known to sensitize multidrug resistant (MDR) tumors with respect to various anticancer agents, particularly, anthracycline antibiotics . After completion of the Phase I clinical trial, the formulation containing doxorubicin and Pluronic, SP1049C, is undergoing Phase II clinical trials . Studies of the mechanism of the sensitization effect of Pluronic suggested an essential role of ATP depletion in MDR tumors by the block copolymer . The ATP depletion phenomenon was further examined using a panel of cells with varying levels of expression of P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) . Cell responses were characterized in terms of EC(50), a concentration of Pluronic P85 resulting in a 50% decrease in ATP intracellular levels . These studies suggested that the cells displaying high responses in ATP depletion with EC(50)<0.01% were strongly sensitized by the block copolymer resulting in drastic increases of doxorubicin cytotoxic activity (over 100-fold) . In contrast, the less responsive cells with EC(50)>ca . 0.02% were practically not sensitized by the block copolymer . The responses of the cells to P85 in ATP depletion studies correlated with the levels of expression of the drug efflux transport proteins, primarily Pgp . This provided initial evidence that Pgp may be useful as a gene expression marker for predicting potential responses to doxorubicin/Pluronic formulation in chemotherapy of cancer.

Indian J Med Res, 2003 Feb, 117, 76 - 80
Rapid detection of mutations in rpoB gene of rifampicin resistant Mycobacterium tuberculosis strains by line probe assay; Sharma M et al.; BACKGROUND & OBJECTIVES: Multidrug resistant (MDR) tuberculosis (TB) is a problem of increasing importance in the world due to limited treatment options . Resistance to rifampicin results from nucleotide changes in the gene encoding the beta subunit of the RNA polymerase (rpoB) of Mycobacterium tuberculosis . Rifampicin resistance is considered as a marker for MDR TB . The nature and frequency of mutations in the rpoB gene of rifampicin resistant clinical isolates vary considerably according to the geographical location and very little information is available on specific mutational patterns in India . This study was undertaken to detect and characterize the rpoB gene mutation associated with rifampicin resistance in M . tuberculosis by line probe assay . METHODS: A total of 36 strains of M . tuberculosis were analysed by INNO-LiPA Rif TB and compared with the results of conventional susceptibility testing method . After PCR amplification of the region of RNA polymerase involved in rifampicin resistance, the amplified product was hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip . From the pattern obtained, the presence or absence of rifampicin resistance in the M . tuberculosis strains was assessed . RESULTS: It was found that the M . tuberculosis probe was 100 per cent specific; the most frequently observed mutation was His-526-Tyr in the rpoB gene; and correlation between the results of the LiPA and those obtained by the classical susceptibility testing was excellent (100%) . INTERPRETATION & CONCLUSION: INNO LiPA was found to be a reliable, simple, rapid and informative tool for the early detection and characterization of rpoB mutation associated with rifampicin resistance in M . tuberculosis in the clinical laboratory setting and may constitute an important molecular method for the control of tuberculosis.

Nature, 2003 Aug 21, 424(6951), 957 - 61
Artemisinins target the SERCA of Plasmodium falciparum; Eckstein-Ludwig U et al.; Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum . Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives . Despite extensive clinical and laboratory experience their molecular target is not yet identified . Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins . Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor) . As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin . Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial . Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6 . Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin . Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner . These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.

Pediatr Res, 2003 Nov, 54(5), 627 - 34 Epub 2003 Aug 20.
Evaluation of MRP1-5 gene expression in cystic fibrosis patients homozygous for the delta F508 mutation; Hurbain I et al.; Cystic fibrosis (CF), due to mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), exhibits a wide range of disease severity, even among deltaF508 homozygous patients, and the mechanisms of this variability have yet to be elucidated . In view of the close structural homology and possible functional overlap between CFTR and Multidrug Resistance-associated Proteins (MRPs), MRPs were investigated as potentially relevant factors in CF pathophysiology . MRP1-5 gene expression was analyzed in nasal respiratory epithelial cells from deltaF508 homozygous patients (n = 19) and control subjects (n = 20) using semiquantitative RT-PCR . Significantly lower MRP1 and MRP5 transcript levels were found in CF patients than in control subjects . MRP1 and MRP5 transcript levels were strongly correlated (r = 0.71) . In CF patients, low MRP1 transcript levels were associated with more severe disease as assessed by the Shwachman score . A relation was also observed between MRP1 levels and presence of a cAMP-independent chloride conductive pathway, as determined by a halide-sensitive fluorescent assay . These results suggest that MRPs, especially MRP1, might play a role in CF phenotype and might therefore constitute a target for a novel pharmacotherapy of CF.

Eur Psychiatry, 2003 Aug, 18(5), 262 - 3
Ciprofloxacin-induced acute psychosis in a patient with multidrug-resistant tuberculosis; Norra C et al.; Tuberculosis (TB) increasingly appears in a multidrug-resistant form (MDR-TB) in Europe, too . Treatment remains difficult due to various side effects of the multi-drug-regimens . Ciprofloxacin is widely used as one of the few TB-second-line drugs . We report on the course of a ciprofloxacin-induced acute psychosis in a patient with MDR(isoniazid, streptomycin)-TB which resolved after cessation of ciprofloxacin treatment and introduction of a novel oxazolidone . Careful treatment considerations particularly in patients with additional predisposing factors to neuropsychiatric symptoms are recommended in the potentially dangerous MDR-TB, thus creating an enormous therapeutic challenge.

Commun Dis Intell, 2003, 27(2), 173 - 80
Tuberculosis in Australia: bacteriologically confirmed cases and drug resistance, 2001; Lumb R et al.; The Australian Mycobacterium Reference Laboratory Network collected and analysed laboratory data on new cases of disease caused by Mycobacterium tuberculosis complex in the year 2001 . A total of 771 cases were identified, representing an annual reporting rate of 4.0 cases of laboratory-confirmed tuberculosis per 100,000 population . The predominant specimen type was sputum, (n=369) and a further 111 were collected at bronchoscopy . Smears were positive for 214 of 369 (58.0%) sputum and 42 of 111 (37.8%) bronchoscopy specimens respectively . Seven children (male n=5, female n=2) under 10 years of age had bacteriologically confirmed tuberculosis . A total of 69 isolates (8.9%), comprising 67 M . tuberculosis, one M . africanum, and one M . bovis, were resistant to at least one of the anti-tuberculosis agents . Excluding the M . bovis isolate, 61 of 64 (93.5%) were classified as having initial resistance, three had acquired resistance, and no data were available on the presence or absence of previous treatment for four patients . Resistance to at least isoniazid and/or rifampicin was noted for 67 isolates (8.7%), with resistance to both isoniazid and rifampicin (i.e . defined as multidrug-resistant disease) observed in 12 (1.6%) isolates . All of the multidrug-resistant isolates were M . tuberculosis, 10 were from the respiratory tract . The country of birth was known for 63 of 68 (92.6%) patients with a drug-resistant strain of M . tuberculosis or M . africanum; five were Australian-born and 58 (92.1%) had migrated from a total of 22 countries . One hundred and seven respiratory specimens had a nucleic acid amplification testing performed; 89 of 90 (98.9%) smear positives were nucleic acid amplification testing positive, whilst only 13 of 17 (76.5%) smear negative specimens were nucleic acid amplification testing positive . The 2001 laboratory data reveals a stable incidence rate and level of drug resistance in isolates from Australian patients with tuberculosis.

Anticancer Res, 2003 Jul-Aug, 23(4), 3295 - 301
Enhanced apoptotic effects of novel paclitaxel analogs on NCI/ADR-RES breast cancer cells; Yang LX et al.; This study aimed to investigate the apoptotic effects of novel paclitaxel analogs on NCI/ADR-RES breast cancer cells . Using the colony formation assay, the cytotoxicity of three novel paclitaxel analogs were evaluated on NCI/ADR-RES cells which overexpress multidrug-resistant gene (MDR1) . All three novel paclitaxel analogs exhibited significantly higher cytotoxicity on NCI/ADR-RES cells than paclitaxel . One analog, TL139, was 140 times more effective than paclitaxel . Using TUNEL and DNA fragmentation assay, remarkably increased apoptosis in the paclitaxel analog-treated cells was observed at 48-72 hours, but not in paclitaxel-treated cells . Caspases-3/7 were dramatically activated at 48-72 hours by the novel paclitaxel analogs . The enhanced activity of caspases-3/7 was evidently verified by the measurement of the cleavage of poly(ADP-ribose) polymerase (PARP) . The increased activity of caspases-3/7 significantly correlated with the enhanced apoptosis and cell survival data . Treatment with paclitaxel analogs resulted in a significant amount of mitotic arrest . Using Western blot, the phosphorylation of Bcl-2 protein was found in palictaxel analog-treated cells in a time-dependent manner similar to that of mitotic arrest, thereby indicating that there existed a close correlation between Bcl-2 phosphorylation and mitotic arrest that preceded apoptosis . We conclude that novel taxane analogs could effectively kill MDR1-positive breast cancer cells via the mode of apoptosis.

Mol Biol Cell, 2003 Aug, 14(8), 3389 - 99 Epub 2003 Apr 17.
Subcellular localization and activity of multidrug resistance proteins; Rajagopal A et al.; The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins . These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time . Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets . The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers . Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin . However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins . Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not . Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.

Biochemistry, 2003 Aug 26, 42(33), 9989 - 10000
Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3; Zhang DW et al.; Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1 . Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity . Unlike MRP1, MRP3 also transports monovalent bile salts . We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport . We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity . Mutation S1229A reduced only methotrexate transport . Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate . Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate . Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested . In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates . On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity . Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix . Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.

Pharmacotherapy, 2003 Aug, 23(8), 979 - 87
Effects of grapefruit juice on intestinal P-glycoprotein: evaluation using digoxin in humans; Parker RB et al.; STUDY OBJECTIVES: To determine the effects of grapefruit juice on the pharmacokinetics of oral digoxin, a P-glycoprotein substrate not metabolized by cytochrome P450 3A4, in healthy volunteers, and to assess whether polymorphic multidrug-resistance-1 (MDR1) expression contributes to interindividual variability in digoxin disposition . DESIGN: Prospective, open-label, unblinded, crossover study . SETTING: University research center . SUBJECTS: Seven healthy adult volunteers (four men, three women) . INTERVENTION: Each subject received a single oral dose of digoxin 1.0 mg with water or grapefruit juice with at least a 2-week washout between treatments . During the grapefruit juice phase, juice was administered 3 times/day for 5 days before digoxin administration to maximize any effect on P-glycoprotein . MEASUREMENTS AND MAIN RESULTS: Digoxin pharmacokinetics in the presence and absence of grapefruit juice were compared . The MDR1 exon 26 C3435T genotype was determined by real-time polymerase chain reaction . Compared with water, grapefruit juice significantly reduced the digoxin absorption rate constant (3.0 +/- 2.4 to 1.2 +/- 1.0 hr(-1), p<0.05) and increased absorption lag time (0.32 +/- 0.12 to 0.53 +/- 0.34 hr, p<0.05) . Grapefruit juice did not affect digoxin maximum concentration (Cmax), area under the curve (AUC), elimination half-life, or renal clearance . The effect of grapefruit juice on digoxin Cmax (-45% to +41%) and AUC(0-4) (-29% to +25%) varied substantially among subjects and was inversely correlated with the values during the water phase . Trends toward higher digoxin Cmax AUC, and absorption rate constant during the water phase were found in CC homozygotes compared with subjects carrying a T allele . CONCLUSION: Inhibition of intestinal P-glycoprotein does not appear to play an important role in drug interactions involving grapefruit juice . Interindividual variability in response to grapefruit juice may be related to the balance of intestinal drug uptake and efflux transport.

Mol Imaging, 2002 Jan-Mar, 1(1), 24 - 35
Characterization of a novel 99mTc-carbonyl complex as a functional probe of MDR1 P-glycoprotein transport activity; Dyszlewski M et al.; Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized barriers to chemotherapy in cancer patients . Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general . Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo . We herein report the Pgp recognition properties of a novel 99mTc(I)-tricarbonyl complex, {99mTc(CO)3(MIBI)3}+ (Tc-CO-MIBI) . Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3-1 cells compared to colchicine-selected drug-resistant KB 8-5 cells . In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM) . Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp . By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1)-expressing H69AR cells, indicating only modest transport by MRP1 . Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(-/-) gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

Mol Pharmacol, 2003 Sep, 64(3), 610 - 8
Breast cancer resistance protein exports sulfated estrogens but not free estrogens; Imai Y et al.; Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer reagents such as mitoxantrone, 7-ethyl-10-hydroxycamptothecin, and topotecan . We reported previously that estrone and 17beta-estradiol reverse BCRP-mediated multidrug resistance . In the present study, we demonstrate that BCRP exports estrogen metabolites . First, we generated BCRP-transduced LLC-PK1 (LLC/BCRP) cells, in which exogenous BCRP is expressed in the apical membrane, and investigated transcellular transport of 3H-labeled compounds using cells plated on microporous filter membranes . The basal-to-apical transport (excretion) of mitoxantrone, estrone, and 17beta-estradiol was greater in LLC/BCRP cells than in LLC-PK1 cells . Thin-layer chromatography of transported steroids revealed that the transport of estrone and 17beta-estradiol was independent of BCRP expression . Alternatively, increased excretion of estrone sulfate and 17beta-estradiol sulfate was observed in LLC/BCRP cells . BCRP inhibitors completely inhibited the increased excretion of sulfated estrogens across the apical membrane . Conversion of estrogens into their sulfate conjugates was similar between LLC/BCRP and LLC-PK1 cells, suggesting that the increased excretion of estrogen sulfates was attributable to BCRP-mediated transport . Next, the uptake of 3H-labeled compounds in membrane vesicles from BCRP-transduced K562 (K562/BCRP) cells was investigated . 3H-labeled estrone sulfate, but not 3H-labeled estrone or 17beta-estradiol, was taken up by membrane vesicles from K562/BCRP cells, and this was ATP-dependent . Additionally, BCRP inhibitors suppressed the transport of estrone sulfate in membrane vesicles from K562/BCRP cells . These results suggest that BCRP does not transport either free estrone or 17beta-estradiol but exports sulfate conjugates of these estrogens.

Epilepsia, 2003 Sep, 44(9), 1166 - 75
Overexpression of the human major vault protein in gangliogliomas; Aronica E et al.; PURPOSE: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR) . We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy . METHODS: Surgical tumor specimens (n = 30), as well as peritumoral and control brain tissues, were examined for the cellular distribution pattern of MVP with immunocytochemistry . Western blot analysis showed a consistent increase in MVP expression in GGs compared with that in control cortex . RESULTS: In normal brain, MVP expression was below detection in glial and neuronal cells, and only low immunoreactivity (IR) levels were detected in blood vessels . MVP expression was observed in the neuronal component of 30 of 30 GGs and in a population of tumor glial cells . In the majority of the tumors, strong MVP IR was found in lesional vessels . Perilesional regions did not show increased staining in vessels or in neuronal and glial cells compared with normal cortex . However, expression of MVP was detected in the hippocampus in cases with dual pathology . CONCLUSIONS: The increased expression of MVP in GGs is another example of an MDR-related protein that is upregulated in patients with refractory epilepsy . Further research is necessary to investigate whether it could play role in the mechanisms underlying drug resistance in chronic human epilepsy.

Probl Tuberk Bolezn Legk, 2003, (6), 20 - 3
{Bacterial factors and decompensation of cor pulmonale in patients with pulmonary tuberculosis}; Zavrazhnov SP et al.; Sputum from 88 patients with different forms of pulmonary tuberculosis complicated by chronic cor pulmonale (CCP) was tested for Mycobacterium tuberculosis (MBT) and nonspecific flora . The high occurrence of multidrug resistance of MBT and nonspecific causative agents are found in patients with signs of heart failure in the presence of decompensatory CCP in 20% of cases, the frequency of concomitant chronic bronchitis was 86%.

J Antimicrob Chemother, 2003 Sep, 52(3), 354 - 8 Epub 2003 Aug 13.
Effect of protease inhibitor-containing regimens on lymphocyte multidrug resistance transporter expression; Ford J et al.; BACKGROUND: Increased expression of multidrug resistance transporters, such as P-glycoprotein (P-gp), has been suggested as a potential mechanism for decreased protease inhibitor (PI) availability at certain intracellular sites and tissue compartments . OBJECTIVES: To investigate the effect of PIs on the surface lymphocyte expression of P-gp in vitro and in vivo . PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects (n = 15) and incubated (72 h) with 10 microM of each PI studied (saquinavir, ritonavir, nelfinavir, indinavir, amprenavir and lopinavir), or dimethyl sulphoxide (DMSO) control . PBMCs were also isolated from HIV-infected subjects (n = 50; viral load <50 copies/mL) on a PI- or a non-PI-containing combination antiretroviral regimen . P-gp expression was analysed by flow cytometry . RESULTS: No differences in surface P-gp expression on lymphocytes, CD4+ or CD8+ lymphocyte subsets were observed following incubation with 10 microM saquinavir, ritonavir, indinavir, amprenavir or lopinavir in vitro . Nelfinavir, however, increased P-gp expression . In vivo, no difference in P-gp expression on total lymphocytes was observed between patients receiving a PI-containing regimen {saquinavir n = 9, ritonavir n = 6, nelfinavir n = 7, indinavir n = 7 and lopinavir/ritonavir n = 13, and two nucleoside reverse transcriptase inhibitors (NRTIs)} and patients receiving a control regimen of three NRTIs alone (n = 8) . CONCLUSION: This study suggests that, of the PIs, only nelfinavir increases P-gp expression in vitro, and in vivo the PI class of antiretrovirals do not increase P-gp expression on lymphocytes . It is clear that factors other than PI induction are important in the inter-individual variability in the lymphocyte expression of P-gp.

Ai Zheng, 2003 Aug, 22(8), 886 - 92
{Research advances on circumventing tumor multidrug resistance}; Miao ZH et al.; Tumor multidrug-resistant(MDR) reverters inhibit the function of drug transporters and thus reverse MDR . In contrast,anti-MDR agents circumvent tumor MDR by directly inhibiting or killing MDR tumor . In recent years, the development of novel anti-MDR agents has become a major focus of research . This review summarized the advances in MDR mechanisms concentrating on the relationships between P-glycoprotein and apoptotic regulation and between ceramide signal system and MDR . With this understanding, the authors classified anti-MDR agents for the first time as agents which are structurally modified from MDR-related drugs, disrupt MDR mechanisms, induce caspase-independent apoptosis, interfere with ceramide metabolism, and are mechanism-unknown . Meanwhile, their mechanisms and major features of actions were also discussed.

Ai Zheng, 2003 Aug, 22(8), 856 - 60
{Significance of functional assay of multidrug resistance for prediction of chemotherapy outcomes in acute leukemia}; Ye X et al.; BACKGROUND & OBJECTIVE: It is well known that the multidrug resistance (MDR) efflux protein, P-glycoprotein (P-gp), plays an important role in mediating MDR in acute leukemia . Several studies have shown that the expression level of P-gp and its encoding gene MDR1 mRNA level are associated with the response of chemotherapy . But what we have observed in clinical practice is in contradiction with this . Besides, several recent reports have shown that the results of MDR functional assay are more predictive of chemotherapy outcomes than P-gp expression level . To examine the prognostic value of P-gp expression and MDR functional assay in acute leukemia, the authors determined the expression levels of P-gp and MDR function in 24 patients with de novo acute leukemia . The predictive value of MDR functional assay for chemotherapy outcomes was discussed . METHODS: The expression percentile of P-gp and the mean fluorescence intensity ratio of rhodamine 123 efflux assay {MIR(RE)} and intracellular daunorubicin accumulation assay {MIR(IDA)} of patients' leukemic cell samples were determined with flow cytometry . The results of remission (CR) and non-remission (NR) patients were compared . Correlative analysis was made between test results and chemotherapy outcomes . RESULTS: The expression levels of P-gp were not significantly different between CR and NR patients (2.16%+/-2.42% versus 15.02%+/-25.88%, P=0.114) . But the MIR (RE) (1.16+/-0.38 versus 1.43+/-0.26, P=0.045) and MIR (IDA) (1.02+/-0.05 versus 1.47+/-0.44,P=0.005) were both significantly lower in CR patients than those in NR patients . Both MIR (RE) (r=0.590, P=0.006) and MIR (IDA) (r=0.867, P=0.000) were significantly correlated with chemotherapy outcomes . But the expression level of P-gp was not correlated with the chemotherapy results . CONCLUSION: The determination of MIR value of MDR functional assay is more valuable than P-gp expression level in predicting chemotherapy outcomes.

Ai Zheng, 2003 Aug, 22(8), 821 - 5
{Sodium butyrate-induced expression of lung resistance-related gene in human myeloid leukemia K562 cells}; Li N et al.; BACKGROUND & OBJECTIVE: Overexpression of lung resistance-related protein(LRP) is involved in multidrug resistance and poor outcome in acute leukemia . This study was designed to investigate the effect of sodium butyrate (NaB) on LRP expression level and the function of LRP in K562 cells . METHODS: Human myeloid leukemia K562 cells, used as an in vitro model, were treated with NaB . The LRP mRNA expression and protein levels in the cells before and after NaB treatment were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry indirect immunofluorescence method,respectively.Intracellular Adriamycin(ADM) location and intracellular daunorubicin(DNR) accumulation were detected by fluorescence microscope and flow cytometry, respectively . RESULTS: Compared with untreated K562 cells, the LRP mRNA level in the cells treated with 2 mmol/L NaB was increased obviously; the protein expression were turned from negative result to positive result and the ratios of positive cells were increased from 1.68% to 35.81% . Intracellular DNR accumulation was decreased in the treated K562 cells and the mean fluorescence of DNR reduced 68.64% compared with the untreated K562 cells . There was strong ADM fluorescence in the nuclei of untreated K562 cells; while the treated K562 cells showed significantly less ADM fluorescence in their nuclei but more fluorescence in the cytoplasm . CONCLUSION: Both the mRNA level and the protein expression of LRP in K562 cells can be increased by NaB induction . LRP induced by NaB is involved in the decreasing anti-cancer drug accumulation and transporting the drugs from the nucleus to the cytoplasm in K562 cells.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Jul, 34(3), 431 - 4
{Internalization of antibody-targeted immunonanoparticles into human hepatoma cells and its reversal effect on MDR}; Liu X et al.; OBJECTIVE: To observe if antibody-targeted immunonanoparticles could internalize into sensitive and multidrug resistance (MDR) cells of human hepatoma; and to study if the immunonanoparticles could reverse the MDR . METHODS: Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles (HAb18-ADR-HSA-NP) were incubated with human hepatoma sensitive cell line (SMMC-7721) or MDR cell line (SMMC-7721/MDR+) and the internalization of immunonanoparticles were observed by laser confocus microscopy, scanning electron microscopy and transmission electron microscopy; MTT colorimetric assay was used for assaying in vitro cytotoxicities of HAb18-ADR-HSA-NP to the resistant variant cells . Then based on these data, IC50 value of the immunonanoparticles and RF (Resistant Factor) of MDR cells were calculated . RESULTS: Laser confocus microscopy showed that many fluorescent particles (labeled immunonanoparticles) tightly adsorbed to SMMC-7721 cells and were also seen in cytoplasm of SMMC-7721 cells . When incubated with immunonanoparticles at 37 degrees C, many of immunonanoparticles were visualized in cytoplasm of SMCC-7721 or SMCC-7721/MDR+ . These immunonanoparticles-contained cells exhibited damaged ultrastructures and the damage degree depended on incubation time . When the human hepatoma cells were pretreated with HAb18 antibody and incubated with immunonanoparticles, few immunonanoparticles were seen in cytoplasm of the cells, suggesting antibody-specific internalization of the immunonanoparticles . Scanning electron microscopy demonstrated specific binding of the immunonanoparticles to the resistant variant cells . That immunonanoparticles exerted enhanced cytotoxicity to the resistant variant cells was demonstrated by a decrease of RF value of MDR cells, compared with free ADR (4.4 vs . 2.1) . CONCLUSION: Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles could specifically internalize into sensitive or multidrug resistance cells of human hepatoma via antibody direction . The immunonanoparticles could enhance the sensitivity of MDR cells to ADR cytotoxicity, suggesting its reverse effect on MDR.

Bull Exp Biol Med, 2003 May, 135(5), 482 - 8
New approach to vital analysis of functional activity in ABC transporters (markers for multidrug resistance) in solid tumors by the method of flow cytofluorometry; Bogush TA et al.; We developed and described a new approach to vital analysis of functional activity of multidrug resistance markers (ABC transporters) in intact biopsy specimens from human solid tumors by the method of flow cytofluorometry . The algorithm of the study underwent revision, and the cell suspension was obtained in the final stage . Intensification of intracellular doxorubicin accumulation (fluorescence) and increase in the number of fluorescent cells and total fluorescence of doxorubicin-accumulating cells produced by ABC transporter inhibitor sodium azide served as the criteria of expression of these transporters in tumor tissue . Informative value of changes in various parameters of doxorubicin fluorescence is discussed . The increase in the count of fluorescent cells in the suspension of tumors cells after treatment with the inhibitor indicates the presence of tumor cells absolutely resistant to this preparation . The proposed method is technically simple, suitable for structurally different tumors, requires small amounts of the biopsy material and, therefore, can be used for routine analysis . The results of our analysis and spectrofluorometric assay of ABC transporters agree very closely, which suggest that this method is adequate for the purpose.

J Biol Chem, 2003 Oct 10, 278(41), 39706 - 10 Epub 2003 Aug 07.
Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein; Loo TW et al.; The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell . The drug-binding pocket of P-gp is located in the transmembrane domains . Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time . In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate . TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil . We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12) . By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone . The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein . These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA . The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.

Am J Physiol Gastrointest Liver Physiol, 2003 Sep, 285(3), G602 - 10
Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells; Li T et al.; The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6 . Enhanced accumulation of F-MTX at 4 degrees C suggests the existence of an active efflux system . MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the accumulation of F-MTX . Transcellular transport of F-MTX from the apical to the basolateral compartment was 2.5 times higher than the opposite direction . This vectorial transport was also reduced by MK-571, indicating the presence of Mrp-type transporter(s) on the basolateral membrane . Mrp3 mRNA was readily detectable, and the protein was localized on the basolateral membrane . Uptake of FMTX into membrane vesicles from IEC-6 cells and Spodoptera frugiperda-9 cells expressing rat Mrp3 were both ATP dependent and saturable as a function of the F-MTX concentration . Similar Km values (11.0 +/- 1.8 and 4.5 +/- 1.1 microM) and inhibition profiles by MK-571, estradiol-17beta-d-glucuronide, and taurocholate for the ATP-dependent transport of F-MTX into these vesicles were obtained . These findings suggest that the efflux of F-MTX is mediated by Mrp3 on the basolateral membrane of IEC-6 cells.

Clin Lab, 2003, 49(7-8), 357 - 65
The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay as rapid colorimetric method for determination of antibiotic susceptibility of clinical Mycobacterium tuberculosis isolates in liquid medium; De Logu A et al.; We investigated the usefulness of a colorimetric method based on the reduction of a tetrazolium salt (XTT) for the susceptibility testing of clinical isolates of Mycobacterium tuberculosis to isoniazid, rifampin, rifabutin, ethambutol hydrochloride, ethionamide and streptomycin . The isolates and the ATCC reference strains reported as susceptible according to the agar dilution method approved by the National Committee for Clinical Laboratory Standards were found to be susceptible by the XTT colorimetric assay after times of incubation ranging between three days for rifampin and rifabutin to eight days for isoniazid . In comparison with other colorimetric methods reviewed in this article, the proposed assay is suitable for determining the susceptibility or resistance to most antituberculous drugs and, as a consequence of the water-solubility of the formazan yielded by reduction of XTT, additional steps such as the addition of extraction buffer and further incubation before the spectrophotometric analysis are not needed . The XTT reduction assay is an inexpensive, rapid and reliable screening method for the detection of susceptible, resistant and multidrug-resistant strains of M . tuberculosis and is an alternative to the costly performance of molecular or radiometric methods.

Cancer Res, 2003 Aug 1, 63(15), 4527 - 32
Transcription factor c-Jun activation represses mdr-1 gene expression; Miao ZH et al.; Expression of mdr-1 is complex and highly regulated . Several lines of evidence indirectly suggest that transcription factor c-Jun may negatively regulate human mdr-1 gene expression . We recently found that salvicine, a novel topoisomerase II inhibitor, is cytotoxic for multidrug resistance (MDR) tumor cells and down-regulates mdr-1 expression in MDR K562/A02 cells . Salvicine also stimulates a significant increase in the level of c-jun mRNA in HL60 cells . This study investigated the relationship between c-Jun activation and down-regulation of mdr-1 expression by salvicine in K562/A02 cells . Reverse-transcription PCR and Western blotting analyses revealed that salvicine suppressed mdr-1 expression in MDR cells and promoted c-jun expression in both MDR and parental K562 cells . Moreover, levels of c-jun expression were enhanced by salvicine before reduction of mdr-1 expression in K562/A02 cells . Furthermore, c-jun antisense oligodeoxynucleotides prevented salvicine-stimulated enhancement of c-Jun protein and reduction of mdr-1 gene expression, but did not affect the increase in c-jun mRNA levels . Salvicine promoted phosphorylation of c-Jun-N-terminal kinase and c-Jun protein in MDR K562/A02 and parental K562 cells . Electrophoretic mobility shift assay analysis showed that salvicine enhanced DNA binding activity of transcription factor activator protein 1 . Additionally, c-jun antisense oligodeoxynucleotides also inhibited salvicine-induced apoptosis and cytotoxicity in MDR and parental K562 cells . A possible pathway emerges from these results: salvicine stimulates c-Jun-N-terminal kinase phosphorylation and activation, resulting in c-Jun phosphorylation and activation . Activated c-Jun promotes expression of c-jun itself, represses mdr-1 transcription, and triggers pro-apoptotic signals, resulting in low mdr-1 expression and cell death . The present results demonstrate that transcription factor c-Jun plays a principal role in down-regulation of mdr-1 expression and induction of apoptosis in salvicine-treated human MDR K562/A02 cells, providing new insights into the complicated mechanisms regulating mdr-1 expression . The findings also suggest that c-Jun might be a potential drug target for circumventing tumor MDR.

Biochem Pharmacol, 2003 Aug 15, 66(4), 613 - 21
Identification of gene expression profiles predicting tumor cell response to L-alanosine; Efferth T et al.; The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion . This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis . L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency . Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway . The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene . Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones . Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine . Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes . None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic . Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes . We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Apr, 24(2), 134 - 9
{Preliminary studies on the mechanisms of a new anti-tumor agent PH II-7 with special preference to multidrug resistant tumor cells}; Tan YH et al.; OBJECTIVE: To determine the anti-tumor activity of PH II-7 in vitro and explore preliminarily its mechanisms . METHODS: The anti-tumor activity was measured using colorimetric MTT assay . Apoptosis was determined with fluorescence-activated cell sorter (FACS), electron microscopy and agarose gel electrophoresis . The expressions of mdr1 and sorcin genes were determined by Northern blot assay . RESULTS: PH II-7 inhibited the proliferation of various human tumor cells derived from different tumor cell lines . The IC50 values varied from 0.34-18.61 mumol/L . Especially, PH II-7 had strong inhibitory effect on multidrug resistant tumor cells, whereas adriamycin (ADR) was resistant . Apoptosis was induced in HL60 and HL60/ADR cells treated with 1 microgram/ml PH II-7, while PH II-7 inhibited the expressions of mdr1 and sorcin genes . CONCLUSIONS: PH II-7 is a new potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells . PH II-7 may increase the intracellular drug concentration in MDR cells by inhibiting the expressions of the MDR-related genes mdr1 and sorcin and induce the apoptosis of MDR cells and their parental tumor cells.

Acta Pharmacol Sin, 2003 Aug, 24(8), 805 - 11
Sensitization and apoptosis augmentation of K562/ADM cells by anti-multidrug resistance gene peptide nucleic acid and antisense oligodeoxyribonucleotide; Wei HL et al.; AIM: To investigate the reversal effect and apoptosis enhancement of peptide nucleic acid (PNA) and antisense oligodeoxyribonucleotide (ASODN) targeted to multidrug resistance gene (mdr1) on human multidrug resistant leukemia K562/ADM cells . METHODS: A 15-mer PNA and the same sequence of ASODN, complementary to the 5' end of the AUG initiator codon-containing region of mdr1 messenger RNA (MDR1-PNA, MDR1-ASODN), were designed and synthesized . Proliferation and sensitivity to adriamycin of K562/ADM cells treated with MDR1-PNA- and MDR1-ASODN were analyzed with a MTT colorimetric assay . Apoptotic morphologies, P-glycoprotein (P-gp) expression, intracellular adriamycin accumulation, and cell cycle were measured . RESULTS: MDR1-PNA 1 to 10 micromol/L and MDR1-ASODN 2 to 20 micromol/L alone had no inhibitory effects on the proliferation of K562/ADM cells, but significantly inhibited the growth of K562/ADM cells cultured in adriamycin-containing medium . After treatment with MDR1-PNA and MDR1-ASODN, intracellular adriamycin accumulation in K562/ADM cells increased greatly and P-gp synthesis was strikingly reduced . The resistance to adriamycin of the drug-resistant cells was partly reversed and the cells were induced to apoptosis by adriamycin . The reversal efficacy of MDR1-PNA was 3.1-fold higher than that of the same sequence of MDR-ASODN, but neither MDR1-PNA nor MDR1-ASODN could completely block the mdr1/P-gp expression . CONCLUSION: Sequence-special PNA targeted to mdr1 gene more effectively than the same sequence of MDR1-ASODN inhibited the expression of P-glycoprotein to overcome the drug-resistance.

Am J Clin Oncol, 2003 Aug, 26(4), e73 - 9
Radiosensitivity in multidrug-resistant and cisplatin-resistant human carcinoma cell lines; Gigante M et al.; The radiosensitivity of a multidrug-resistant (MDR) clone and a cisplatin-resistant clone was compared with that of their parental chemosensitive cell lines . The LoVo cell line was derived from a human colon carcinoma, and LoVo-R was the MDR clone . The MDR phenotype is attributable to an increased drug efflux mediated by the P-glycoprotein and involves several classes of structurally unrelated drugs . The 2008 cell line was derived from a human ovary carcinoma and C13 was the cisplatin-resistant clone . Reduced cisplatin accumulation and elevated plasma membrane potential partially account for the drug resistance of C13 cells . The chemoresistance of LoVo-R and C13 cells was confirmed by cytotoxicity tests consisting of 24-hour paclitaxel and 1-hour cisplatin incubation, respectively . The radiosensitivity was evaluated by a clonogenic test . The dose-reducing cell survival fraction from 1 to 0.37 (D(0)), the quasi-threshold dose (Dq), and the survival fraction (SF) after 2 or 4 Gy were determined for each cell line . D(0), Dq, and SF(2) were 1.3 +/- 0.4 Gy, 2.1 +/- 0.6 Gy, and 43 +/- 4% for the LoVo cell line and 1.0 +/- 0.2 Gy, 1.7 +/- 0.4 Gy, and 45 +/- 8%, respectively, for the LoVo-R cell line . D(0), Dq, and SF(4) were 1.7 +/- 0.3 Gy, 3.1 +/- 0.4 Gy, and 43 +/- 12% for 2008 cells and 2.6 +/- 0.5 Gy, 4.3 +/- 0.6 Gy, and 53 +/- 11%, respectively for C13 cells . No significant differences were found between LoVo and LoVo-R cells, whereas C13 cells showed a significantly greater D(0,) Dq, and SF(4) than 2008 cells (p <0.05) . Incubation of 2008 and C13 cells with subcytotoxic buthionine (BSO) before and after irradiation partially restored C13 radiosensitivity . In fact, D(0) dropped from 2.8 +/- 0.1 to 2.0 +/- 0.3 Gy in C13 cells with and without BSO, whereas it was 1.9 +/- 0.2 Gy in 2008 cells in the absence and presence of BSO . The total glutathione content (GSH) of C13 cells was 1.5-fold higher than that of 2008 cells . BSO treatment caused a partial depletion of GSH in 2008 and C13 cells, but their radiosensitivity did not change accordingly.

J Nucl Med, 2003 Aug, 44(8), 1330 - 9
Biodistribution, radiation dose estimates, and in vivo Pgp modulation studies of 18F-paclitaxel in nonhuman primates; Kurdziel KA et al.; Multidrug resistance (MDR) associated with increased expression and function of the P-glycoprotein (Pgp) efflux pump often causes chemotherapeutic failure in cancer . To provide insight into both the dynamics of the pump and the effects of MDR, we radiolabeled paclitaxel, a substrate for the Pgp pump, with (18)F to study MDR in vivo with PET . We obtained biodistribution and radiation dose estimates for (18)F-paclitaxel (FPAC) in monkeys and studied the effects of a Pgp blocker (XR9576, tariquidar) on FPAC kinetics . METHODS: Paired baseline and Pgp modulation (2 mg/kg XR9576) 4-h whole-body dynamic PET scans were obtained in 3 rhesus monkeys after injection of FPAC . Measured residence times were extrapolated to humans and radiation dose estimates were obtained using MIRDOSE3.1 . The postmodulator area under the time-activity curves (AUCs) and Logan plot slopes, a measure of tracer distribution volume (equilibrium tissue-to-plasma ratio) that is inversely proportional to tracer efflux, were compared with baseline values to determine changes in FPAC distribution . RESULTS: Cumulative activities of the organs sampled accounted for 80% of the injected dose . The critical organ is gallbladder wall (0.19 mGy/MBq {0.69 rad/mCi}), followed by liver (0.14 mGy/MBq {0.52 rad/mCi}); the effective dose is 0.022 mSv/MBq (0.083 rem/mCi) . XR9576 preinfusion changed the Logan plot slope for liver by +104% (P = 0.02), lung by +87% (P = 0.11), and kidney by -14% (P = 0.08) . Changes in the mean AUC (normalized to the plasma AUC) were +54% (P = 0.08), +97% (P = 0.04), and -12% (P = 0.02), respectively, for liver, lung, and kidney . No significant difference was found in the metabolite-corrected plasma AUC (normalized to the injected dose) between the baseline and XR9576 modulator studies (P = 0.69) . CONCLUSION: Under Radioactive Drug Research Committee guidelines, 266 MBq (7.2 mCi) FPAC can be administered to humans up to 3 times a year . The increase in FPAC accumulation in liver and lung after XR9576 is consistent with Pgp inhibition and demonstrates the potential of FPAC to evaluate MDR.

Pharmacol Res, 2003 Oct, 48(4), 347 - 59
P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement; Varma MV et al.; Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to possess functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route . Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux . Broad substrate recognition by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors . In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest . Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination . Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors . In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.

Nucl Med Biol, 2003 Aug, 30(6), 627 - 32
To predict response chemotherapy using technetium-99m tetrofosmin chest images in patients with untreated small cell lung cancer and compare with p-glycoprotein, multidrug resistance related protein-1, and lung resistance-related protein expression; Kuo TH et al.; Our preliminary studies found technetium-99m tetrofosmin (Tc- TF) chest imaging was related to Pgp or MRP1 expression and successfully predict chemotherapy response and in SCLC in human . However, there was no published literature to study relationship of Tc-TF chest images and LRP expression in SCLC patients . Therefore, the aim of this study was to investigate the relationships among Tc- TF accumulation in untreated small cell lung cancer (SCLC), the expression of P-glycoprotein (Pgp), multidrug resistance related protein-1 (MRP1), and lung resistance-related protein (LRP), as well as the response to chemotherapy in patients with untreated SCLC . Thirty patients with SCLC were studied with chest images 15 to 30 minutes after intravenous injection of Tc-TF before chemotherapeutic induction . Tumor-to-background (T/B) ratios were obtained on the static and plantar Tc-TF chest images . The response to chemotherapy was evaluated upon completion of chemotherapy by clinical and radiological methods . These patients were separated into 15 patients with good response and 15 patients with poor response . No significant differences of prognostic factors (Karnofsky performance status, tumor size, or tumor stage) were found between the patients with good and poor responses . Immunohistochemical analyses were performed on multiple nonconsecutive sections of biopsy specimens to detect Pgp, MRP1, and LRP expression . The difference in T/B ratios on the Tc-TF chest images of the patients with good versus poor response was significant . The differences in T/B ratios of the patients with positive versus negative Pgp expression and with positive versus negative MRP1 expression were significant . The difference in T/B ratios of the patients with positive versus negative LRP expression was not significant . We concluded that Tc-TF chest images could accurately predict chemotherapy response of patients with SCLC . In addition, The Tc-TF tumor uptake was related to Pgp or MRP1 but not LPR expression in SCLC.

Trends Mol Med, 2003 Jul, 9(7), 307 - 12
Targeting cancer cells by exploiting their resistance; Blagosklonny MV; Cancer cells are intrinsically resistant to growth arrest and can further acquire multidrug resistance . Current approaches to this problem are intended to reverse, overcome or prevent the drug resistance . However, the resistance of cancer cells can be exploited to kill resistant cells selectively, while sparing sensitive normal cells . As the simplest example, multidrug-resistant cells pump out protectors, such as pharmacological inhibitors of apoptosis . A sequence of at least two agents must include an exclusive protector, which is ineffective in resistant cancer cells, and an inclusive cytotoxic drug, which kills unprotected cells . By abolishing several dose-limiting side effects of chemotherapy, this strategy provides a means to treat selectively most deranged, aggressive and resistant cancers.

Probl Tuberk Bolezn Legk, 2003, (5), 14 - 6
{Therapy strategy in the treatment of patients with drug-resistant pulmonary tuberculosis}; Ivanova LA et al.; The article contains a clinical- X-ray- and laboratory description of 56 patients with tuberculosis (disease length ranging from 2 to 5 years), who, during the previous treatment stage, received a therapy in compliance with the generally accepted methods and without any effect . All patients discharged bacteria . Drug-resistance to 3 and more preparations was registered in all patients . A complex approach to the therapy of patients with destructive pulmonary tuberculosis is suggested, within the study, with regard for the presence of multidrug-resistance of Mycobacterium tuberculosis (MBT), for a degree of inactivation of isoniazid and for a degree of bacterial-static activity of patient's blood, which provides for a higher treatment efficiency.

Probl Tuberk Bolezn Legk, 2003, (5), 9 - 14
{Experience with using anti-TB preparations (rifabutin)}; Borisov SE et al.; The study deals with the first experience of practically using a computer program designed to monitor the treatment process with a subsequent analysis of the efficiency of an anti-TB therapy with regard for different characteristics of patients . The efficiency of chemotherapy was compared (by using the clinical, microbiological and laboratory criteria) in two patients' groups, i.e . a control group (standard chemotherapy regimens) and a group, whose patients received rifabutin . Subgroups were isolated from among the last mentioned patients according to the below factors: primary and secondary therapy courses, completed and interrupted chemotherapy courses, and patients with multidrug resistance who discharged M . tuberculosis . Statistically reliable advantages of rifabutin were shown in respect to the arrest of bacterial discharge during a sufficiently prolonged (at leas 4 months) treatment course applicable to primarily diagnosed patients including those with multidrug resistance and M . tuberculosis . Disadvantages related with the application of rifabutin (a lack of clear-cut indications and abuse of treatment terms virtually in 50% of cases), which reduces the efficiency of its prescription, were equally detected.

Parasitol Res, 2003 Sep, 91(1), 79 - 85 Epub 2003 Jul 29.
Detection of P-glycoprotein-mediated multidrug resistance against anthelmintics in Haemonchus contortus using anti-human mdr1 monoclonal antibodies; Kerboeuf D et al.; The "multidrug resistance" (MDR) system involves the action of transmembrane P-glycoproteins (Pgp) which may be responsible for failure of chemotherapy in both invertebrates and vertebrates . We previously obtained partial reversion of anthelmintic resistance in nematodes subjected to both anthelmintics and inhibitors of this system . The results presented here are able to describe more accurately the presence of Pgp in nematodes because of the use of C219 and UIC2 monoclonal antibodies, which are used for the detection of human and mouse mdr1 gene products . These antibodies demonstrated the presence of Pgp in eggshells . Their role in these structures, which are considered to be passive barriers, remains to be determined . Flow cytometry analyses of the UIC2 staining allowed determination of the resistance of individuals, which varied within the parasite population . UIC2 demonstrated both the presence and activity of Pgp in nematodes as has previously been shown in tumour cells . Resistance seems to be due to an increase in both the number of Pgp sites and parasites with high levels of Pgp.

Can J Physiol Pharmacol, 2003 Aug, 81(8), 800 - 5
Verapamil metabolites: potential P-glycoprotein-mediated multidrug resistance reversal agents; Woodland C et al.; Multidrug resistance in cancer chemotherapy frequently correlates with overexpression of the P-glycoprotein drug transporter . Attempts to reverse P-glycoprotein-mediated multidrug resistance with racemic verapamil or its less toxic (R)-enantiomer have been complicated by cardiotoxicity . The objective of this study was to investigate the effects of the major verapamil metabolite, norverapamil, as well as the PR-22 and D-620 metabolites, on P-glycoprotein-mediated drug transport . We measured the basolateral-to-apical fluxes of the P-glycoprotein substrates digoxin and vinblastine in the presence and absence of verapamil, (R)-norverapamil, (S)-norverapamil, racemic norverapamil, PR-22, or D-620 across confluent monolayers of Madin-Darby canine kidney (MDCK) cells that express P-glycoprotein on their apical membranes . Verapamil and norverapamil nonstereospecifically inhibited the renal tubular secretion of digoxin and vinblastine similarly in a dose-dependent manner . However, there was no decrease in the cellular accumulation of digoxin and vinblastine, suggesting that neither verapamil nor norverapamil prevent the substrates from entering the MDCK cells . Furthermore, the norverapamil metabolite P-22 also inhibited the secretion of these P-glycoprotein substrates . Our results suggest that the verapamil metabolites norverapamil and PR-22, which are less cardiotoxic than the parent compound, have comparable inhibitory abilities to verapamil (norverapamil greater than PR-22) and may be useful in reversing resistance to P-glycoprotein substrates.

Biofactors, 2003, 17(1-4), 103 - 14
Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate; Homolya L et al.; Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space . The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters . A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs) . The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors . The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate . MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH . MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH . MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level . Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting . The human MRP4, MRP5 and MRP6 have only partially been characterized . However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs . MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes . Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown . In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates and GSSG in the cytoplasm, and play, therefore, an essential role in detoxification and defense against oxidative stress.

Zhonghua Fu Chan Ke Za Zhi, 2003 May, 38(5), 294 - 7
{In vivo reversal of multidrug resistance by transduction of human tumor necrosis factor-alpha into drug resistant cell line of choriocarcinoma}; Feng FZ et al.; OBJECTIVE: To investigate in vivo reversal of multidrug resistance and biological properties of a drug resistant cell line of choriocarcinoma transduced by human tumor necrosis factor-alpha (TNF-alpha) gene via the establishment of its animal model . METHODS: Choriocarcinoma cell line JEG-3, drug-resistant choriocarcinoma cell line JEG-3/VP2, and human TNF-alpha-transduced drug-resistant choriocarcinoma cell line JEG-3/VP2/TNF-alpha were injected subcutaneously in the neck of nude mices . Tumor size and weight were routinely measured, tumor histological structure was observed and its chemosensitivity was tested . Expression of multidrug resistance (MDR1) mRNA was investigated using reverse transcription-polymerase chain reaction (RT-PCR) and P-glycoprotein (P-gp) expression was determined by immunohistochemistry with the monoclonal antibody MDR1 . RESULTS: The rate of inoculation for all three tested cell lines was 100%, the latent period was 10 to 14 days . Tumor growth rates and weights were significantly different among three cell lines (P < 0.05), with the lowest in JEG-3/VP2/TNF-alpha cell line . Tumor inhibition rate after treatment with etopside (VP-16) was significantly higher in JEG-3/VP2/TNF-alpha (41.0% - 42.5%) (P < 0.05), compared with JEG-3/VP2 (24.3% - 28%), and similar to JEG-3 (46.7% -47.7%) . Transduction and expression of human TNF-alpha in drug-resistant choriocarcinoma cell line JEG-3/VP2 was found to reverse MDR1 on the mRNA and P-gp levels . CONCLUSION: Transduction and expression of human TNF-alpha in drug-resistant choriocarcinoma cell line JEG-3/VP2 can reverse expressions of MDR1 mRNA and P-gp, enhance the susceptibility of the JEG-3/VP2 to the cytotoxic drugs, and lower its tumorigenesis.

Paediatr Drugs, 2003, 5(8), 533 - 43
Childhood ependymoma: a systematic review of treatment options and strategies; Grill J et al.; Childhood intracranial ependymoma have a dismal prognosis, especially in young children and when a gross total resection cannot be performed . Even in the absence of a radiologically proven residuum, around two-thirds of these young children will have a recurrence . Adjuvant therapy is therefore necessary for most, if not all, patients . Despite some indication that benign ependymoma (WHO grade II) could show a better outcome, histology cannot be used at present to stratify treatment protocols.Craniospinal irradiation combined with posterior fossa boost has deleterious adverse effects on cognition . Consequently, pediatric oncology teams have, firstly, tried to use chemotherapy to delay or avoid irradiation, and secondly, progressively reduced irradiation fields to the tumor bed without altering the prognosis . Cisplatin, at a dose of 120 mg/m(2) (cumulated response rate of 34% {95% CI 19-54%}) is the only single agent that has reproducibly shown some efficacy in ependymoma . Despite some combinations showing efficacy in the adjuvant setting, childhood intracranial ependymomas can, in general, be considered as chemoresistant . The overexpression of the multidrug resistance-1 gene and the 06-methylguanine-DNA methyltransferase have been implicated as possible mechanisms for this phenomenon . As the use of chemotherapy with current agents is questionable, phase II studies with new agents and combinations are necessary.Since the main problem of this disease is local relapse, it may not be necessary to irradiate the whole posterior fossa . However, local control of the disease by irradiation has to be improved . In this respect, hyperfractionation or radiosensitizers may be valuable therapeutic options.The treatment of children with ependymoma is a challenge for all caregivers . There is no doubt that any possible improvement in the management of this rare tumor will only be the result of well designed cooperative trials.

Anticancer Res, 2003 May-Jun, 23(3B), 2735 - 40
MDR1 single nucleotide polymorphism C3435T in normal colorectal tissue and colorectal carcinomas detected by MALDI-TOF mass spectrometry; Humeny A et al.; Single nucleotide polymorphisms (SNPs) may contribute to the malignant process and may show clinicopathological importance as prognostic markers . The multidrug resistance gene MDR1 encodes a membrane transporter which confers cytostatic drug resistance in tumors and protects normal tissues from xenobiotics . We analyzed the C3435T SNP in the MDR1 gene which is associated with altered cellular drug uptake in matched tumor and normal tissues of 45 patients suffering from colorectal carcinoma . We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method to survey the C3435T polymorphism in PCR-amplified fragments of the MDR1 gene . Thirteen patients were homozygous for C/C (29%), 15 were heterozygous (33%) and 17 were homozygous for T/T (38%) . None of the tumor samples showed an altered SNP compared to their matched normal tissue samples . As analyzed by the Kruskall-Wallis test, none of the clinicopathological parameters was significantly associated with homo- or heterozygosity . The combination of PCR, allele-specific primer extension reactions and MALDI-TOF-MS offers a promising alternative method for genotyping the MDR1 gene especially for heterozygous situations . The inherent advantages of MALDI-TOF-MS based genotyping include its high molecular resolution, high signal-to-noise-ratios and reproducibility, combined with an excellent sensitivity . As none of the tumor samples showed an altered state compared to their matched normal tissue samples, the genotypic frequency of this polymorphism seems not to be altered during colorectal tumorigenesis.

Anticancer Res, 2003 May-Jun, 23(3B), 2681 - 90
Effect of MDR1 phosphorothioate antisense oligodeoxynucleotides in multidrug-resistant human tumor cell lines and xenografts; Ramachandran C et al.; The effect of MDR1 antisense phosphorothiate oligodeoxynucleotides (S-ODNs) on resistant phenotype was investigated in multidrug-resistant human colon carcinoma and breast carcinoma cells in vitro and in vivo . Drug resistance in human colon carcinoma (SW620 Ad300) and breast carcinoma (MCF-7/INT500, MCF-7/AD150 and MCF-7/TH) cell lines is predominantly due to overexpression of P-glycoprotein (P-gp) resulting in decreased daunorubicin (DNR) accumulation . Two MDR1 antisense S-ODNs, one complementary to the initial 15 bases of first exon (S-ODN I) and the other a loop forming sequence (S-ODN II) complementary to bases from 993-1007 of MDR1 gene, were tested for enhancing the doxorubicin (DOX) cytotoxicity in vitro and the efficiency of chemotherapy in human tumor xenografts . MDR1 antisense S-ODN I reduced the DOX IC50 value 9-fold in multidrug-resistant SW620 Ad300 human colon carcinoma cells and 7 to 10-fold in breast carcinoma cells in vitro . The increase in DOX cytotoxicity correlated with a significant reduction of MDR1 mRNA in antisense S-ODN I-treated SW620 Ad300 cells . Even though the P-gp level was reduced at the end of the third day in antisense S-ODN I-treated cells, the rate of reduction was only partial compared to mRNA . The combination treatment of MDR1 antisense S-ODN I or II for three days and DOX for four days significantly controlled tumor growth rate in human tumors developed in nude mice . Our results suggest that MDR1 antisense S-ODN treatment can increase the efficiency of chemotherapy by suppressing gene expression and resistant phenotype.

Anticancer Res, 2003 May-Jun, 23(3B), 2657 - 64
Chemo- and radiation sensitivity of xenografted acute lymphoblastic leukemias--correlation to the expression of multidrug resistance proteins; Fichtner I et al.; The aim of our study was to characterise, for the first time, the chemo- and radiation sensitivity of seven pediatric acute lymphoblastic leukemias xenotransplanted into immunodeficient NOD/SCID mice and to correlate the findings with the expression of three drug resistance proteins, P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and lung resistance protein (LRP) . Mice were treated with single drugs used in clinical protocols: daunorubicin, doxorubicin, cyclophosphamide, vincristine, cytarabine, asparaginase and methotrexate . Two ALL samples, established from primarily diagnosed patients, responded to 5 or 6 of the tested cytostatics, respectively, while 3 out of 5 ALLs from relapse patients were only sensitive towards 2-4 drugs tested . Daunorubicin was more efficient than doxorubicin . The response of xenografted ALL toward vincristine and cyclophosphamide was inversely correlated with the expression of P-gp, LRP and MRP1 (R2 = 0.71, 0.70 and 0.64 for vincristine and 0.44, 0.70 and 0.60 for cyclophosphamide) . A good correlation could be detected between the expression of P-gp and LRP (R2 = 0.88), P-gp and MRP1 (R2 = 0.75) and LRP and MRP1 (R2 = 0.90) . The highest co-expression of the drug resistance proteins in the leukemia ALL-SCID 6 coincided with a high resistance to radiation and chemotherapy . Prediction of the individual drug resistance profile of a patient on the basis of results from the ALL-SCID xenograft studies was not possible because of the relatively long time necessary and because of the changes in the expression of P-gp, LRP and MRP1 during the murine generations . We conclude that in the drug resistance phenotype of ALL not only the above mentioned proteins but a variety of different molecules are involved.

Anticancer Res, 2003 May-Jun, 23(3B), 2363 - 75
Modulation by the ATP/GTP ratio of the phosphorylation level of P-glycoprotein and of various plasma membrane proteins of KB-V1 multidrug resistant cells; Lelong-Rebel IH et al.; The level of protein phosphorylation is known to affect the properties of various membrane proteins . We have previously shown that GTP is capable of greatly enhancing the phosphorylation by {gamma-32P}ATP of P-glycoprotein (Pgp) from KB-V1 cells (3) . Investigating the possibility of a general modulation of {gamma-32P}ATP plasma membrane protein phosphorylation, we found that phosphorylation of other membrane proteins are also modulated by various combinations of {ATP + GTP} . The ATP/GTP ratio giving the highest phosphorylation level depended on the protein studied . Modulation of the {gamma-32P}ATP-mediated phosphorylation of numerous membrane proteins requires hydrolysis of both ATP and GTP . ADP and GDP also increased {gamma-32P}ATP-driven phosphorylation but to a lesser extent than GTP . This plasma membrane endogenous phosphorylation activity was neither inhibited by specific inhibitors of protein kinase C, nor by inhibitors of cAMP- or cGMP-dependent protein kinases or of casein kinase II, respectively . Mastoparan, a G-protein regulator, increased the phosphorylation of some proteins that were already enhanced by the presence of {ATP + GTP} mixtures, especially proteins migrating in gels at the same position as P-glycoprotein.

Anticancer Res, 2003 May-Jun, 23(3B), 2339 - 48
Expression profiling of tumor necrosis factor alpha-induced apoptosis-associated genes in human solid tumor cell lines; Yanai Y et al.; BACKGROUND: The induction of genes associated with cellular apoptosis by tumor necrosis factor-alpha (TNF-alpha) in human cancer cell line sof various tissue origins may characterize TNF-alpha responder cell lines/cancers . MATERIALS AND METHODS: Using quantitative real-time polymerase chain reaction (PCR), the comprehensive molecular profiling of genes downstream of the TNF-alpha receptor genes in 91 well-defined human cancer cell lines allowed us to elucidate relationships between TNF-alpha response and the genetic expression profiles of the target cell lines . RESULTS: Among the 52 genes tested, the above average expression of Akt mRNA showed significant correlation with TNF-alpha-induced susceptibility to apoptosis . In addition, multidrug resistance protein 5 (MRP5) and tumor necrosis factor receptor type 1 (TNFR1) mRNA expressions also appear to be possible markers for responsiveness to TNF-alpha . CONCLUSION: These results provide a preliminary basis for the screening for genetic markers that may help to predict a favorable therapeutic outcome, and also to identify patients who may benefit from cytokine therapy.

Pharmacogenetics, 2003 Aug, 13(8), 481 - 94
Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene; Kroetz DL et al.; OBJECTIVES: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates . The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein . METHODS AND RESULTS: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples . These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed . The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes . Significant population substructure was detected at both the nucleotide and haplotype level . Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred . The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function . CONCLUSION: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

J Pharmacol Exp Ther, 2003 Oct, 307(1), 314 - 21 Epub 2003 Jul 31.
Metabolism of flavonoids via enteric recycling: mechanistic studies of disposition of apigenin in the Caco-2 cell culture model; Hu M et al.; The purpose of this study was to determine the mechanisms responsible for intestinal disposition of apigenin in the human Caco-2 cell culture model . The results indicated that most of the absorbed apigenin (10 microM) were conjugated and only a small fraction was transported intact . The amounts of conjugates excreted, especially that of the sulfate, were dependent on days-post-seeding . Apical efflux of apigenin sulfate did not change with concentration of apigenin (4 to 40 microM), whereas its basolateral efflux increased (p < 0.01) with concentration and plateaued at about 25 microM . In contrast, sulfate formation rates in cell lysate increased with concentration and plateaued at 25 microM and were 4 to 6 times faster than the corresponding excretion rates . Formation and polarized excretion rates of glucuronidated apigenin increased with apigenin concentration but formation rates were usually 2.5 to 6 times faster than the corresponding excretion rates . Inhibitors of multidrug resistance-related proteins (MRPs) such as leukotriene C4 and MK-571, which inhibited glucuronidation of apigenin at a high concentration (>or=25 microM), significantly decreased excretion of both apigenin conjugates, and higher concentrations of MK-571 increased the extent of inhibition . In contrast, an organic anion transporter (OAT) inhibitor estrone sulfate only inhibited excretion of apigenin sulfate . In conclusion, we have shown for the first time that intestinal efflux is the rate-limiting step in the intestinal excretion of phase II conjugates of flavones . Furthermore, MRP and OAT are involved in the intestinal efflux of these hydrophilic phase II conjugates.

J Pharmacol Exp Ther, 2003 Oct, 307(1), 282 - 90 Epub 2003 Jul 31.
Functional expression of the multidrug resistance protein 1 in microglia; Dallas S et al.; Brain expression of the multidrug resistance proteins (MRPs), a collection of membrane-associated ATP-dependent efflux transporters, is poorly understood . Although several studies have examined the expression of these proteins within the brain barriers (i.e., the blood-brain barrier and choroid plexus), little information is available with respect to brain parenchyma cells such as microglia and astrocytes . Because microglia are the primary brain cells infected by the human immunodeficiency virus type 1 (HIV-1), MRP1 expression within microglia may contribute to lower brain accumulation of anti-HIV drugs . To examine the expression pattern of MRP1 within microglia, we performed reverse transcriptase-polymerase chain reaction analysis and Western blotting on a rat brain microglia cell line MLS-9, and in primary cultures of rat microglia . Both rat MRP1 (rMPR1) mRNA and protein were expressed in the cell line, as well as the primary cultures . We then characterized rMRP1-mediated transport properties in MLS-9 cells using {3H}vincristine, a known MRP1 substrate . Vincristine accumulation by monolayers of MLS-9 cells increased significantly in the presence of several well established MRP1 inhibitors (MK571, genistein, sulfinpyrazone, probenecid, and indomethacin), protease inhibitors, or the ATPase inhibitor sodium azide . In addition, vincristine accumulation was significantly modulated by altering the intracellular concentration of the reduced form of glutathione, further suggesting the involvement of rMRP1-mediated transport . These results provide strong evidence that the MRP1 protein is both expressed and functional in microglia cells . They also suggest that brain parenchyma can act as a "second" barrier to drug permeability and regulate brain distribution/accumulation of various xenobiotics, including protease inhibitors.

J Pharmacol Exp Ther, 2003 Oct, 307(1), 306 - 13 Epub 2003 Jul 31.
Biliary secretion of glutathione in estradiol 17beta-D-glucuronide-induced cholestasis; Mottino AD et al.; Estradiol-17beta-D-glucuronide (E2-17G) induces an acute but reversible inhibition of bile flow after its intravenous administration to rats, due in part to the endocytic retrieval of the canalicular multidrug resistance-associated transporter protein 2 and the bile salt export pump, transporters that contribute to bile flow . Decreased bile salt-independent bile flow (BSIF) is also involved and persists during the phase of recovery from cholestasis . Because glutathione and HCO3- are major contributors to BSIF, we evaluated changes in their biliary excretion and the hepatic content of total glutathione during E2-17G-induced cholestasis . E2-17G acutely decreased bile flow and biliary excretion of total glutathione by about 80%; glutathione excretion was still inhibited at 80 min and 120 min, even though bile flow was partially and totally restored, respectively . Neither liver glutathione content nor the proportions of oxidized glutathione in bile and liver were affected by E2-17G at any time . HCO3- concentrations in bile were unchanged, so that secretion paralleled variations in bile flow . In the isolated perfused liver, addition of E2-17G decreased both bile flow and the biliary concentration of glutathione, whereas addition of its noncholestatic isomer estradiol-3-D-glucuronide (E2-3G) did not inhibit bile flow, but significantly reduced the concentration of glutathione in bile . The bile:liver concentration ratios of glutathione were significantly decreased in vivo by E2-17G and in the perfused liver by E2-17G and E2-3G . These data indicate that E2-17G cis-inhibits the canalicular transport of glutathione and thus contributes to the cholestatic effect by inhibiting BSIF.

Am J Physiol Gastrointest Liver Physiol, 2003 Dec, 285(6), G1335 - 44 Epub 2003 Jul 31.
Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells; Lou H et al.; Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates . Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions . Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH . Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2 . Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid . Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL . Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.

Cancer Lett, 2003 Jul 30, 198(1), 21 - 7
Effect of PSC 833, a potent inhibitor of P-glycoprotein, on the growth of astrocytoma cells in vitro; Sadanand V et al.; Malignant astrocytomas have been found to express P-glycoprotein (Pgp, mdr1 gene product) . It was hypothesized that in addition to conferring multidrug resistance, Pgp is intimately associated with the development of astrocytomas . Accordingly, we studied the effect of PSC 833 (PSC, Novartis), a potent inhibitor of Pgp, on the growth of Pgp-expressing astrocytoma cells . The results showed that in all the cell lines tested, PSC (10-60 microM) inhibited the growth as well as induced cell death . Cells exposed to PSC exhibited DNA ladder characteristic of apoptosis . PSC-induced cell death could be reversed by Z-VAD-fmk, a general caspase inhibitor, indicating that PSC-induced cell death was characteristic of caspase-mediated apoptosis . These results suggest a novel therapeutic strategy in the treatment of malignant astrocytomas by inhibitors of Pgp.

Acta Pharm Hung, 2003, 73(1), 29 - 39
{The multidrug resistance modulators heterocondensed quinazolones}; Kokosi J et al.; Exploration for new MDR-modulators utilizing pyrazino{2,1-b}quinazolones as scaffolds disclosed after systematic synthetic investigation highly hydrophobic N-substituted derivatives as a readily accessible active tricyclic compounds . A versatile synthesis of 2-substituted-1,2,3,4-tetrahydro-6H-pyrazino{2,1-b}quinazoline-3,6-diones is presented starting from 2,3-substituted quinazolines . The new compounds have been characterized by elemental analyses, 1H nmr and in some cases by 13C ruler, and X-ray investigations.

Eur J Haematol, 2003 Aug, 71(2), 119 - 23
Quantitative determination of the human MRP1 and MRP2 mRNA expression in FACS-sorted peripheral blood CD4+, CD8+, CD19+, and CD56+ cells; Oselin K et al.; OBJECTIVES: ATP-binding cassette (ABC) transporters extrude a wide variety of endogenous and exogenous compounds . In cancer cells, they are known to confer multidrug resistance . The aim of the present study was to determine the expression of the multidrug resistance-associated protein 1 (MRP1) and 2 (MRP2), which are members of the subfamily C of the ABC transporters family, in human hematopoietic cells . METHODS: CD4+, CD8+, CD19+, and CD56+ cells were isolated from whole blood by FACS-sort in 20 healthy volunteers . MRP1 and MRP2 mRNA levels were quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays on a LightCycler (Roche, Mannheim, Germany) . RESULTS: The MRP1 mRNA exhibited the highest abundance in CD4+ cells (7.4 x 10(3)+/-3.19 x 10(3) molecules/ng of total RNA), followed by CD8+ > CD19+ > CD56+ cells . The MRP2 mRNA expression was highest in CD4+ cells (6.7 x 10(2)+/-2.84 x 10(2)), followed by CD8+ > CD56+ > CD19+ cells . No correlation between the MRP1 and MRP2 mRNA expression was observed . Interestingly, beta2-microglobulin mRNA expression in CD19+ cells was found to be twofold lower in comparison with other cells . CONCLUSIONS: On an mRNA level both MRP1 and MRP2 were expressed in peripheral blood cells, with more than sevenfold higher MRP1 expression in all cell populations investigated . The impact of the MRP1 and MRP2 transcription in these cells remains to study . The use of beta2-microglobulin as a housekeeping gene could have a critical impact on the interpretation of RT-PCR data.

Neuropsychopharmacology, 2003 Nov, 28(11), 1991 - 9
ABCB1 (MDR1)-type P-glycoproteins at the blood-brain barrier modulate the activity of the hypothalamic-pituitary-adrenocortical system: implications for affective disorder; Muller MB et al.; Multidrug-resistance gene 1-type P-glycoproteins (ABCB1-type P-gps) protect the brain against the accumulation of many toxic xenobiotics and drugs . We recently could show that the access of the endogenous glucocorticoids corticosterone and cortisol to the brain are regulated by ABCB1-type P-gps in vivo . ABCB1-type P-gp function, therefore, is likely to exert a profound influence on the regulation of the hypothalamic-pituitary-adrenocortical (HPA) system . Hyperactivity of the HPA system is frequently observed in human affective disorder, and a considerable amount of evidence has been accumulated suggesting that normalization of the HPA system might be the final step necessary for stable remission of the disease . To examine whether blood-brain barrier (BBB) function influences neuroendocrine regulation, we investigated HPA system activity in abcb1ab (-/-) mice under basal conditions and following stress . Abcb1ab (-/-) mice showed consistently lower plasma ACTH levels and lower evening plasma corticosterone levels . CRH mRNA expression in the hypothalamic paraventricular nucleus was decreased and pituitary POMC mRNA expressing cells were significantly reduced in number in abcb1ab (-/-) mutants; however, they showed a normal activation of the HPA system following CRH stimulation . Lower doses of dexamethasone were required to suppress plasma corticosterone levels in mutants . Our data thus provide evidence for a sustained suppression of the HPA system at the hypothalamic level in abcb1ab (-/-) mice, suggesting that BBB function significantly regulates HPA system activity . Whether naturally occurring polymorphisms in the human ABCB1 gene might result in persistent changes in the responsiveness and regulation of the HPA system will be the subject of future investigations, correlating both genetic information with individual characteristics of the neuroendocrine phenotype.

Leukemia, 2003 Aug, 17(8), 1544 - 50
Low expression of the myeloid differentiation antigen CD65s, a feature of poorly differentiated AML in older adults: study of 711 patients enrolled in ECOG trials; Paietta E et al.; CD65s appears when the progenitor antigen CD34 disappears, suggesting that this sialylated carbohydrate antigen marks a turning point in normal myeloid differentiation . We characterized acute myeloid leukemia (AML) with low CD65s expression (CD65s(low) AML) in 711 patients entered on seven Eastern Cooperative Oncology Group AML treatment trials (1986-1999) . Of those, 198 (28%) qualified as having CD65s(low) AML . Morphologically, CD65s(low) AML was more common in FAB subgroups with minimal differentiation, M0/M1 (P=<0.0001) . Early precursor antigens CD34, CD117 and terminal transferase were more frequent in CD65s(low) than CD65s(high) AML (P=<0.0001) . Myeloperoxidase was present in fewer CD65s(low) myeloblasts, and the more mature myeloid antigens, CD15 and CD11b, were rarely detected (P=<0.0001) . Yet, the two diagnoses did not differ in the distribution of cytogenetic prognostic groups or the occurrence of the multidrug-resistance mediator, P-glycoprotein . CD65s(low) AML patients were significantly older than CD65s(high) cases (P<0.0001) . Furthermore, the incidence of CD65s(low) cases increased with age, from 20% in patients under the age of 50 years to 67% in patients older than 80 years (P<0.0001) . Overall, complete remission (CR) rate and overall survival were comparable in CD65s(low) and CD65s(high) AML . However, among patients >55 years of age, CD65s(low) AML had a decreased CR rate of 33 vs 44% in CD65s(high) AML (P=0.055) . Thus, CD65s(low) AML represents immunophenotypically undifferentiated disease and occurs predominantly in older adults . Although not statistically significant, the observed association between low CD65s expression and decreased CR rate only in patients over the age of 55 is intriguing.

FEBS Lett, 2003 Jul 31, 548(1-3), 28 - 32
Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines; Dijkhuis AJ et al.; The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established . SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells . These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein . SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides . In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function.

J Hum Genet, 2003, 48(8), 425 - 9 Epub 2003 Jul 22.
Identification of a novel 2026G-->C mutation of the MRP2 gene in a Japanese patient with Dubin-Johnson syndrome; Wakusawa S et al.; Dubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes . A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members . In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found . In the third patient, a -24C-->T polymorphism and the two mutations c.1901del67 and 2026G-->C were detected . The 2026G-->C mutation was a novel mutation in exon 16 affecting the conversion of Gly(676) to Arg(676) (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers . The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein . It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case.

Oncol Rep, 2003 Sep-Oct, 10(5), 1593 - 9
Induction chemotherapy for bone sarcoma in adults: correlation of results with erbB-4 expression; Merimsky O et al.; Tumor response to preoperative chemotherapy is an important prognostic factor for localized, operable extremity osteosarcoma . Other clinical variables include tumor size and location, age and sex, and serum enzymes . Advances in molecular oncology yielded a second group of factors such as multidrug resistance status, loss of heterozygosity of RB gene, and HER2/erbB-2 expression . The aim of this study was to investigate the expression and the prognostic value of the newly described erbB-4 receptor in specimens from adults with bone sarcomas treated by pre- and postoperative chemotherapy . Thirty-three patients with non-metastatic bone sarcoma have been treated by two doxorubicin-based induction chemotherapy regimen, followed by limb sparing surgery and tailored adjuvant chemotherapy . Pre-chemotherapy tissue specimens were investigated for the expression of erbB-4 receptor and post-induction specimens were assessed for pathological response . The clinical response rates were 32-36% . The degree of induced necrosis was correlated with the disease-free survival (DFS) . Patients achieving >/=90% necrosis had an improved DFS over patients with poor histological response . ErbB-4 expression was significantly associated with poor histologic response and shorter DFS . ErbB-4 expression may be used for prognostication of adults with bone sarcomas.

Hepatology, 2003 Aug, 38(2), 374 - 84
Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane; Rius M et al.; The liver is the major source of reduced glutathione (GSH) in blood plasma . The transport protein mediating the efflux of GSH across the basolateral membrane of human hepatocytes has not been identified so far . In this study we have localized the multidrug resistance protein 4 (MRP4; ABCC4) to the basolateral membrane of human, rat, and mouse hepatocytes and human hepatoma HepG2 cells . Recombinant human MRP4, expressed in V79 hamster fibroblasts and studied in membrane vesicles, mediated ATP-dependent cotransport of GSH or S-methyl-glutathione together with cholyltaurine, cholylglycine, or cholate . Several monoanionic bile salts and the quinoline derivative MK571 were potent inhibitors of this unidirectional transport . The K(m) values were 2.7 mmol/L for GSH and 1.2 mmol/L for the nonreducing S-methyl-glutathione in the presence of 5 micromol/L cholyltaurine, and 3.8 micromol/L for cholyltaurine in the presence of 5 mmol/L S-methyl-glutathione . Transport of bile salts by MRP4 was negligible in the absence of ATP or without S-methyl-glutathione . These findings identify a novel pathway for the efflux of GSH across the basolateral hepatocyte membrane into blood where it may serve as an antioxidant and as a source of cysteine for other organs . Moreover, MRP4-mediated bile salt transport across the basolateral membrane may function as an overflow pathway during impaired bile salt secretion across the canalicular membrane into bile . In conclusion, MRP4 can mediate the efflux of GSH from hepatocytes into blood by cotransport with monoanionic bile salts.

Mol Cancer Ther, 2003 Jul, 2(7), 597 - 605
Mutations in alpha- and beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine; Hari M et al.; Single-step selections were used to obtain Chinese hamster ovary cell lines resistant to Colcemid and vinblastine . Verapamil was included in the selections to circumvent the isolation of cells with P-glycoprotein-mediated multidrug resistance and thereby enrich for cells with tubulin alterations . The isolated cell lines were 2-fold resistant to the selecting drug, exhibited cross-resistance to other drugs that inhibit microtubule assembly, and had enhanced sensitivity to the microtubule-stabilizing drug paclitaxel . The concomitant resistance to microtubule-destabilizing drugs and enhanced sensitivity to paclitaxel suggested that these cell lines have changes in microtubule assembly . Consistent with this interpretation, drug-resistant cell lines exhibited altered alpha- or beta-tubulin mobility on two-dimensional gels and higher levels (47-54%) of assembled tubulin compared with wild-type (39%) or paclitaxel-resistant cells (25%) . Some drug-resistant cells also had bundled microtubules as judged by immunofluorescence . Genomic sequencing of 11 drug-resistant cell lines predicted five different alterations (D45Y, C211F, D224N, S234N, and K350N) in beta-tubulin and four different alterations (H283Y, E55K, A383V, and R390C) in alpha-tubulin . The amino acid substitutions are dispersed on the primary and tertiary structures of tubulin and, together with the other mutant properties, argue against a mechanism involving changes in drug binding . Rather, we propose that the alterations in alpha- and beta-tubulin increase microtubule stability by promoting longitudinal interdimer and intradimer interactions and/or lateral interactions between protofilaments . This enhanced stability of microtubules increases their resistance to drugs that inhibit assembly.

J Biol Chem, 2003 Oct 3, 278(40), 38537 - 47 Epub 2003 Jul 27.
Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1); Payen LF et al.; MRP1 belongs to subfamily "C" of the ABC transporter superfamily . The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins . They also differ in their ability to bind and hydrolyze ATP . In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active . Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state . Little is known of the structural basis for these functional differences . One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2 . We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide . An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity . In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect . The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states . In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP . This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide . These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.

Biotechnol Lett, 2003 Feb, 25(4), 291 - 4
Quantification of doxorubicin and validation of reversal effect of tea polyphenols on multidrug resistance in human carcinoma cells; Wei D et al.; HPLC was used to analyze doxorubicin in multidrug-resistance (MDR) human carcinoma cells . This method is novel, simple, sensitive, linear, accurate and precise . The minimal detectable concentration is 0.2 microg ml(-1) . The reversal effects of tea polyphenols on MDR are elucidated by this method . The results indicate that the tea polyphenol, (-)-epigallocatechin gallate, is a potential modulator of MDR.

Prog Nucleic Acid Res Mol Biol, 2003, 73, 251 - 79
Transcriptional control of multidrug resistance in the yeast Saccharomyces; Moye-Rowley WS; A major problem in chemotherapeutic treatment of many pathological conditions including cancer and fungal infections is the development of a multidrug-resistant state in the target cell . Saccharomyces cerevisiae cells can be isolated that have single genetic alterations that cause the resulting mutant strains to become tolerant of a wide range of compounds that would otherwise be toxic . These mutant cells are referred to as having a pleiotropic drug-resistant (Pdr) phenotype . Studies of these Pdr cells have demonstrated that mutations either within genes encoding transcriptional regulators or in their regulatory inputs lead to overexpression of downstream transporter proteins with associated multidrug resistance . This review is aimed at providing a framework for understanding the networks modulating expression of PDR genes in S . cerevisiae.

EMBO J, 2003 Aug 1, 22(15), 3833 - 43
N-terminal transmembrane domain of the SUR controls trafficking and gating of Kir6 channel subunits; Chan KW et al.; The sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP-sensitive potassium channel (K(ATP)) complex . Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is still unknown . All functional ABC proteins contain two transmembrane domains but some, including SUR and MRP1 (multidrug resistance protein 1), contain an extra N-terminal transmembrane domain called TMD0 . The functions of any TMD0s are largely unclear . Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single-oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its trafficking and gating . Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2 . These results underscore the importance of TMD0 in K(ATP) channel function, explaining how specific mutations within this domain result in disease, and suggest how an ABC protein has evolved to regulate a potassium channel.

Cancer Lett, 2003 Jul 18, 197(1-2), 93 - 8
The p53 pathway and its inactivation in neuroblastoma; Tweddle DA et al.; Early studies of p53 in neuroblastoma reported infrequent mutations in tumours and cell lines . Cytoplasmic sequestration was later proposed as an alternative mechanism of inactivation, but many studies have since reported an intact p53 pathway in neuroblastoma cell lines, as detected by nuclear p53 accumulation after DNA damage, intact DNA binding, transcriptional activation of target genes and the induction of apoptosis . In some MYCN amplified cell lines however, an irradiation induced G(1) arrest does not occur, despite the presence of normal p53 . Neuroblastoma usually responds to chemotherapy but frequently relapses, and there is evidence from tumours, and cell lines that p53 inactivation via mutation or MDM2 amplification occurs at relapse and is sometimes associated with multidrug resistance . If p53 inactivation occurs frequently in relapsed tumours it may be appropriate to include p53 independent therapies in the initial management of high-risk neuroblastoma.

J Invest Dermatol, 2003 Aug, 121(2), 390 - 8
Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation; Smith G et al.; There are unpredictable inter-individual differences in response to ultraviolet radiation, used in the treatment of psoriasis and other common skin diseases . It is therefore essential that we attempt to identify phenotypic markers that correlate with individual treatment outcomes . Exposure of human skin to ultraviolet radiation results in the generation of reactive intermediates and oxidative stress . Hepatic drug metabolizing and cytoprotective genes are induced as an adaptive response to xenobiotics and reactive intermediates; as several of these genes are present in skin, we hypothesized that their cutaneous expression and regulation may be implicated in responses to ultraviolet radiation . We used quantitative real-time reverse transcription-polymerase chain reaction to investigate interindividual differences in the cutaneous expression of a variety of drug metabolizing and cytoprotective genes, including cytochrome P450s, glutathione S-transferases and drug transporters, and investigated the regulation of gene expression by ultraviolet radiation and in lesional psoriatic skin . We confirmed significant induction of cyclooxygenase 2 (mean 3.63-fold, range 0.14-22.6, p<0.0001) by ultraviolet radiation and showed more modest (approximately 2-fold) inductions of glutathione peroxidase, and novel inductions of glutathione S-transferase P1 and the drug transporter multidrug resistance associated protein-1 . Glutathione S-transferase P1 (3.74-fold, 1.3-33.1, p<0.0001) and multidrug resistance associated protein-1 (4.06-fold, 1.3-24.8, p<0.0001) were also significantly increased in psoriatic plaque, as were P450 CYP2E1 (3.64-fold, 1-28.9 p<0.0001) and heme oxygenase-1 (10.19-fold, 2.9-49.7, p<0.0001), implying a differential adaptive response to oxidant exposure in lesional psoriatic skin . We found considerable interindividual variation in constitutive gene expression and inducibility, indicating that these genes may be associated with individuality in response to ultraviolet radiation.

J Nat Prod, 2003 Jul, 66(7), 999 - 1001
Dihydrolindbladiones, three new naphthoquinone pigments from a myxomycete Lindbladia tubulina; Misono Y et al.; Three new naphthoquinone pigments, 6,7-dimethoxydihydrolindbladione (1), dihydrolindbladione (2), and 6-methoxydihydrolindbladione (3), have been isolated from a myxomycete Lindbladia tubulina, and their structures were elucidated by spectral data . Compound 3 appreciably exhibited a reversal effect of multidrug resistance . Lindbladione (4), the major pigment of this myxomycete, was also isolated from Cribraria intricata.

J Nat Prod, 2003 Jul, 66(7), 976 - 9
Jatrophane diterpenoids from Euphorbia mongolica as modulators of the multidrug resistance of L5128 mouse lymphoma cells; Hohmann J et al.; The dried aerial parts of Euphorbia mongolica afforded three new acylated polyhydroxy diterpenoids based on the jatrophane framework . The structures were established by means of a combination of 1D and 2D NMR techniques and mass spectrometry as (2S,3S,4R,5R,7S,8R,13S,15R)-5alpha,7beta,8alpha-triacetoxy-3beta-benzoyloxy-15beta-hydroxyjatropha-6(17),11E-diene-9,14-dione (1), (2S,3S,4R,5R,7S,8S,9S13S,15R)-5alpha,7beta,8alpha,9alpha,15beta-pentaacetoxy-3beta-benzoyloxyjatropha-6(17),11E-dien-14-one (2), and (2S,3S,4R,5R,7S,8S,9S13S,15R)-3beta,7beta,8alpha,9alpha,15beta-pentaacetoxy-5alpha-benzoyloxyjatropha-6(17),11E-dien-14-one (3) . When the isolates were assayed for multidrug resistance-reversing activity in a rhodamine 123 exclusion test using L5178 mouse lymphoma cells, all compounds demonstrated a concentration-dependent effect in inhibiting the efflux pump activity of these tumor cells in the range 11.2-112 microM.

J Comp Physiol {B}, 2003 Sep, 173(7), 559 - 64 Epub 2003 Jul 22.
P-glycoprotein-like protein contributes to cadmium resistance in Euglena gracilis; Einicker-Lamas M et al.; Selective pressures from polluted environments have led to the development of resistance systems in aquatic organisms . Using different techniques, this study examined a cadmium defense mechanism of the freshwater unicellular protozoa Euglena gracilis, and found it to be an efflux pump similar to the multidrug resistance P-glycoprotein . Cd(2+)-treated E . gracilis were able to extrude Rhodamine 123 at 21 degrees C, but not at 4 degrees C . Furthermore, verapamil, a P-glycoprotein modulator, partially blocked the efflux process (at 21 degrees C), and enhanced the Cd(2+) toxic effects on these cells . Western immunoblots of cell lysates, using the anti-P-glycoprotein antibody JSB-1, revealed a 120-KDa protein, which was expressed, in high amounts on Cd(2+)-exposed cells (74% above the control values) . Moreover, cells treated with JSB-1 became more sensitive to the harmful effects of cadmium, showing a decreased survival rate . Taken together, these results suggest that a MDR phenotype has evolved in Euglena as one of the mechanisms for cadmium detoxification.

J Membr Biol, 2003 May 15, 193(2), 79 - 90
The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane; Rychlik B et al.; We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2',7'-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL) . We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane . Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds . Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity . Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters . BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport . Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye . No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.

Antimicrob Agents Chemother, 2003 Aug, 47(8), 2393 - 6
Reversal of mefloquine and quinine resistance in Plasmodium falciparum with NP30; Ciach M et al.; Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells . We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors . We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD) . NP30 was able to sensitize (reversal, >80%) P . falciparum to MF, QN, QD, and, to a lesser extent, CQ . The presence of 2 micro M verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ . Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity . We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

Eur J Pharmacol, 2003 Jul 18, 473(1), 9 - 17
Reversal of multidrug resistance in cancer cells by pyranocoumarins isolated from Radix Peucedani; Wu JY et al.; The pyranocoumarins, (+/-)-3'-angeloyl-4'-acetoxy-cis-khellactone, were isolated from Radix Peucedani, the dry root of Peucedanum praeruptorum Dunn, through bioassay-guided fractionation . The chemical structure of pyranocoumarins was determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy . X-ray crystallography showed that there are eight molecules (i.e . two each of four conformers) in each unit cell with their optical activities equally cancelled out . The four conformers are 3'(R)-angeloyl-4'(R)-acetoxy-khellactone in two conformational forms, and 3'(S)-angeloyl-4'(S)-acetoxy-khellactone in two conformational forms . Pyranocoumarins caused apoptotic cell death with IC50 of 41.9+/-2.8 and 17.3+/-8.2 microM for drug-sensitive KB-3-1 and multidrug resistant (MDR) KB-V1, respectively . The two- to threefold sensitivity difference between the two cell lines is interesting considering that the same ratio for doxorubicin is 50-300 . Strong synergistic interactions were demonstrated when pyranocoumarins were combined with common anti-tumor drugs including doxorubicin, paclitaxel, puromycin or vincristine in MDR KB-V1 cell line, but not in drug-sensitive KB-3-1 cells . Pyranocoumarins increased doxorubicin accumulation in KB-V1 cells by about 25% after 6 h of incubation . Pyranocoumarins treatment for 24 h down-regulated the expression of P-glycoprotein in KB-V1 cells at both protein and mRNA levels . Pyranocoumarins also transiently reduced the cellular ATP contents in KB-V1 cells in a dose-dependent manner . Our results suggest that pyranocoumarins could be a potential MDR reversing agent.

An Sist Sanit Navar, 2001 May, 24(2), 197 - 207
{New recommendations and perspectives for the control of tuberculosis}; Arevalo JM; This report summarises the ATC/CDC recommendations on the diagnosis and treatment of latent tuberculosis infection . Based on the sensitivity and specificity of the tuberculin skin test and the prevalence of TB in different groups, 3 cut-off levels have been proposed for defining a positive tuberculin reaction: > or =5 mm, >/=10 mm and > or =15 mm of induration . Treatment can be carried out mainly with Isoniazid, but there are alternatives such as Rifampin plus Pyrazinamide in the following cases: when Isoniazid-resistant M . tuberculosis appears and when the treatment is to be shortened; Rifampin alone for persons who cannot tolerate Pyrazinamide . Rifabutin can be substituted for Rifampin in HIV+ patients taking some protease inhibitors and non-nucleoside reverse transcriptase inhibitors . In persons who have been infected with multidrug resistant strains, Ethambutol and Pyrazinamide or Pyrazinamide and a fluoroquinolone can be prescribed . On the other hand, for a general prophylaxis BCG vaccination is not usually recommended due to its variable effectiveness, except in children and health care workers exposed continually to an untreated patient or to one with multidrug resistant M . tuberculosis strains . In order to achieve a new vaccine, a programme is currently being developed in the European Union called "The cluster for Tuberculosis vaccine development", which involves four main projects: optimisation and preclinical evaluation of TB vaccines, development of new live attenuated vaccines, non-protein antigens for TB vaccine and identification of mechanisms and indicators of protective immunity.

Curr Opin Oncol, 2003 Jul, 15(4), 300 - 3
Hyperthermic isolated limb perfusion in the management of extremity sarcoma; Hoekstra HJ et al.; High local drug concentrations can be achieved in a limb with minimal systemic toxicity with the technique of hyperthermic isolated limb perfusion (HILP) . The currently most successful drugs are still Tumor Necrosis Factor alpha (TNFalpha) and melphalan . With HILP, as an induction chemotherapy treatment of locally advanced primarily irresectable soft tissue sarcomas of a limb, a limb salvage rate of 71% can be achieved, with a minimal treatment related morbidity . For the HILP is no upper age limit . Systemic inflammatory response syndrome is currently seldom seen . The exact working mechanisms of TNFalpha are still unknown . Experimental work is now directed to the development of drugs sensitizing the tumor vasculature to the effects of TNFalpha . In the clinical HILP setting are currently lower doses of TNFalpha in combination with melphalan investigated . Although multidrug resistance (MDR) is a major issue in effectiveness of chemotherapy in human cancer treatment, HILPs with TNFalpha and melphalan did not induce MDR in sarcomas . The future research in HILP with TNFalpha is directed in increasing tumor sensitivity for TNF with lowering the dosage without decreasing tumor response.

What Is Genome?, What Is Cell Biology?, What Is Biofilm?, What Is Pcr?, What Is Staphylococcus Aureus?, n, Microbes, r, Bacterium, n, Bacteria, s, Microbe, i, Microorganisms, n, Escherichia coli, s, Brevibacteria, e, Streptomycin, s, S. cerevisiae, r, Citrobacter, a, Photobacteria, s, Yeasts, c, Agrobacterium, c, Microbial, i, Bacteriological, i, Microbial, e, Pichia, s, Erythromycin, a, Microorganisms, s, Biodegradation, e, Streptomycin, i, Salmonella, o, Bacillus subtilis, n, Escherichia coli, s, Bacteria, c, Multidrug resistant

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005