Multidrug resistant

Biol Pharm Bull, 2001 May, 24(5), 555 - 7
Increase in doxorubicin cytotoxicity by inhibition of P-glycoprotein activity with lomerizine; Shiraki N et al.; Acquired resistance to chemotherapy is a major problem during cancer treatment . One mechanism for drug resistance is overexpression of the MDR (multidrug resistance)1 gene encoding the transmembrane efflux pump, P-glycoprotein (P-gp) . Calcium channel blockers such as verapamil, nifedipine and nicardipine have been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux . This study evaluated whether a new calcium channel blocker, lomerizine, influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline . Verapamil, and even more markedly, lomerizine, increased cellular uptake of calcein transported by P-gp in a P-gp-expressing erythroleukemia cell line, K562-Dox . Ten microM of lomerizine reduced the IC50 of doxorubicin in the K562-Dox from 60000 ng/ml to 800 ng/ml, whereas the IC50 of doxorubicin in the K562 subline was only marginally affected by these drugs . Lomerizine showed greater reduction in P-gp efflux than verapamil at an equimolar concentration . These results suggest that lomerizine has the clinical potential to reverse tumor MDR involving the efflux protein P-gp.

Biol Pharm Bull, 2001 May, 24(5), 474 - 9
Mechanism of resistance to oxidative stress in doxorubicin resistant cells; Furusawa S et al.; Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells . The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line . Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells . The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide . Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death . Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells . Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells . Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells . In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide . Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.

Brain Res, 2001 May 25, 902(1), 131 - 4
Inhibition of human multidrug resistance P-glycoprotein 1 by analogues of a potent delta-opioid antagonist; Lovekamp T et al.; Analogues Dmt-Tic (2',6'-dimethyl-L-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) pharmacophore, a potent delta-opioid receptor antagonist, inhibited hMDR1 P-GP expressed in a G-185 fibroblast cell line in a manner similar to verapamil . N,N(Me)2-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-NH-1-adamantane, H-Dmt-Tic-Ala-NH-1-adamantane and N,N(Me)2-Dmt-Tic-NH-tBut were highly effective inhibitors . Weaker inhibition was observed with N,N(Et)2-Dmt-Tic-OH, H-Dmt-Tic-Ala-NH-tert-butyl amide and cyclo(Dmt-Tic) . Results demonstrate that N- and C-terminal hydrophobic/lipophilic analogues of the Dmt-Tic pharmacophore inhibit hMDR1 and point to a potential role as chemosensitizing agents in chemotherapy for cancers containing hMDR1.

J Clin Microbiol, 2001 Jun, 39(6), 2213 - 8
Molecular epidemiology study of exogenous reinfection in an area with a low incidence of tuberculosis; Bandera A et al.; In geographical areas with a low incidence of tuberculosis, recurrent tuberculosis is generally due to reactivation of the disease . However, the relative contribution of tuberculosis reinfection increases in parallel with the incidence of disease and is likely to depend on the epidemiological context: factors such as the spread of multidrug resistance, human immunodeficiency virus (HIV) infection, and immigration from developing countries could modify disease transmission in areas at low risk for tuberculosis . A molecular epidemiology study was performed in Lombardy, Northern Italy, where the incidence of tuberculosis is 17.5 cases per 100,000 persons . A total of 2,452 cases of culture-confirmed tuberculosis in 2,127 patients were studied . A group of 32 patients (1.5%), each of whom had two episodes of tuberculosis with cure as the outcome of the first episode and with more than 6 months between the two episodes, were studied by means of restriction fragment length polymorphism DNA fingerprinting analysis . For 5 of the 32 patients (16%), the DNA fingerprinting patterns of Mycobacterium tuberculosis strains responsible for the second episode did not match those of the corresponding isolates of the first episode, indicating exogenous reinfection . Two of these patients developed multidrug-resistant tuberculosis during the second episode, and in three cases the isolates belonged to clusters of M . tuberculosis strains spreading in the community . A fourfold-increased risk for reinfection was observed in immigrant patients compared to Italian subjects . In contrast, a higher risk of relapse rather than reinfection was evidenced in HIV-positive subjects and in patients infected with multidrug-resistant tuberculosis . Episodes of tuberculosis reinfection in areas with a low incidence of tuberculosis are rare compared to those in high-incidence geographical regions . In populations that have immigrated from high-risk areas, reinfection may represent a considerable contributor to the rate of recurrent tuberculosis . This finding emphasizes the importance of containing the spread of epidemic strains in close communities, in order to prevent changes in global tuberculosis trends for developed countries.

J Biol Chem, 2001 Jul 27, 276(30), 27846 - 54 Epub 2001 May 25.
Transport of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug resistance protein 1 (MRP1) . Requirement for glutathione or a non-sulfur-containing analog; Leslie EM et al.; Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds . In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export . MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH . Using membrane vesicles prepared from transfected cells, we found that MRP1 transports {3H}NNAL-O-glucuronide but is dependent on the presence of GSH (Km 39 microm, Vmax 48 pmol x mg(-1) x min(-1)) . We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, gamma-glutamyl-alpha-aminobutyryl-glycine . Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of {3H}GSH transport . Moreover, whereas the MRP1 substrates leukotriene C4 (LTC4) and 17beta-estradiol 17beta-(d-glucuronide) (E(2)17betaG) inhibited GSH-dependent uptake of {3H}NNAL-O-glucuronide, only {3H}LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex . A mutant form of MRP1, which transports LTC4 but not E(2)17betaG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition . Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake . These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.

Clin Chem, 2001 Jun, 47(6), 1048 - 52
No influence of the MDR-1 C3435T polymorphism or a CYP3A4 promoter polymorphism (CYP3A4-V allele) on dose-adjusted cyclosporin A trough concentrations or rejection incidence in stable renal transplant recipients; von Ahsen N et al.; BACKGROUND: A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration . A polymorphism in the CYP3A4 promoter region, termed "variant" allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity . A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp . METHODS: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant . Real-time, rapid-cycle PCR methods were developed and used for genotyping . RESULTS: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations . No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes . Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection . CONCLUSIONS: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.

MMW Fortschr Med, 2001 Apr 2, 143 Suppl 1, 14 - 7
{New antiretroviral drugs . Improved pharmacokinetics and simpler dose schedules}; Arasteh K et al.; Despite of 18 licensed antiretroviral drugs in Germany, there is an increased pressure for development of new substances and new targets in the treatment of HIV positive patients . Multidrug resistance and poor pharmacokinetics are the biggest problems we have to fight with . But there is hope for resolving these challenges . Second generation drugs like FTC and DPC 083 with once a day dosage and a high resistance barrier are tested in clinical trials . In addition new classes of HIV drugs are available in phase III trials (T-20 fusion inhibitor) . So the future will provide us with some new effective arms in the battle against HIV.

Zhonghua Fu Chan Ke Za Zhi, 2000 Oct, 35(10), 617 - 20
{Establishment of cisplatin-induced multidrug resistant human epithelial ovarian cancer cell line 3AO/cDDP and its expressions of multidrug resistance proteins}; Chen J et al.; OBJECTIVE: To establish multidrug resistant ovarian cancer cell line 3AO/cDDP by exposing parent cell line intervally and repeatedly to high-level concentration of cisplatin, and to detect expressions of lung resistance protein (LRP), multidrug resistance-associated protein(MRP) and P-glycoprotein (P-gp) . METHODS: Using the corresponding dose calculated from clinical chemotherapy, we established 3AO/cDDP, with 3AO exposed intervally and repeatedly to high-level concentration of cisplatin at 10 micrograms/ml for 24 hours each time; LRP, MRP and P-gp expressions were detected with flow cytometry(FCM) . RESULTS: 3AO/cDDP was established after 4.5 months with stable resistance and the drug resistant index was 1.62 which was similar to clinical resistance degree . Low expression levels of MRP and P-gp were found in both 3AO and 3AO/cDDP, and no statistical difference between the two lines (P > 0.05) . However, LRP expression level in 3AO/cDDP was significantly higher than that in 3AO with FCM detection (P < 0.01) . CONCLUSIONS: 3AO/cDDP, induced by exposing 3AO to high-level concentration of cisplatin intervally and repeatedly, is an ideal model for investigation of cisplatin resistance . Cisplatin resistance in 3AO/cDDP could be related to high LRP expression and be not associated with MRP or P-gp.

Clin Pharmacol Ther, 2001 May, 69(5), 308 - 16
Pharmacokinetic and prognostic significance of intestinal MDR1 expression in recipients of living-donor liver transplantation; Hashida T et al.; BACKGROUND: Living-donor liver transplantation (LDLT) and subsequent immunosuppressive therapy with tacrolimus have been cornerstones in the recovery of patients from end-stage liver failure, but there has been no critical dosage regimen for tacrolimus therapy, especially the initial dosage . In this study, we examined whether the absorptive barriers, multidrug resistance protein (MDR1), or cytochrome P450 IIIA4 (CYP3A4) are important pharmacokinetic factors for tacrolimus and are prognostic indicators for LDLT outcome . METHODS: We used competitive polymerase chain reaction to evaluate the messenger ribonucleic acid (mRNA) expression levels of MDRL And Cyp3A4 in mucosal cells of the upper jejunum from a part of the Rroux-en- Y limb for biliary reconstruction during LDLT of recipients (n = 48) . The tacrolimus dosage was started at an oral dose of 0.075 mg/kg every 12 hours and adjusted on the basis of its whole-blood trough level by use of a semiautomated microparticle enzyme immunoassay . RESULTS: The mRNA expression level of MDR1 (r = -0.776), but not CYP3A4 (r = -0.094), was inversely related to the concentration/dose ratio of tacrolimus . High levels of MDR1, but not CYP3A4, were strongly associated with reductions in survival rates after LDLT with the Kaplan-Meier method and log-rank statistics (P =.020 and P =.135, respectively) . With use of a Cox regression procedure, high levels of MDR1 (relative risk, 12.99; 95% confidence interval, 1.64-103.23), but not CYP3A4 (relative risk, 0.93; 95% confidence interval, 0.87-1.00) appeared to be a significant prognostic indicator for poor survival . CONCLUSIONS: Intestinal MDR1 is not only a good probe with which to predict the interindividual variation in tacrolimus pharmacokinetics after LDLT but also a powerful prognostic indicator for the outcome of LDLT.

Curr Opin Pulm Med, 2001 May, 7(3), 170 - 2
Science, society, and disease: emerging perspectives; Gandy M; The understanding of tuberculosis (TB) requires effective links to be made between advances in biomedical knowledge and the wider social and economic dynamics of disease epidemiology . This review highlights some recent advances in contemporary TB research with particular emphasis on the spread of multidrug-resistant tuberculosis (MDR-TB); the prevalence of TB in prisons, urban ghettoes, and the marginalized communities of the South; and the relation between TB control and general developments in public policy making . The article also draws attention to the significance of new historical insights into the epidemiological transition of the twentieth century and the implications of new and emerging diseases for the future of public health . It is concluded that the real obstacles to the effective control of TB are political rather than scientific.

Curr Opin Pulm Med, 2001 May, 7(3), 154 - 9
Molecular epidemiology of tuberculosis: recent developments and applications; Fletcher HA; The standard method for the typing of Mycobacterium tuberculosis is still IS6110 restriction fragment length polymorphism (RFLP) . This method has been widely used and has provided information on the variety and distribution of tuberculosis strain types across the globe . Recently, IS6110 RFLP has been used to investigate the question of reinfection versus reactivation, examine the existence of multiple infection, and track the spread of multidrug-resistant tuberculosis . There have also been efforts to increase our understanding of the biologic characteristics of IS6110 . These studies have resulted in a clearer understanding of fingerprinting data and increased our understanding of the evolution and pathogenicity of this organism.

Curr Opin Pulm Med, 2001 May, 7(3), 148 - 53
Drug-resistant tuberculosis: worldwide trends, problems specific to Eastern Europe and other hotspots, and the threat to developing countries; Willcox PA; Although it had been appreciated that high levels of antituberculous drug resistance existed in some regions of the world, the full extent of the problem was not known . A combined initiative by the World Health Organization and the International Union Against Tuberculosis and Lung Disease was launched in 1994 to address this . A second report was issued in March 2000, in which surveillance of drug resistance had been extended to 72 countries and regions . A number of drug resistant "hotspots," where there are high levels of combined multidrug-resistant tuberculosis (> 3% prevalence), have been identified . Particular areas of concern are countries of the former Soviet Union, India, and China, because these countries have the highest burden of multidrug-resistant tuberculosis . For the first time, information on trends in global drug resistance is available.

AIDS, 2001 Apr 13, 15(6), 675 - 81
Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport; Jones K et al.; OBJECTIVES: To investigate the intracellular accumulation of HIV protease inhibitors (PI) and to assess the effect of active transport on this accumulation . METHODS: CEM cells were incubated with a PI for 18 h and the intracellular concentration determined using cell number and radioactivity . The effect of active transport was investigated using cells expressing P-glycoprotein (CEM(VBL)) and cells expressing multidrug resistance-associated protein 1 (MRP1; CEM(E1000)) . Incubations were also carried out at 4 degrees C and in the presence of 2-deoxyglucose plus rotenone to examine the effect of inhibiting active transport . RESULTS: Nelfinavir (NFV) accumulated to the greatest extent (> 80-fold) followed by saquinavir (SQV; approximately 30-fold), ritonavir (RTV; 3-7-fold) and finally indinavir (IDV; extracellular equivalent to intracellular) . In CEM(VBL) cells there was a significant reduction in the intracellular accumulation of NFV, SQV and RTV and in CEM(E1000) cells there was reduced accumulation of SQV and RTV . Inhibition of active transport processes caused a reduction in SQV and RTV accumulation but had no effect on IDV accumulation in all cell types . NFV accumulation was increased in CEM(VBL) cells as a result of inhibition of active transport . CONCLUSIONS: Marked differences can be detected in the intracellular accumulation of HIV PI drugs in vitro . Both P-glycoprotein and MRP1 may play a role in limiting the intracellular concentration of the PI and active influx mechanisms may contribute to drug accumulation.

Ann Med, 2001 Apr, 33(3), 167 - 71
Multidrug-resistant bacteria: overcoming antibiotic permeability barriers of gram-negative bacteria; Savage PB; Because of the permeability barrier provided by the outer membrane (OM), gram-negative bacteria are inherently resistant to many hydrophobic antibiotics . This resistance limits the arsenal of antibiotics that are effective in treating gram-negative bacterial infections . Compounding this problem, strains of gram-negative bacteria have emerged that display specific resistance mechanisms for effective antibiotics . As a means of expanding the arsenal of effective antibiotics for gram-negative bacteria, compounds that permeabilize the OM to hydrophobic substances have been developed . These compounds are typically cationic, amphiphilic molecules that can be prepared from peptides or steroids . Effective OM permeabilizers sensitize gram-negative bacteria to hydrophobic antibiotics, including erythromycin, fusidic acid, novobiocin and rifampin . These antibiotics are generally not useful in treating gram-negative bacterial infections because they traverse the OM ineffectively . The use of OM permeabilizers, in combination with hydrophobic antibiotics, may provide additional means of controlling growth of gram-negative bacteria . This review describes classes of permeabilizers, including those derived from peptides, and recently reported examples based on steroids.

AIDS Clin Care, 1995 Apr, 7(4), 27 - 9, 36
Diagnosis and detection of drug-resistant strains of M . tuberculosis; Ferraro MJ et al.; AIDS: Factors contributing to the recent upswing in tuberculosis (TB) cases in the United States include the shifting of management of TB cases from specialized hospital units to lesser-equipped outpatient settings; reduced medical school teaching of TB-related issues; large-scale immigration from regions endemic for TB; and the homelessness and AIDS cases that have created a pocket of disease in high-risk populations . At least 33 percent of current cases can be attributed to new rather than reactivated infection, and many of these are caused by the ever-increasing number of drug-resistant strains . Multidrug-resistant TB has a higher overall fatality rate and disproportionately affects immunocompromised and HIV-infected individuals . Clearly, expedient diagnosis is important in controlling the spread of tuberculosis . Sputum samples, evaluated first by direct microscopic evaluation (smear), are visualized with either the easily detected acid-fast fluorochrome dye auramine O, or the more specific Ziehl-Neelsen stain . Specimens are cultured on either solid media (Lowenstein-Jensen slant), or are grown in a liquid medium, such as the BACTEC automated radiometric system . Next, biochemical or nucleic acid probe testing is used to identify various strains . Isolates are tested for resistance to commonly used antituberculosis drugs, often by using the new method of susceptibility testing in liquid broth rather than the traditional agar dilution method . Clinical laboratories await FDA approval of two new methods employing hybridization with rRNA genes or polymerase chain reaction (PCR) that will further speed up diagnosis of TB .

J Biol Chem, 2001 Jul 27, 276(30), 28562 - 9 Epub 2001 May 21.
Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters; Stein U et al.; Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1 . Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes . Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116 . Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity . However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells . These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines . The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

Cancer Lett, 2001 Jun 26, 167(2), 157 - 62
Induction of apoptosis in MDR1 expressing cells by daunorubicin with combinations of suboptimal concentrations of P-glycoprotein modulators; Aszalos A et al.; The application of most agents with the capacity to reverse multidrug resistance (MDR) via modulation of the multidrug transporter P-glycoprotein (Pgp) was shown to be associated with toxic side-effects . For this reason, we have investigated the effect of combinations of suboptimal concentrations of Pgp blockers on the induction of apoptosis and growth arrest in daunorubicin (D) treated, MDR1 gene transfected cells . We used verapamil, PSC833 and Cremophor EL as Pgp modulators, which affect the function of Pgp by different mechanisms . Treatment of NIH3T3/MDR1 cells with combinations of suboptimal concentrations of Pgp modulators in the presence of D caused apoptosis and G(2) arrest to the same extent as optimal concentrations of singly used blockers . We conclude that combinations of suboptimal concentrations of Pgp modulators may cause effective sensitization of resistant tumor cells, and at the same time, may avoid the frequently observed toxic effects experienced in clinical trials with a single modifier applied at the optimal dose.

Biochem J, 2001 Jun 1, 356(Pt 2), 317 - 25
Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine; Voshol PJ et al.; Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol . The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption . SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice . Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient {Mdr2((-/-))} mice that produce phospholipid/cholesterol-free bile, and cholesterol 7alpha-hydroxylase-deficient {Cyp7a((-/-))} mice, which exhibit qualitative and quantitative changes in the bile-salt pool . Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method . SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine . In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by approximately 50% . Bile-diverted rats, Mdr2((-/-)) mice and Cyp7a((-/-)) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected . Cholesterol absorption was reduced by >90% in bile-duct-ligated and bile-diverted animals and in Cyp7a((-/-)) mice, whereas Mdr2((-/-)) mice showed an approximately 50% reduction . This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression . Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression . The role of SR-BI in the cholesterol-absorption process remains to be defined.

Leukemia, 2001 May, 15(5), 764 - 71
Combined action of PSC 833 (Valspodar), a novel MDR reversing agent, with mitoxantrone, etoposide and cytarabine in poor-prognosis acute myeloid leukemia; Visani G et al.; PSC 833 (Valspodar) can reverse multidrug resistance (MDR) in patients with hematologic malignancies, but alters the pharmacokinetics of concomitant anticancer agents . A phase I, dose-finding study was initiated to define a safe and effective regimen of mitoxantrone, etoposide, and cytarabine (MEC) when administered with PSC 833 to patients with early relapsed or refractory acute myeloid leukemia (AML) . Poor-prognosis AML patients refractory to first-line induction therapy or relapsing within 9 months of attaining complete remission (CR) were treated with cytarabine (1.0 g/m2/day), etoposide (30 mg/m2/day), and mitoxantrone at a dose of either 3.0 mg/m2/day (cohort 1) or 4.5 mg/m2/day (cohorts 2 and 3) for 6 days plus continuous-infusion PSC 833 (10 mg/kg/24 h with a 2.0 mg/kg loading dose) for 6 or 7 days each 21-day cycle . Patients achieving CR were given a 4-day MEC plus PSC 833 consolidation cycle . Twenty-three patients were enrolled (eight with primary refractory AML and 15 in relapse) . Dose-limiting toxicity occurred in one of six patients in cohort 2 (grade 4 mucositis) and one of seven patients in cohort 3 (grade 4 hyperbilirubinemia) . The maximum tolerated dose of mitoxantrone was defined as 4.5 mg/m2/day . Clinically significant grade 4 hyperbilirubinemia, possibly related to PSC 833, occurred in four patients . Hematologic toxicities were as expected in this patient population, but were not dose limiting . Mild to moderate cerebellar ataxia and paresthesia occurred in six (26%) and five (22%) patients, respectively, but were not dose limiting . Overall, six of 23 (26%) patients achieved CR, including five patients with demonstrated P-glycoprotein expression and/or function . The median overall survival was 4 months . All six patients with a CR were alive and four (17%) patients were disease free at 12 months . Blood levels of PSC 833 were well above the target level of 1000 ng/ml, a concentration that is known to reverse MDR in vitro . PSC 833 reduced the clearance of etoposide by approximately two-fold . No correlation was observed between the mitoxantrone or etoposide area under the curve and response . In conclusion, the MEC plus PSC 833 tested regimen was well tolerated and the 26% CR rate warrants further testing of this regimen in a randomized, phase III trial.

AIDS Treat News, 1999 Oct 1, (No 328), 1 - 2
ABT-378 early access program begins; Raising the stakes at Retro '99 or amplified diversity; AIDS: The 6th Conference on Retroviruses and Opportunistic Infections raised serious concerns about the development of multidrug-resistant strains of HIV . There are press reports of HIV-positive men increasingly engaging in unprotected anal sex and who are seeking thrills by engaging in high risk behavior . This behavior may be attributed to the view that acquiring the virus no longer seems like a death sentence . Data from studies of HIV-positive people demonstrate how the disease spreads within a population and show that close adherence correlates with viral suppression in patients taking protease inhibitors (PIs) . Other topics covered at the conference included the importance of the thymus in HAART responders, treatment interruption failures, IL-2 trials and uses, stopping cytomegalovirus (CMV) and Pneumocystis carinii pneumonia (PCP) treatment, and CD4+ cell survival versus viral fitness . Also addressed were risk factors associated with PIs, and salvage regimens . Results from a number of drug trials are summarized . Contact information is provided .

J Int Assoc Physicians AIDS Care, 1999 Jan, 5(1), 10 - 30
Therapeutic options or antiretroviral anarchy?
Mascolini M.
AIDS: The number of drug combinations being used to treat HIV infections may produce a public health nightmare by creating multidrug resistance . The issue of whether to use aggressive multidrug combinations early in the infectious cycle or whether to delay treatment as long as possible remains controversial and unresolved . The use of viral markers to track disease progression is discussed . Tables illustrate the relationships between CD4+ counts and opportunistic infections, disease progression, and long-term viral load reduction . The effectiveness of different drugs combinations is discussed . Results of various clinical trials are presented .

BETA . 1998 Oct;:18-25.
Tuberculosis; Highleyman L; AIDS: Approximately one-third of the world's population is infected with tuberculosis (TB), and TB kills more people worldwide than any other infectious disease . TB infection, however, is not the same as active TB disease . Nine out of ten people with healthy immune systems who are infected with TB do not develop active TB disease; the rate at which people who are coinfected with HIV and TB develop active TB is 100 times higher . The history and epidemiology of the disease are outlined . In 1990, epidemiologists began seeing new strains of TB that are drug-resistant . This multidrug-resistant TB (MDRTB) is especially dangerous for HIV-positive persons . Symptoms of active TB disease include lung fluid expulsion, fatigue, weakness, malaise, fever, and chills . Untreated TB can lead to severe wasting and death . TB prevention, transmission, and treatment are described . A table is included which lists the major drugs used to prevent and treat TB, and describes the primary side effects associated with each . A resource list is provided .

AIDS Alert . 1998 Jun;13(6):68.
Reporting of co-infection still limited; Looking down the drug pipeline; AIDS: Reports at the 5th Conference on Retroviruses and Opportunistic Infections addressed new anti-HIV agents in primary phases of development that offer treatment alternatives to people with little or no treatment history and individuals with few treatment choices . FTC, a new nucleoside analog produced by Triangle Pharmaceuticals, is an alternative to 3TC . F-ddA (lodenosine), a nucleoside analog licensed by US Bioscience, is structurally similar to ddI and is reported to have good bioavailability, once-a-day dosing, and no bone marrow suppression . F-ddA has also shown in vitro activity against multidrug-resistant strains of HIV . Adult and pediatric studies are currently being conducted by the National Cancer Institute (NCI) and US Bioscience . Oral versus IV PMPA shows promising results as a possible alternative for 3TC- and AZT-experienced patients . Further testing is being done by Gilead Sciences and HIV Network for Prevention Trials (HIVNET) . Abbott Laboratories is developing a second-generation protease inhibitor, ABT-378, which has a ten-fold greater antiviral activity in vitro than the original, ritonavir . It is administered with ritonavir to increase ABT-378 levels in the blood, but has no food requirements, and less severe side effects . Two trials are being conducted: one for patients who are treatment-naive and the second for patients who are failing other protease inhibitors . Immune-based therapies, such as Leukine (GM-CSF), are used to handle neutropenia and offset bone marrow toxicities from drugs . Concerns that GM-CSF may increase viral replication may be balanced by using highly active antiretroviral therapy . FP-21399, developed by Lexigen Pharmaceuticals, is being tested as an HIV fusion inhibitor .

AIDS Alert, 1995 Jul, 10(7), 91 - 3
Prolonging survival among MDR-TB AIDS patients; Second National Retrovirus conference: a further report; AIDS: It was reported at the 1995 Second National Retrovirus Conference that AIDS has now surpassed unintentional injury as the leading cause of death for male Americans between the ages of 25 to 44, and for women, AIDS is fourth behind unintentional injury . A study of multidrug resistant tuberculosis that showed improved survival rates as long as appropriate therapy began within four weeks of diagnosis was also presented . The current recommendation is to consider the PPD skin test positive in persons with HIV if the bump that appears is over five millimeters in diameter . A new ganciclovir implant study demonstrated the implant's effectiveness in preventing CMV disease progression with low rates of complications, suggesting implants should probably replace intravenous ganciclovir as maintenance therapy . Another study demonstrated the effectiveness of cidofovir as a treatment for CMV infection, indicating that cidofovir was appropriate as a salvage therapy for those failing ganciclovir and foscarnet . In vitro studies involving Taxol and Kaposi's sarcoma (KS) show partial responses (55 percent), and some disease stabilization (40 percent) . Four of five patients with pulmonary KS responded with clearance of tumor lesions . The first randomized trial involving Loviride with zidovudine has shown a sustained increase in CD4 cells with headache, nausea, and diarrhea as the most common side effects . Preliminary assessments reveal a reduction in viral load using the combination as opposed to monotherapy . Additional Loviride combination trials are being planned in Europe .

AIDS Alert, 1995 Apr, 10(4), 58 - 9
Tuberculosis rising in areas with high HIV rates; Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice; Faculty of Pharmacy, University of Toronto, Ontario, CanadaP-glycoprotein (PGP), an ATP-dependent membrane transporter is found in epithelial tissues of the liver, kidneys, intestine and blood-brain barrier . In tumor cells, PGP is often overexpressed and confers multidrug resistance toward cancer chemotherapeutics . It has been previously shown in rats that induction of an inflammatory response evokes a decrease in hepatic expression of PGP . In order to identify the inflammatory mediators involved in this phenomenon, we examined the influence of experimentally induced inflammation and pro-inflammatory cytokines (interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha) on the hepatic expression of PGP in mice . A significant reduction in the hepatic expression of mdr1a, mdr1b, mdr2 and spgp genes were seen in endotoxin (lipopolysaccharide (LPS)) and turpentine-treated mice . Similarly, IL-6-treated mice displayed a 70% reduction in protein expression and a 40-70% reduction in the mRNA levels of all PGP mdr isoforms . Administration IL-1beta caused an increase in both mdr1b mRNA and protein expression, however, mRNA levels of mdr1a, mdr2 and spgp were significantly reduced . Administration of TNF-alpha also caused increases in mdr1b mRNA . These findings indicate that IL-6 plays a principal role in the downregulation of PGP that is observed in the livers of mice during an inflammatory response.

Cancer Res, 2001 May 15, 61(10), 4030 - 7
Pervilleine A, a novel tropane alkaloid that reverses the multidrug-resistance phenotype; Mi Q et al.; P-Glycoprotein-mediated drug efflux can yield a multidrug-resistance (MDR) phenotype that is associated with a poor response to cancer chemotherapy . Pervilleine A, a novel tropane alkaloid obtained from a chloroform extract of Erythroxylum pervillei as the result of bioactivity-guided fractionation, was found to restore the vinblastine sensitivity of cultured multidrug-resistant KB-V1 and CEM/VLB(100) cells, with IC(50) values of 0.36 and 0.02 microM, respectively . Similarly, the chemosensitivity of KB-8-5 cells to colchicine was restored with an IC(50) value of 0.61 microM . The mechanism of this response was evaluated with a number of model systems . First, incubation of multidrug-resistant KB-V1 and CEM/VLB(100) cells with up to 45 microM pervilleine A for 72 h did not significantly affect either the transcription of MDR1, as revealed by reverse transcriptional-PCR-based analysis of MDR1 mRNA, or levels of P-glycoprotein, as shown by Western blots . ATP-dependent binding of {(3)H}vinblastine observed with isolated multidrug-resistant KB-V1 cell membrane vesicles was inhibited by pervilleine A in a dose-dependent manner, and kinetic analysis indicted competitive inhibition with respect to vinblastine binding with a K(i) of 7.3 microM . Consistent with this effect, intracellular accumulation of {(3)H}vinblastine was increased from 0.18 pmol {(3)H}vinblastine/50 x 10(4) cells to approximately 5 pmol {(3)H}vinblastine/50 x 10(4) cells in the presence of 40 microM pervilleine A . To explore the potential relevance of these responses, KB-V1 or KB-8-5 cells were placed in hollow fibers and implanted into NCr nu/nu mice . Cell growth was not significantly inhibited when vinblastine or pervilleine A were administered as single agents, but when used in combination, inhibition of up to 75% was observed . Equimolar doses of verapamil were less effective . These data suggest that pervilleine A is an effective inhibitor of P-glycoprotein and should be further evaluated for clinical utility.

Blood Cells Mol Dis, 2001 Jan-Feb, 27(1), 165 - 80
Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis; Abraham EH et al.; In addition to the better-known roles of the erythrocyte in the transport of oxygen and carbon dioxide, the concept that the red blood cell is involved in the transport and release of ATP has been evolving (J . Luthje, Blut 59, 367, 1989; G . R . Bergfeld and T . Forrester, Cardiovasc . Res . 26, 40, 1992; M . L . Ellsworth et al., Am . J . Physiol . 269, H2155, 1995; R . S . Sprague et al., Am . J . Physiol . 275, H1726, 1998) . Membrane proteins involved in the release of ATP from erythrocytes now appear to include members of the ATP binding cassette (ABC) family (C . F . Higgins, Annu . Rev . Cell Biol . 8, 67, 1992; C . F . Higgins, Cell 82, 693, 1995) . In addition to defining physiologically the presence of ABC proteins in RBCs, accumulating gel electrophoretic evidence suggests that the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance-associated protein (MRP1), respectively, constitute significant proteins in the red blood cell membrane . As such, this finding makes the mature erythrocyte compartment a major mammalian repository of these important ABC proteins . Because of its relative structural simplicity and ready accessibility, the erythrocyte offers an ideal system to explore details of the physiological functions of ABC proteins . Moreover, the presence of different ABC proteins in a single membrane implies that interaction among these proteins and with other membrane proteins may be the norm and not the exception in terms of modulation of their functions .

Urology, 2001 May, 57(5), 993 - 8
Increased intracellular doxorubicin by anti-FAS monoclonal antibody: a mechanism that enhances the cytotoxicity in renal cell carcinoma cells; Wu X et al.; OBJECTIVES: To investigate the effect of anti-Fas monoclonal antibody (mAb) on the intracellular concentration of doxorubicin in renal cell carcinoma (RCC) cells . Little is known about the influence of anti-Fas mAb on the intracellular concentration of chemotherapeutic agents . METHODS: The concentration of intracellular doxorubicin was determined by high-performance liquid chromatography . The mRNA and protein levels of multidrug resistance-associated protein gene were evaluated by reverse transcriptase-polymerase chain reaction and immunocytochemistry, respectively . RESULTS: An increased concentration of doxorubicin inside the cells was found: 2.4-fold in ACHN cells (a human RCC cell line) after treatment with doxorubicin combined with anti-Fas mAb compared with doxorubicin alone . Of the five cases of freshly derived RCC cells treated with doxorubicin and anti-Fas mAb, the intracellular concentration of doxorubicin was increased 2.3 and 2.7-fold in two of them, respectively . Furthermore, both the mRNA and the protein levels of the multidrug resistance-associated protein gene were downregulated after treatment of ACHN cells with anti-Fas mAb . Treatment of ACHN cells with a combination of anti-Fas mAb and doxorubicin resulted in a potentiation of the doxorubicin-mediated cytotoxicity . CONCLUSIONS: The increased intracellular concentration of doxorubicin by anti-Fas mAb might be one of the mechanisms responsible for the enhancement of doxorubicin-mediated cytotoxicity in RCC cells.

J Pharmacol Exp Ther, 2001 Jun, 297(3), 1137 - 43
Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene; Tanabe M et al.; To evaluate whether mutations in the human multidrug resistance (MDR)-1 gene correlate with placental P-glycoprotein (PGP) expression, we sequenced the MDR-1 cDNA and measured PGP expression by Western blotting in 100 placentas obtained from Japanese women . Nine single nucleotide polymorphisms (SNPs) were observed with an allelic frequency of 0.005 to 0.420 . Of these SNPs, G2677A (allelic frequency = 0.18) and G2677T (0.39) in exon 21 were associated with an amino acid conversion from Ala to Thr and to Ser, respectively . Sixty-one of 65 samples (93.8%), which had a C3435T allele, also had a mutant G2677(A,T) allele, suggesting an association between the two SNPs . Correlations of mutations with expression levels were observed; individuals having the G2677(A,T) and/or T-129C (p < 0.05) allele had less placental PGP . Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based genotyping tests were developed for the detection of these SNPs . The PCR, in which genomic DNAs obtained from healthy subjects (n = 48) are used as samples, was successful . The frequency of mutations in placental cDNA was identical with that in genomic DNA . When genotype results were compared between Caucasians and Japanese, ethnic differences in the frequency of polymorphism in the MDR-1 gene were suspected . Although it remains to be determined whether these SNPs influence the pharmacokinetic and dynamic properties of clinically useful drugs that are substrates of PGP, the polymorphism of the MDR-1 gene presented here may provide useful information in in vivo study of these issues.

Endocrinology, 2001 Jun, 142(6), 2686 - 94
Multidrug resistance P-glycoprotein hampers the access of cortisol but not of corticosterone to mouse and human brain; Karssen AM et al.; In the present study, we investigated the role of the multidrug resistance (mdr) P-glycoprotein (Pgp) at the blood-brain barrier in the control of access of cortisol and corticosterone to the mouse and human brain . {(3)H}Cortisol poorly penetrated the brain of adrenalectomized wild-type mice, but the uptake was 3.5-fold enhanced after disruption of Pgp expression in mdr 1a(-/-) mice . In sharp contrast, treatment with {(3)H}corticosterone revealed high labeling of brain tissue without difference between both genotypes . Interestingly, human MDR1 Pgp also differentially transported cortisol and corticosterone . LLC-PK1 monolayers stably transfected with MDR1 complementary DNA showed polar transport of {(3)H}cortisol that could be blocked by a specific Pgp blocker, whereas {(3)H}corticosterone transport did not differ between transfected and host cells . Determination of the concentration of both steroids in extracts of human postmortem brain tissue using liquid chromatography mass spectrometry revealed that the ratio of corticosterone over cortisol in the brain was significantly increased relative to plasma . In conclusion, the data demonstrate that in both mouse and human brain the penetration of cortisol is less than that of corticosterone . This finding suggests a more prominent role for corticosterone in control of human brain function than hitherto recognized.

Best Pract Res Clin Haematol, 2001 Mar, 14(1), 211 - 33
Chemotherapy resistance in acute myeloid leukaemia; Sonneveld P et al.; The development of refractory disease in acute myeloid leukaemia (AML) is frequently associated with the expression of one or several multidrug resistance (MDR) genes . MDR1, MRP1 and LRP have been identified as important adverse prognostic factors in AML . Recently it has become possible to reverse clinical multidrug resistance by blocking P-glycoprotein-mediated drug efflux . The potential relevance of MDR and new approaches to treat refractory disease, are discussed .

J Cancer Res Clin Oncol, 2001 May, 127(5), 301 - 13
Exploring the mechanisms of action of FB642 at the cellular level; Hammond LA et al.; FB642(methyl-2-benzimidazolecarbamate, carbendazim) is a systemic fungicide belonging to the benzimidazole family with antitumor activity against a broad spectrum of tumors both in vitro and in vivo such as pancreas, prostate, colon, and breast . Although the preclinical antitumor activity of FB642 has been well explored, its mechanism of action has not been as well delineated . Previous studies indicate that FB642 may interfere with mitosis and thus may disrupt or inhibit microtubule function resulting in apoptosis . This study seeks to determine if FB642 is a sufficiently novel agent worthy of further development by examining the effect of FB642 on apoptosis, the cell cycle, p53-positive and -negative tumors, and drug-resistant and MDR cell lines . The results of this present study indicate that FB642 increases the degree of apoptosis in all examined tumor cell lines, may induce G2/M uncoupling, may selectively kill p53 abnormal cells, and exhibits antitumor activity in drug- and multidrug-resistant cell lines . The induction of apoptosis by FB642, particularly in p53-deficient cells, its impressive in vivo activity against a broad spectrum of murine and human tumors, as well as an acceptable toxicity profile in animals, make FB642 an excellent candidate for further evaluation in clinical trials in cancer patients.

J Gastroenterol Hepatol, 2001 Apr, 16(4), 460 - 6
Modulation of doxorubicin sensitivity by cyclosporine A in hepatocellular carcinoma cells and their doxorubicin-resistant sublines; Shiraga K et al.; BACKGROUND AND AIMS: Cyclosporine A (Cys) and verapamil (Ver) sensitize multidrug-resistant (MDR) cells to various anticancer drugs by interacting with membrane glycoproteins involved in the drug efflux . In the present study, we assessed the effect of Cys on the modulation of doxorubicin (DOR) sensitivity in hepatocellular carcinoma (HCC) cell lines, and their DOR-resistant sublines . METHODS: The sensitivity to DOR and the chemosensitizing effects of Cys were assessed by using two human HCC cell lines, PLC/PRF/5 and Hep-3B, and their DOR-resistant sublines, PLC/DOR and 3B/DOR . The expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein (MRP) mRNA in these cells were detected by using a RT-PCR . The HCC cell lines grown in individual wells of 24-well plates were incubated with DOR that were sequentially diluted in culture medium in combination with 5 micromol/L Cys for 24 h . The cell viability in each well was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay . RESULTS: The mRNA of MDR1 and that of MRP were readily detectable in the HCC cell lines by RT-PCR . When 5 micromol/L Cys was added to the culture, the 50% inhibiting concentration (IC50) of DOR was reduced from 0.93 +/- 0.29 microg/mL to 0.32 +/- 0.10 microg/mL in PLC/PRF/5, and from 0.25 +/- 0.07 microg/mL to 0.09 +/- 0.04 microg/mL in Hep-3B . Furthermore, in the presence of 5 micromol/L Cys, the IC50 of DOR was reduced from 48.63 +/- 17.04 microg/mL to 0.49 +/- 0.14 microg/mL in PLC/DOR, and from 4.60 +/- 1.22 microg/mL to 0.15 +/- 0.06 microg/mL in 3B/DOR . The amounts of PCR products of MDR1 mRNA in PLC/DOR and 3B/DOR were greater than those in PLC/PRF/5 and Hep-3B, respectively . CONCLUSIONS: In HCC, the amplification of MDR1 mRNA is probably the main mechanism underlying acquired DOR resistance . Cyclosporine is also indicated to be highly active in potentiating the anticancer activity of DOR in HCC cells and their DOR-resistant sublines.

Mol Pharmacol, 2001 Jun, 59(6), 1433 - 40
Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux; Terlouw SA et al.; We previously used killifish proximal tubules, fluorescent substrates, and confocal microscopy to demonstrate that transport mediated by the multidrug resistance protein (Mrp2) and by P-glycoprotein was reduced by nanomolar concentrations of endothelin-1 (ET), acting through a basolateral B-type ET receptor and protein kinase C (PKC) . Here we show that representatives of two classes of nephrotoxicants decrease transport by activating the endothelin-PKC signaling pathway . Exposing tubules to radiocontrast agents (iohexol, diatrizoate) or aminoglycoside antibiotics (gentamicin, amikacin) reduced Mrp2-mediated fluorescein methotrexate (FL-MTX) transport from cell to tubular lumen . Pretreating the tubules with an ET(B)-receptor antagonist or with PKC-selective inhibitors abolished these effects . The nephrotoxicants activated signaling by inducing release of ET from the tubules, because adding of an antibody against ET to the medium abolished the effects . Elevating medium Ca(2+) also reduced FL-MTX transport; this reduction was abolished when tubules were pretreated with an ET antibody, an ET(B)-receptor antagonist, PKC-selective inhibitors, or the Ca(2+) channel blocker, nifedipine . None of these drugs by themselves affected FL-MTX transport . Importantly, nifedipine also blocked the ET(B)-receptor/PKC-dependent reduction in FL-MTX transport caused by gentamicin and diatrizoate . These results for two classes of structurally unrelated nephrotoxicants suggest that Ca(2+)-dependent ET release and subsequent action through an autocrine mechanism may be an early response to tubular injury.

Antimicrob Agents Chemother, 2001 Jun, 45(6), 1836 - 42
Genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors; Masquelier B et al.; Genomic rearrangements in the 5' part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI) . We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements . Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories . Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay . Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5' part of the RT, with an extensive molecular variation . The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each . In four cases, codon 67 was deleted . High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively . Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations . Resistance to 3TC could develop in the absence of specific mutations . Low-level resistance to ddI was noticed in 40% of the patients . The deletions of codon 67 seemed to have little effect on NRTI sensitivity . Most of the rearrangements were shown to contribute to cross-resistance to NRTI . The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.

Antimicrob Agents Chemother, 2001 Jun, 45(6), 1746 - 50
In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron; Pradines B et al.; The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test . Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P . falciparum, which increased after a prolonged exposure (96 or 144 h) . In the presence of iron (FeCl(3)), only the activities of tetracyclines and norfloxacin were decreased . Their in vitro activity against intraerythrocytic stages of multidrug-resistant P . falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs . However, due to their slow antimalarial action and to the fact that they act better after prolonged contact, they probably need to be administered in conjunction with a rapidly acting antimalarial drug, such as a short course of chloroquine or quinine.

Am J Physiol Gastrointest Liver Physiol, 2001 Jun, 280(6), G1261 - 73
Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats; Mottino AD et al.; We analyzed the expression of multidrug resistance-associated protein 2 (mrp2) in the small intestine of control female rats and in rats during late pregnancy (19-20 days of pregnancy) and lactation (2-4, 10-14, and 21 days after delivery) . Western blot analysis was performed on brush-border membranes prepared from different regions of the small intestine . Expression of mrp2 was maximal in the proximal segments for all experimental groups, was preserved in pregnant rats, and increased by 100% in postpartum rats by late lactation with respect to control animals . Northern blot analysis of mrp2 mRNA revealed a positive correlation with protein levels . Transport of S-glutathione-dinitrophenol (DNP-SG) from the intestinal cell to the lumen was analyzed in the everted intestinal sac model . Secretion of DNP-SG was not altered in pregnant rats but increased in lactating animals by late lactation . Intestinal mrp2 mRNA, protein, and transport activity are increased in lactating rats, suggesting that this may represent an adaptive mechanism to minimize the toxicity of dietary xenobiotics in response to increased postpartum food consumption.

Int J Cancer, 2001 Jun 15, 92(6), 777 - 83
Role of glutathione S-transferase P1, P-glycoprotein and multidrug resistance-associated protein 1 in acquired doxorubicin resistance; Harbottle A et al.; While P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) are known to be important in acquired doxorubicin resistance, the role of glutathione S-transferases (GST) remains unclear . Our study assessed roles of these 3 factors in a human drug-sensitive carcinoma cell line (HEp2), a subclone made resistant by prolonged incubation in doxorubicin (HEp2A), and HEp2 cells stably transfected with human GSTP1 . Drug-resistant HEp2A cells showed greater total GST activity, GSTP class enzyme expression, Pgp expression, MRP1 transcript expression, drug efflux and at least 13-fold greater resistance to doxorubicin than the parent HEp2 cell line . GSTM class enzyme expression was similar in both cell types, while GSTA class enzymes were not detected . In the resistant HEp2A cells, cytotoxicity was markedly enhanced by the Pgp/MRP inhibitor verapamil at low doxorubicin concentrations . The GST inhibitor curcumin also enhanced cytotoxicity in HEp2A cells when the Pgp/MRP efflux barrier had been reversed by verapamil or overcome by high doxorubicin concentrations . In addition, curcumin had a chemosensitising effect at low doxorubicin concentrations in HEp2 cells . Stable transfection of HEp2 cells with human GSTP1 increases doxorubicin resistance 3-fold over control cells . Our study indicates involvement of GSTP enzymes as well as efflux mechanisms in the acquired doxorubicin-resistance phenotype .

Clin Cancer Res, 2001 May, 7(5), 1429 - 37
BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy; Lee FY et al.; BMS-247550, a novel epothilone derivative, is being developed by Bristol-Myers Squibb Company (BMS) as an anticancer agent for the treatment of patients with malignant tumors . BMS-247550 is a semisynthetic analogue of the natural product epothilone B and has a mode of action analogous to that of paclitaxel (i.e., microtubule stabilization) . In vitro, it is twice as potent as paclitaxel in inducing tubulin polymerization . Like paclitaxel, BMS-247550 is a highly potent cytotoxic agent capable of killing cancer cells at low nanomolar concentrations . Importantly, BMS-247550 retains its antineoplastic activity against human cancers that are naturally insensitive to paclitaxel or that have developed resistance to paclitaxel, both in vitro and in vivo . Tumors for which BMS-247550 demonstrated significant antitumor activity encompass both paclitaxel-sensitive and -refractory categories, i.e., (a) paclitaxel-resistant: HCT116/VM46 colorectal (multidrug resistant), Pat-21 breast and Pat-7 ovarian carcinoma (clinical isolates; mechanisms of resistance not fully known), and A2780Tax ovarian carcinoma (tubulin mutation); (b) paclitaxel-insensitive: Pat-26 human pancreatic carcinoma (clinical isolate) and M5076 murine fibrosarcoma; and (c) paclitaxel sensitive: A2780 ovarian, LS174T, and HCT116 human colon carcinoma . In addition, BMS-247550 is p.o . efficacious against preclinical human tumor xenografts grown in immunocompromised mice or rats . Schedule optimization studies indicate that BMS-247550 is efficacious when administered frequently (every 2 days x 5) or intermittently (every 4 days x 3 or every 8 days x 2) . These efficacy data demonstrate that BMS-247550 has the potential to surpass Taxol in both clinical efficacy and ease of use (i.e., less frequent treatment schedule and/or oral administration).

Clin Cancer Res, 2001 May, 7(5), 1221 - 9
A phase I trial of doxorubicin, paclitaxel, and valspodar (PSC 833), a modulator of multidrug resistance; Advani R et al.; PURPOSE: P-glycoprotein is an efflux pump for many drugs including doxorubicin and paclitaxel . This study evaluated the coadministration of these drugs with the P-glycoprotein inhibitor valspodar (PSC 833) with the aim of determining: (a) maximum tolerated doses (MTDs) of doxorubicin followed by paclitaxel (DP); (b) the MTD of DP combined with PSC 833 (DPV), without and with filgrastim (G-CSF); and (c) the pharmacokinetic interactions of PSC 833 with doxorubicin and paclitaxel . EXPERIMENTAL DESIGN: For the first cycle, patients received doxorubicin as a 15-min infusion followed by paclitaxel as a 1-h infusion . For the second cycle, patients received reduced doses of DP with PSC 833 at 5 mg/kg p.o., four times a day for 12 doses . RESULTS: Thirty-three patients with various refractory malignancies were enrolled and assessable . The MTD of DP without PSC 833 was 35 mg/m(2) doxorubicin and 150 mg/m(2) paclitaxel . The MTD of DPV without G-CSF was 12.5 mg/m(2) doxorubicin and 70 mg/m(2) paclitaxel . The dose-limiting toxicity for both DP and DPV was neutropenia without thrombocytopenia . With G-CSF, the MTD for DPV was 20 mg/m(2) doxorubicin and 90 mg/m(2) paclitaxel . No grade 4 nonhematological toxicities were observed . Five partial and two minor tumor remissions were observed . Paired pharmacokinetics with and without PSC 833 revealed substantial drug interactions with both doxorubicin and paclitaxel . CONCLUSIONS: PSC 833 can be administered safely with doxorubicin and paclitaxel . The pharmacokinetic profiles of these drugs are significantly affected by PSC 833, requiring approximately 60% dose reductions for equivalent degrees of myelosuppression.

Br J Pharmacol, 2001 May, 133(2), 306 - 14
Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines; Tolomeo M et al.; Recently discovered chemically modified tetracyclines (CMTs) have shown in vitro and in vivo anti-proliferative and anti-tumour activities . Here, we evaluated in vitro the anti-proliferative and apoptotic activity of six different dedimethylamino chemically modified tetracyclines (CMT-1, CMT-3, CMT-5, CMT-6, CMT-7 and CMT-8) in sensitive and multidrug resistant myeloid leukaemia cells (HL60 and HL60R) in vitro . Three of these compounds (CMT-5, CMT-6, CMT-7) showed low cytotoxic activity both in sensitive and in resistant cells, CMT-3 was endowed with a high anti-proliferative activity only in sensitive cells and was moderately effective as apoptosis inducing agent, with an activity similar to that shown by doxycycline . On the contrary, CMT-1 and CMT-8 were very effective as programmed cell death inducing agents . The apoptotic pathway activated by these compounds involved the activation of caspases, especially caspase-9 and, for CMT-1, also the activation of FAS: Interestingly CMT-8, but not CMT-1, was able to induce apoptosis in multidrug resistant HL60R and in Fas-ligand resistant HUT78B1 cell lines . These properties, together with others previously described (e.g . anti-metastatic and anti-osteolytic activities), suggest that CMT-8 may have important applications in the clinical management of cancer . The comparative analysis of structure-activity relationship of CMT-8 and doxycycline suggests that the C-5 hydroxy moiety may play an important role in conferring activity in multidrug resistant cells . These findings appear to support the hypothesis that CMT-8 may represent an interesting lead for the development of a new class of potent apoptosis inducer agents active in multidrug resistant and Fas-ligand resistant malignancies.

Hepatol Res, 2001 Jun, 20(2), 221 - 231
Biliary excretion of phenolphthalein glucuronide in the rat; Ogasawara T et al.; To examine the substrate specificity of an ATP-dependent organic anion transporter, the multidrug resistance protein 2, we examined the effects of various bile acid conjugates and organic anions on the biliary excretion of phenolphthalein glucuronide, a hydrophilic glucuronide conjugate, in rats . Biliary phenolphthalein glucuronide excretion was markedly inhibited by taurolithocholate-3-sulfate and ursodeoxycholate-3-O-glucuronide . In contrast, ursodeoxycholate-3,7-disulfate and pravastatin only slightly inhibited and cefpiramide did not inhibit biliary phenolphthalein glucuronide excretion . Biliary excretion of sulfobromophthalein, leukotriene C(4) and pravastatin was inhibited by phenolphthalein glucuronide infusion to some extent . These findings suggest that phenolphthalein glucuronide is a relatively low affinity substrate for the multidrug resistance protein 2.

Hepatol Res, 2001 Jun, 20(2), 216 - 220
Biliary excretion of temocapril in zone 1- and zone 3-injured rat; Takikawa H et al.; Temocapril is a prodrug of an angiotensin-converting enzyme inhibitor, temocaprilat, a substrate of multidrug resistance protein 2 . Hepatocytes in zone 1 play a role in the uptake and biliary excretion of bile acids under physiological condition, and those in zone 3 may play a role only with their high-dose load . To investigate the pharmacokinetics of temocapril in liver injury, biliary excretion of temocapril was studied in zone 1- and zone 3-injured rats, caused by allyl alcohol and bromobenzene, respectively . Biliary excretion of a tracer dose of radiolabeled temocapril was delayed both in zone 1 and the zone 3 injury, but the extent of inhibition was more prominent in zone 3 injury . Since biliary excretion of organic anions was decreased only in zone 1 injury in our previous study, the present findings indicate that decreased biliary excretion of temocaprilat in zone 3 injury is caused by the inhibition of the metabolism from temocapril to temocaprilat.

Farmaco, 2001 Jan-Feb, 56(1-2), 145 - 8
Physicochemical properties in pharmacokinetic lead optimization; Kramer SD et al.; The ADME (absorption, distribution in the body, metabolism and elimination from the body) profile of a drug determines its pharmacokinetics in the body . Modern drug design includes the modeling of pharmacokinetically favorable behavior . The pharmacokinetic parameters of most interest concern intestinal absorption, blood-brain barrier (BBB) passage and metabolism . Traditionally, experimental parameters such as partition coefficients and chromatographic capacity factors have been used for the estimation of intestinal absorption or BBB passage of newly synthesized compounds . Several studies have shown a sigmoidal relationship between intestinal absorption and lipophilicity . The latter is usually expressed by the apparent partition coefficient log D in a biphasic system at physiological pH or by the affinity to a lipophilic phase determined by chromatographic techniques . In contrast, structure-based descriptors need no experimental investigation of the compound studied . The most relevant descriptors give information on hydrogen-bonding characteristics and molecular volume . In recent years, attempts have been made to recognize substrates for multidrug resistance proteins by their structure characteristics without crucial success . There is evidence that multidrug resistance is not only driven by direct protein-substrate recognition, but also by the behavior of the compound in the lipid environment of the protein.

Folia Med (Plovdiv), 2000, 42(3), 5 - 10
Cytogenetic abnormalities in chronic lymphocytic leukemia; Karnolsky IN; Trisomy of chromosome 12 is one of the commonest cytogenetic abnormalities in the karyotype in chronic lymphocytic leukemia (CLL) . It is associated with atypical morphology of lymphocytes, progressing disease and poor survival . A high incidence abnormality in the B-cell CLL is deletion of chromosome 13 (13q14) detected by using modern diagnostic methods such as southern blot hybridization and fluorescence in situ hybridization . It occurs in 51% of the CLL patients and in as much as 70% in mantle-cell lymphoma . The deletion of 13q14.3 affects a locus telomeric to the RB1 gene (retinoblastoma gene) and the marker D13S25 which bear relation to a candidate tumour suppressor gene . Also common are the chromosome 14 abnormalities which are expressed as the translocation t(11;14)(q13;q32) and which correlate with a high leukocytes count, adverse response to cytostatic therapy and increased risk of prolymphocytic proliferation . The oncogene BCL-1 is activated in this translocation . Deletions of the long arm of chromosome 18 (18q21)(q32;q13.1) activate the BCL-2 oncogene, while the translocation t(14;19)(q32;q13.1) activates the BCL-3 oncogene . Essential role in the pathogenesis of CLL is played by the aberrations in chromosome 17 and the p53 mutations (17p13.1) . The gene p53 is defined as a tumour suppressor gene; mutations of this gene leads to a CLL characterized with rapid progression, aggressive course, poor prognosis and low survival . The deletions in chromosome 7 are associated with the multidrug resistance gene which causes resistance to doxorubicin, vinblastine and colchicine . All these abnormalities are characteristic of the B-cell chronic lymphocytic leukemia . In the T-cell leukemia characteristic deletions are 11q22-q23, a.14q23.1, as well as the inversion inv(14)(11q32) and some rarer aberrations.

Cancer, 2001 May 15, 91(10), 1940 - 8
Expression of P-glycoprotein, multidrug resistance-associated protein 1, and lung resistance-related protein in human soft tissue sarcomas before and after hyperthermic isolated limb perfusion with tumor necrosis factor-alpha and melphalan; Komdeur R et al.; BACKGROUND: Multidrug resistance (MDR) is associated with expression of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) . Tumor necrosis factor (TNF-alpha) is able to modify the expression of these three proteins in different cell types . The effect of TNF-alpha in the clinical situation on patients with soft tissue sarcomas (STS) is indeterminate . METHODS: Thirty-seven patients with a locally advanced extremity STS underwent hyperthermic isolated limb perfusion (HILP) with TNF-alpha and melphalan; 15 patients received additional interferon gamma . Clinical and histologic responses were documented and used to define the overall response . Samples before and after HILP were analyzed immunohistochemically for P-gp, MRP1, and LRP . Samples were scored as negative or positive (< or = 5% or > 5% positive tumor cells) . RESULTS: Six patients had an overall complete response, 25 patients had a partial response, and 4 patients with STS revealed no change; in 2 patients, the response remained unclear . The percentage STS samples that were positive for all three proteins dropped from 92% before HILP to 85% after HILP . P-gp positive samples were encountered more often than MRP1 positive samples (P < 0.05) . The percentage of samples that were negative for all three MDR proteins increased after HILP from 6% to 16% . MDR status had no significant correlation with tumor response . CONCLUSIONS: HILP with TNF-alpha and melphalan results in excellent overall tumor response in patients with locally advanced STS . STS more often are positive for P-gp than for MRP1 . MDR status in patients with STS is not predictive for tumor response after HILP . Data from the current study suggest that the combination of TNF-alpha and melphalan does not induce MDR positive STS: a result with clinical importance when consecutive, adjuvant, doxorubicin-containing chemotherapy is considered .

Jpn J Cancer Res, 2001 Apr, 92(4), 452 - 8
Expression of multidrug resistance-related transporters in human breast carcinoma; Kanzaki A et al.; The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin-based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo . The mRNA levels were determined using a semi-quantitative reverse-transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells . The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene . The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes . These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor . The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin-based chemotherapy . P-glycoprotein (P-gp) / MDR1, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.

Cancer Chemother Pharmacol, 2001 Apr, 47(4), 327 - 32
Continuous infusion prochlorperazine: pharmacokinetics, antiemetic efficacy, and feasibility of high-dose therapy; Morgan RJ Jr et al.; PURPOSE: The purpose of these sequential phase I studies was to evaluate the antiemetic efficacy and pharmacokinetics of high-dose continuous infusion prochlorperazine . METHODS: A total of 52 patients with advanced cancer were treated in two sequential phase I studies utilizing high-dose prochlorperazine . In study 1, designed to investigate the antiemetic effects of dose-intensive prochlorperazine, various cisplatin-based multiagent chemotherapeutic regimens were administered in combination with escalating doses of prochlorperazine . In study 2, a fixed dose of cisplatin (60 mg/m2) was administered over 24 h as a continuous intravenous infusion in combination with infusional high-dose prochlorperazine . Antiemetic efficacy in the first trial was assessed in terms of the number of episodes of nausea, retching, and/or emesis during the 24 h following cisplatin administration . The pharmacokinetics of high-dose prochlorperazine were evaluated in eight patients treated in study 2 at the two dose levels below those at which dose-limiting toxicity was noted . RESULTS: The maximally tolerated dose of prochlorperazine in combination with cisplatin (60 mg/m2 administered as a continuous infusion over 24 h) was 24 mg/h . The dose-limiting toxicity was grade 4 agitation and confusion noted in one patient treated at 26 mg/h . This patient died 3 days following cessation of chemotherapy due to the toxicity of the regimen in combination with the debilitating pulmonary effects of the disease . The mean end of infusion prochlorperazine level at the 24 mg/h dose level was 1.1 microM, a concentration previously reported to be consistent with the reversal of the multidrug resistance phenotype . Two partial responses were observed in study 2 . CONCLUSIONS: We conclude that the antiemetic efficacy of high-dose infusional prochlorperazine does not appear to be improved over more convenient bolus administration . However, prochlorperazine levels consistent with those required in vitro for drug resistance reversal are attainable within the dose range having a tolerable toxicity profile.

Hepatology, 2001 May, 33(5), 1194 - 205
Etiologic significance of defects in cholesterol, phospholipid, and bile acid metabolism in the liver of patients with intrahepatic calculi; Shoda J et al.; Intrahepatic calculi, highly prevalent in the Far East, including Japan, are characterized clinically by chronic proliferative cholangitis with frequent stone recurrences . Intrahepatic calculi consist of 2 groups, i.e., brown pigment stones, including a high cholesterol content, and cholesterol stones, with the former predominating . To gain insights into the pathogenesis of intrahepatic calculi, cholesterol and bile acid biosynthesis, as well as alterations in intracellular transport and/or canalicular secretion of phospholipid and bile acid were investigated in liver of patients with intrahepatic calculi . Enzyme activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase were increased (12.8 +/- 1.9 pmol/min/mg protein, mean +/- SEM vs . 5.5 +/- 0.4 in controls; P < .01) and cholesterol 7 alpha-hydroxylase activities were decreased (1.3 +/- 0.4 vs . 4.9 +/- 0.6; P < .01) in liver specimens of patients with brown pigment stones . In addition, messenger RNA (mRNA) levels of multidrug resistance P-glycoprotein 3 (MDR3 Pgp) and phosphatidylcholine transfer protein (PCTP) were markedly low in the liver specimens compared with the levels in specimens of control subjects, gallbladder stone patients, and patients with obstructive cholestasis . The protein levels and the immunohistochemical staining were decreased for MDR3 Pgp and PCTP in the liver . Consistently, the concentrations of phospholipid were markedly reduced in the hepatic bile from both affected and unaffected hepatic segments . In patients with intrahepatic calculi, biliary cholesterol supersaturation and the formation of cholesterol-rich brown pigment as well as cholesterol stones may be attributed to decreased hepatic transport and biliary secretion of phospholipids, in the setting of increased cholesterogenesis and decreased bile acid synthesis.

J Infect Dis, 2001 Jun 1, 183(11), 1688 - 93 Epub 2001 Apr 23.
Vertical transmission of multidrug-resistant human immunodeficiency virus type 1 (HIV-1) and continued evolution of drug resistance in an HIV-1-infected infant; Johnson VA et al.; To confirm the vertical transmission of multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) and to assess its impact on further evolution of drug-resistant virus in an infant, proviral DNA amplified from infected peripheral blood mononuclear cell cultures was sequenced to identify reverse transcriptase (RT) and protease (PR) mutations . The infant had proviral DNA with evidence of RT mutations (M41L, L74V, and T215Y) and 3 PR substitutions (K20R, M36I, and V82A) . After delivery, the mother's proviral DNA had the same substitutions . Phylogenetic analyses of these HIV-1 RT and PR sequences indicated epidemiological linkage . Plasma drug susceptibility was determined by using a recombinant virus assay . Plasma HIV-1 obtained after the infant's birth demonstrated reduced susceptibility to zidovudine and ritonavir . Thus, vertical transmission of MDR HIV-1 was demonstrated in the setting of detectable maternal plasma viremia . Further accumulation of broad MDR in the infant's virus to 3 antiretroviral classes occurred, despite postnatal therapy.

Biochim Biophys Acta, 2001 Feb 9, 1510(1-2), 414 - 25
Trifluoperazine induces domain formation in zwitterionic phosphatidylcholine but not in charged phosphatidylglycerol bilayers; Hendrich AB et al.; The interaction of trifluoperazine with the zwitterionic lipids dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine and with anionic dimyristoylphosphatidylglycerol was studied by means of microcalorimetry and fluorescence spectroscopy . Intercalation of drug molecules into the lipid bilayers was confirmed by the observed differential scanning calorimetry peak broadening and the decrease in chain-melting temperatures . For trifluoperazine:lipid mole ratios higher than 0.4 and 0.6 (for dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, respectively) the deconvolution of transition profiles into two Gaussian components was possible, which suggests phase separation in the studied mixtures . Deconvolution of the thermograms was not possible for any of the drug:dimyristoylphosphatidylglycerol mole ratios studied . To confirm the existence of phase separation in trifluoperazine-phosphatidylcholine mixtures fluorescence spectroscopy experiments were performed using Laurdan as a probe . From the generalised polarisation versus excitation wavelength dependences, recorded at different temperatures, we conclude that a phase separation occurs in the gel state of the studied trifluoperazine-phosphatidylcholine mixtures . We attribute the existence of domains in the bilayer to the dissimilar interactions of two protonation forms of trifluoperazine with phosphatidylcholine molecules . Structural defects present at domain boundaries could be related to the trifluoperazine induced increase of membrane permeability and fluidity . This may partially explain the mechanism of multidrug resistance modulation by trifluoperazine.

Orv Hetil, 2001 Apr 22, 142(16), 833 - 7
{Molecular biology examination in chronic lymphocytic leukemia}; Telek B et al.; Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia characterised by the accumulation of monoclonal CD5 + B-lymphocytes . The pathogenesis and the biology of CLL is complex and many details are still unknown . Several molecular biological methods have been used in the investigation of CLL, among them the study of apoptosis appears to be one of the most important . Initial experiences obtained by the spontaneous and fludarabine induced apoptosis, multidrug resistance (MDR)-test and fluorescent in situ hybridization (FISH) are reported by the authors . Apoptosis of CLL cells could be induced by fludarabine, while more studies should be performed to determine the exact role of MDR-test and FISH.

Pharmacogenetics, 2001 Apr, 11(3), 217 - 21
MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity; Ameyaw MM et al.; P-glycoprotein (PGP), the product of the multidrug resistance gene (MDR1), acts as an energy-dependent efflux pump that exports its substrates out of the cell . PGP expression is an important factor regulating absorption of a wide variety of medications . It has also been associated with intrinsic and acquired cross resistance to a number of structurally unrelated anticancer drugs . A single nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene, C3435T, was recently correlated with PGP protein levels and substrate uptake . Individuals homozygous for the T allele have more than four-fold lower PGP expression compared with CC individuals . As overexpression of PGP has been associated with altered drug absorption, therapy-resistant malignancies, and lower concentrations of HIV-1 protease inhibitors, this SNP may provide a useful approach to individualize therapy . To facilitate clinical application throughout the world, 1280 subjects from 10 different ethnic groups were evaluated for this SNP using the polymerase chain reaction-restriction fragment length polymorphism assay and the genotype and allele frequency for each group were ascertained . Marked differences in genotype and allele frequency were apparent between the African populations and the Caucasian/Asian populations (P < 0.0001) . The Ghanaian, Kenyan, African American and Sudanese populations studied had frequencies of 83%, 83%, 84% and 73%, respectively, for the C allele . The British Caucasian, Portuguese, South-west Asian, Chinese, Filipino and Saudi populations had lower frequencies of the C allele compared to the African group (48%, 43%, 34%, 53%, 59%, and 55%, respectively) . The high frequency of the C allele in the African group implies overexpression of PGP and may have important therapeutic and prognostic implications for use of PGP dependent drugs in individuals of African origin.

J Nucl Med, 2001 Apr, 42(4), 646 - 54
In vitro and in vivo tracer characteristics of an established multidrug-resistant human colon cancer cell line; Lorke DE et al.; 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) has been suggested as a tracer for the scintigraphic detection of multidrug resistance (MDR) . The aim of this study was to compare MDR characteristics in vitro and in vivo by immunohistochemic and functional uptake assays in established tumor cell lines cultured and grown in severe combined immunodeficient (SCID) mice . METHODS: The presence of MDR was assessed in vitro in drug-resistant HT-29(mdr1) colon carcinoma cells and in nonresistant HT-29(par) cells by JSB-1 immunohistochemistry, uptake of the fluorescent dye Rhodamine 123, and quantitative measurement of 99mTc-MIBI accumulation . For in vivo imaging, SCID mice bearing subcutaneous xenografts of these cell lines were injected with 99mTc-MIBI and 18F-FDG for scintigraphic and PET examination . After imaging, tumors were analyzed by immunohistochemistry and electron microscopy . RESULTS: All HT-29(mdr1) cells cultured in vitro exhibited distinct JSB-1 immunoreactivity, although to a variable degree, whereas HT-29(par) cells were completely devoid of JSB-1 staining . Rhodamine 123 accumulated poorly in HT-29(mdr1) cells but strongly in HT-29(par) cells . Accumulation of 99mTc-MIBI was 0.05% +/- 0.01% of the activity of the external medium in HT-29(mdr1) cells, but about eight times higher in HT-29(par) cells (0.40% +/- 0.09%), a very low percentage compared with other tumor cell lines . No difference in 201TlCl accumulation was observed between both cell lines . In vivo, neither HT-29(par) nor HT-29(mdr1) tumors grown in SCID mice could be detected by 99mTc-MIBI scintigraphy . In FDG PET, both HT-29(mdr1) and HT-29(par) tumors were clearly visible . FDG uptake was, however, markedly higher in HT-29(par) than in HT-29(mdr1) tumors . Both tumor types were poorly vascularized, as shown histologically . JSB-1 immunoreactivity was absent in all HT-29(par) tumors examined, whereas the majority of HT-29(par) tumor cells were stained . Electron microscopy showed that HT-29(par) tumors contained significantly less mitochondria than hepatocytes of the SCID mouse liver, which displayed high 99mTc-MIBI uptake in our scintigraphy studies . CONCLUSION: Sufficient 99mTc-MIBI uptake is the major prerequisite for distinguishing successfully between drug-resistant and sensitive cells . Negative 99mTc-MIBI scintigrams are not necessarily associated with MDR expression . In some tumors, FDG may be an in vivo marker for MDR as suggested by PET.

Biochem J, 2001 May 15, 356(Pt 1), 71 - 5
Role of glycine-534 and glycine-1179 of human multidrug resistance protein (MDR1) in drug-mediated control of ATP hydrolysis; Szakacs G et al.; The human multidrug resistance protein (MDR1) (P-glycoprotein), a member of the ATP-binding cassette (ABC) family, causes multidrug resistance by an active transport mechanism, which keeps the intracellular level of hydrophobic compounds below a cell-killing threshold . Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D/G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes . These mutations caused a loss of measurable ATPase activity but still allowed ATP binding and the formation of a transition-state intermediate (nucleotide trapping) . In contrast with the wild-type protein, in which substrate drugs accelerate nucleotide trapping, in the ABC signature mutants nucleotide trapping was inhibited by MDR1-substrate drugs, suggesting a miscommunication between the drug-binding site(s) and the catalytic domains . Equivalent mutations of the two catalytic sites resulted in a similar effect, indicating the functional equivalence of the two sites . On the basis of these results and recent structural information on an ABC-ABC dimer {Hopfner, Karcher, Shin, Craig, Arthur, Carney and Tainer (2000) Cell 101, 789-800}, we propose a key role of these glycine residues in the interdomain communication regulating drug-induced ATP hydrolysis.

Pharm Res, 2001 Jan, 18(1), 39 - 44
A human lymphocyte based ex vivo assay to study the effect of drugs on P-glycoprotein (P-gp) function; Parasrampuria DA et al.; PURPOSE: The effect of drugs on P-glycoprotein (P-gp) is normally studied in transfected or overexpressing cell lines derived from tumor cells or animal tissue . We wanted to develop an assay using normal healthy human tissue to study and characterize the drug-transporter interaction . METHODS: Lymphocytes were isolated from healthy human blood . The effect of inhibitors of P-gp (cyclosporine, tacrolimus, verapamil, quinidine, vinblastine) and of other transporters (indomethacin, probenecid, sulfinpyrazone) on intracellular accumulation of rhodamine 123 was evaluated by flow cytometry . RESULTS: The efflux of rhodamine 123 was inhibited by P-gp inhibitors in a saturable, concentration-dependent manner . The potency of inhibition of P-gp was cyclosporine > tacrolimus > quinidine > verapamil > vinblastine . Vinblastine inhibited P-gp at lower concentrations, whereas at high concentrations, there was an activation of rhodamine 123 efflux from lymphocytes . The multidrug resistance associated protein (MRP) inhibitors, sulfinpyrazone and probenecid, did not have any significant effect on intracellular accumulation of rhodamine 123, but indomethacin caused a concentration-dependent increase in retention of rhodamine 123, indicating the involvement of other uncharacterized transporters . CONCLUSIONS: Lymphocytes can serve as a model tissue for studying modulation of P-gp activity by drugs . Both inhibitors and inducers of P-gp activity can be evaluated.

Int J Tuberc Lung Dis, 2001 May, 5(5), 413 - 8
Risk factors for nosocomial transmission of multidrug-resistant tuberculosis due to Mycobacterium bovis among HIV-infected patients; Cobo J et al.; OBJECTIVE: To identify risk factors for transmission of multidrug-resistant tuberculosis (MDR-TB) among hospitalized human immunodeficiency virus (HIV) infected patients exposed during a nosocomial outbreak . DESIGN: Case control study . Cases were HIV-infected patients with MDR-TB due to Mycobacterium bovis (MDR-TBMb) who acquired the disease after exposure to an MDR-TBMb patient in an hospital ward . Controls were HIV-infected patients exposed to a case of MDR-TBMb in an hospital ward but who did not develop MDR-TBMb during the follow-up . RESULTS: Nineteen cases and 31 controls were included . CD4 cell counts were significantly lower among cases . Exposure in the infectious diseases ward or exposure to the index patient were associated with development of MDR-TBMb, while exposure during a single-room hospital stay and exposure in the respiratory isolation ward were associated with non-development of MDR-TBMb . A multiple regression logistic model showed that only a CD4 cell count below 50/microL and exposure to the index patient increased the risk of developing MDR-TBMb (P < 0.05) . Hospitalization in a single room seemed to protect HIV-infected patients from developing nosocomial MDR-TBMb (P = 0.052) . CONCLUSIONS: Over classic risk factors, such as length of exposure or sharing a room with a case, severe immunosuppression independently increases the risk of MDR-TB transmission in the context of a nosocomial MDR-TB outbreak among HIV-infected patients . This information must be considered in the management of tuberculosis outbreaks . Patients with CD4 cell counts below 50/microL should be the principal group targeted for prevention strategies in nosocomial outbreaks.

Anticancer Drugs, 2001 Apr, 12(4), 359 - 67
Antitumor activity of XR5944, a novel and potent topoisomerase poison; Stewart AJ et al.; Inhibitors of topoisomerases are widely used in the treatment of cancer, including inhibitors of topoisomerase I (camptothecin analogs such as irinotecan and topotecan) and topoisomerase II (etoposide and doxorubicin) . The novel bis-phenazine, XR5944, is a joint inhibitor of topoisomerase I and II as shown by the stabilization of topoisomerase-dependent cleavable complexes . XR5944 demonstrated exceptional activity against human and murine tumor cells in vitro and in vivo . In a range of cell lines XR5944 (IC50 0.04-0.4 nM) was significantly more potent than TAS-103, originally proposed as a joint topoisomerase I and II inhibitor, as well as agents specific for topoisomerase I or II (topotecan, doxorubicin and etoposide) . In addition, XR5944 was unaffected by atypical drug resistance and retained significant activity in cells overexpressing P-glycoprotein or multidrug resistance-associated protein . Antitumor efficacy of XR5944 was demonstrated in human carcinoma xenograft models (H69 small cell lung cancer and HT29 colon) . In the HT29 model, which is relatively unresponsive to chemotherapy, XR5944 (15 mg/kg i.v., q4dx3) induced tumor regression in the majority of animals (six of eight), whereas TAS-103, dosed at its maximum tolerated dose (45 mg/kg i.v., q7dx3), only induced a delay in tumor growth compared with control animals . In the H69 model, low doses of XR5944 (5 mg/kg i.v., qdx5/week for 2 weeks or 10-15 mg/kg i.v., q4dx3), induced complete tumor regression in the majority of animals . In contrast, topotecan (20 mg/kg i.v., q4dx3) or etoposide (30 mg/kg i.v., q5dx5) only slowed the tumor growth rate . These studies show that XR5944 is a highly active novel anticancer agent that is well tolerated at efficacious doses.

Eur J Cancer, 2001 May, 37(8), 1041 - 52
Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness; Liang Y et al.; A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650Ml) and a paclitaxel-resistant variant (RPMI-2650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650, were established . The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated . Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650Ml cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant . Increased expression of alpha(2), alpha(5), alpha(6), beta(1) and beta(4) integrin subunits, decreased expression of alpha(4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-2650Ml cells . Decreased expression of the alpha(2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant . These results suggest that melphalan exposure can result in not only a MDR phenotype, but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells.

Int J Tuberc Lung Dis, 2001 Apr, 5(4), 329 - 38
Evaluation of tuberculosis control by periodic or routine susceptibility testing in previously treated cases; Van Deun A et al.; SETTING: A national tuberculosis control programme (NTP) disposing of baseline drug resistance rates and using 2EHRZ/6TH in the treatment of new cases . OBJECTIVE: To estimate the extent of drug resistance created by the NTP . DESIGN: Resistance rates in 2EHRZ/6TH failure and relapse cases were compared to baseline, and resistance profiles of repeat isolates were checked . Numbers of observed resistant failures were compared to numbers expected due to pre-existing resistance . Trends of resistance in combined new and previously treated cases were extrapolated . RESULTS: High drug resistance rates were observed . Changes in resistance to streptomycin, the virtual absence of documented acquired resistance and a close match of observed with expected resistant failures all indicated accumulation of primary drug resistance as the main mechanism . Resistance in relapse/failure cases showed a significantly declining trend, and estimated combined drug resistance decreased rapidly . CONCLUSIONS: Drug resistance in previously treated cases seems to consist of passed-on primary rather than true acquired resistance . A one-time survey is thus confusing, but continuous routine testing may constitute the best drug resistance monitoring method . Cases previously treated with short-course chemotherapy may show drug resistance much more frequently than generally assumed, and all should receive a re-treatment regimen . The 2EHRZ/6TH regimen proved very safe under field conditions, causing no 'amplification' towards multidrug resistance and almost no acquired isoniazid resistance . Implementation of this regimen, together with a standardised re-treatment regimen, seemed to rapidly reduce isoniazid as well as multidrug resistance levels, despite the fact that directly observed treatment was not strictly applied.

Int J Tuberc Lung Dis, 2001 Apr, 5(4), 321 - 8
Infection and disease among household contacts of patients with multidrug-resistant tuberculosis; Teixeira L et al.; SETTING: Urban public teaching and referral hospital in Espirito Santo, Brazil . OBJECTIVE: To assess whether rates of infection and progression to active tuberculosis (TB) differed between household contacts of patients with multidrug-resistant (MDR) and drug susceptible (DS) pulmonary tuberculosis . DESIGN: Household contacts were assessed for evidence of TB infection and disease by purified protein derivative (PPD) skin testing, physical examination, chest X-ray, and sputum smear and culture . RESULTS: Among 133 close contacts of patients with MDR-TB, 44% were PPD-positive (> or =10 mm) compared to 37% of 231 contacts of the DS-TB cases (P = 0.18, chi2 test, OR 1.2, 95%CI 0.8-2) . In a multivariate logistic regression analysis, after allowance for between-household variation in PPD responses, PPD positivity among household contacts of patients with MDR-TB remained comparable to PPD positivity in contacts of patients with DS-TB (OR 2.1, 95%CI 0.7-6.5) . Respectively six (4%) and 11 (4%) contacts of the MDR- and DS-TB cases were found to have active TB at the time of initial evaluation or during follow-up (P = 0.78, chi2 test) . Five of six contacts of MDR-TB cases and nine of nine contacts of DS-TB cases who developed TB, and for whom drug susceptibility test results were available, had the same bacterial susceptibility profiles as their index cases . DNA fingerprinting analysis of Mycobacterium tuberculosis isolates was identical between household contacts with active TB and the index MDR or DS-TB case for all 14 pairs compared . CONCLUSION: Our data suggest that the prevalence of tuberculous infection and progression to active TB among household contacts exposed to DS and MDR-TB cases is comparable, despite a longer duration of exposure of contacts to the index case in patients with MDR-TB.

Int J Tuberc Lung Dis, 2001 Apr, 5(4), 313 - 20
A clinic-based molecular epidemiologic study of tuberculosis in Monterrey, Mexico; Yang ZH et al.; SETTING: A tuberculosis clinic associated with a university hospital in Monterrey, Mexico, an urban community with high tuberculosis incidence . OBJECTIVE: To determine the diversity of DNA fingerprint patterns and the extent of drug resistance of Mycobacterium tuberculosis isolates from patients who attended the clinic . DESIGN: Isolates of M . tuberculosis obtained from 186 patients during the period from 31 January 1996 to 31 March 1998 were tested for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin . Demographic data and the social history of each patient were obtained prospectively by interview . The IS6110 DNA fingerprints were obtained for 166 of the 186 isolates . Secondary typing was carried out on isolates with fewer than six copies of IS6110 . RESULTS: Thirty-two per cent of the tested isolates (60/ 186) were drug-resistant, and 18% (33/186) were multidrug-resistant . Approximately 55% of the resistant isolates (33/60) were attributed to acquired resistance . A total of 106 different IS6110 fingerprint patterns were observed among the 166 fingerprinted isolates . Based on both IS6110 and pTBN12 fingerprinting, 65 (39%) of the 166 isolates were part of 22 DNA fingerprint clusters . Various drug susceptibility patterns were seen in most clusters . CONCLUSION: Fingerprint clustering indicates extensive recent transmission of tuberculosis in patients attending the clinic . The prevalence of drug-resistant tuberculosis is high.

Cancer Gene Ther, 2001 Mar, 8(3), 185 - 92
Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance-associated ABC transporter BCRP/MXR/ABCG2; Kowalski P et al.; Breast cancer resistance protein (BCRP) is a recently identified new member of the superfamily of ATP-binding cassette transporters . BCRP is a "half transporter" that may homo- or heterodimerize to form an active transport complex . A considerable overexpression of BCRP was reported from various atypical multidrug-resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone . Thus, BCRP represents a very interesting candidate molecule for reversal of a drug-resistant phenotype . Six hammerhead ribozymes directed against the BCRP-encoding mRNA were designed and tested for their ability to cleave their target molecule . The anti-BCRP ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence . BCRP-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using total RNA prepared from the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV exhibiting a high BCRP mRNA expression level . One anti-BCRP ribozyme was found to show a very high endoribonucleolytic cleavage activity at physiologic pH and temperature . This ribozyme was characterized in a cell-free system with regard to its specific kinetic parameters using large target molecules.

Int J Lepr Other Mycobact Dis, 2000 Dec, 68(4), 452 - 5
A Mycobacterium leprae isolate resistant to dapsone, rifampin, ofloxacin and sparfloxacin; Matsuoka M et al.; Mycobacterium leprae were isolated from a Japanese patient, and susceptibility to antileprosy drugs was examined by the mouse foot pad method . The isolate was susceptible to clofazimine and clarithromycin, and resistant to dapsone, rifampin, ofloxacin and sparfloxacin . Mutations were identified in the genes associated with resistance to these drugs . The risk of the emergence of leprosy with multidrug resistance is emphasized.

Biochem Pharmacol, 2001 Jun 1, 61(11), 1401 - 8
Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines; Bergman AM et al.; Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR) . However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C) . H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively . MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively . dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C . In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells . The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants . These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells) . Ara-CTP accumulation was not detectable in either H69 variant . The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger . The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants . Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase . This might be related to the lack of ara-CTP in H69 variants . In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation . In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.

Biochem Pharmacol, 2001 Jun 1, 61(11), 1393 - 9
Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells; Green LJ et al.; Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins . The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp) . The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-{4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl}-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes . We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells . Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR . Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM) . We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood . These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.

Biochem Pharmacol, 2001 Jun 1, 61(11), 1387 - 91
Resistance of human multidrug resistance-associated protein 1-overexpressing lung tumor cells to the anticancer drug arsenic trioxide; Vernhet L et al.; The human multidrug-resistance protein (MRP1) confers resistance to some heavy metals such as arsenic and antimony, mainly through mediating an increased cellular efflux of metal . However, it was recently suggested that arsenic, used under its trioxide derivative form as anticancer drug, is not handled by MRP1 . The aim of the present study was to test this hypothesis in MRP1-overexpressing human lung tumor GLC4/Sb30 cells . Using the cytotoxicity MTT assay, GLC4/Sb30 cells were found to be 10.8-fold more resistant to arsenic trioxide (As2O3) than parental GLC4 cells . MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells, but did not alter the sensitivity of GLC4 cells . Moreover, As2O3-loaded GLC4/Sb30 cells poorly accumulated arsenic through an increased MK571-sensitive efflux of metal . Finally, depletion of cellular glutathione levels in buthionine sulfoximine-treated GLC4/Sb30 cells was found to result in increased accumulation and reduced efflux of arsenic in cells exposed to As2O3, outlining the glutathione-dependence of MRP1-mediated transport of the metal . These results indicate that MRP1 overexpression in human tumor cells can confer resistance to As2O3, which may limit the clinical use of this anticancer drug for treatment of MRP1-positive tumors.

J Interferon Cytokine Res, 2001 Mar, 21(3), 187 - 96
Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M . avium complex; Takii T et al.; Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB) . In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines . When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M . tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence . Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria . MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined . The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts . This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M . avium complex (MAC) . MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity . Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13) . Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

Biochemistry, 2001 May 8, 40(18), 5542 - 7
Hypersensitization of tumor cells to glycolytic inhibitors; Liu H et al.; The slow growth of cells in the inner core of solid tumors presents a form of multidrug resistance to most of the standard chemotherapeutic agents, which target the outer more rapidly dividing cells . However, the anaerobic environment of the more centrally located tumor cells also provides an opportunity to exploit their dependence on glycolysis for therapeutic gain . We have developed two in vitro models to investigate this possibility . Model A represents osteosarcoma wild-type (wt) cells treated with agents which inhibit mitochondrial oxidative phosphorylation (Oxphos) by interacting with complexes I, III, and V of the electron transport chain in different ways, i.e., rhodamine 123 (Rho 123), rotenone, antimycin A, and oligomycin . All of these agents were found to hypersensitize wt cells to the glycolytic inhibitor 2-deoxyglucose . Cells treated with Rho 123 also become hypersensitive to oxamate, an analogue of pyruvate, which blocks the step of glycolysis that converts pyruvate to lactic acid . Model B is rho(0) cells which have lost their mitochondrial DNA and therefore cannot undergo Oxphos . These cells are 10 and 4.9 times more sensitive to 2-deoxyglucose and oxamate, respectively, than wt cells . Lactic acid levels, which are a measure of anaerobic metabolism, were found to be > 3 times higher in rho(0) than in wt cells . Moreover, when wt cells were treated with Rho 123, lactic acid amounts increased as a function of increasing Rho 123 doses . Under similar Rho 123 treatment, rho(0) cells did not increase their lactic acid levels . These data confirm that cell models A and B are similarly sensitive to glycolytic inhibitors due to their dependence on anaerobic metabolism . Overall, our in vitro results suggest that glycolytic inhibitors could be used to specifically target the slow-growing cells of a tumor and thereby increase the efficacy of current chemotherapeutic and irradiation protocols designed to kill rapidly dividing cells . Moreover, glycolytic inhibitors could be particularly useful in combination with anti-angiogenic agents, which, a priori, should make tumors more anaerobic.

Gan To Kagaku Ryoho, 2001 Apr, 28(4), 505 - 9
{Fundamental study of combination chemotherapy with THP, 5-FU and CDDP for human KB carcinoma cell line and its multidrug resistant cell line KB-C1--usefulness of treatment with 5-FU preceding CDDP}; Kishimoto H et al.; This study was designed to investigate the usefulness of treatment with 5-FU preceding CDDP in combination chemotherapy with THP, 5-FU and CDDP . Using the human KB carcinoma cell line and its multidrug resistant cell line KB-C1, the difference in the antitumor effect due to the sequence of administration of CDDP and 5-FU (TCF or TFC) was examined on cultured cells and nude mouse tumor xenografts . When KB and KB-C1 were treated with THP (0.01 microgram/ml) on day 1, CDDP (0.05 microgram/ml) on day 2 or day 4 and 5-FU (0.25 microgram/ml) on day 3 and 4 or day 2 and 3, TFC suppressed the cell proliferation more strongly than TCF (p < 0.05), though there was no difference between KB and KB-C1 . In nude mouse xenografts, intraperitoneal administrations of THP (0.5 mg/kg) on day 1, CDDP (2 mg/kg) on day 2 or day 5, and 5-FU (10 mg/kg) on day 3-5 or day 2-4 inhibited tumor growth more effectively in KB than in KB-C1 . At three weeks postadministration, growth inhibition by TCF and TFC was 29.9% and 57.4% in KB and 25.2% and 49.8% in KB-C1, respectively . These results indicate that TFC was superior to TCF in cytocidal and antitumor effects for KB and KB-C1.

J Antimicrob Chemother, 2001 May, 47(5), 505 - 11
Activity of phenothiazines against antibiotic-resistant Mycobacterium tuberculosis: a review supporting further studies that may elucidate the potential use of thioridazine as anti-tuberculosis therapy; Amaral L et al.; The in vitro and in vivo anti-mycobacterial activities of a number of phenothiazine compounds are reviewed . These compounds, normally employed for the management of psychosis, inhibit the growth in vitro of Mycobacterium tuberculosis at concentrations that are significantly greater than those that can safely be achieved in a patient harbouring these infections . Nevertheless, one of these phenothiazines, chlorpromazine, is concentrated by human macrophages to 10-100 times its concentration in plasma, and has activity against mycobacteria that have been phagocytosed by these cells . Phenothiazines have significant in vitro activity against susceptible, polydrug- and multidrug-resistant strains of M . tuberculosis, as well as enhancing the activity of some agents employed for first-line treatment . Because thioridazine, the very mild anti-psychotic agent whose most common side effect is drowsiness, has equal anti-tuberculosis properties in vitro to chlorpromazine, we recommend that thioridazine be studied as an adjuvant to the four- or five-drug regimens employed for the management of a freshly diagnosed tuberculosis infection of unknown antibiotic susceptibility, at least during the period required for the assessment of antibiotic susceptibility . Because it also enhances the activity of rifampicin and streptomycin, antibiotics that frequently have adverse effects, additional studies evaluating the use of thioridazine as an adjuvant may eventually allow a reduction in the dosages of these antibiotics and result in a decreased frequency of adverse effects . It is important to note that whereas the management of patients with thioridazine for periods in excess of many months will result in the appearance of some undesirable side effects, its use for a limited period of 2-3 months should not produce side effects that are more severe than simple drowsiness . Nevertheless, further in vitro and in vivo studies are essential before thioridazine may be recommended for the management of select cases of pulmonary tuberculosis.

Biochemistry, 2001 Feb 27, 40(8), 2564 - 71
Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity; Conseil G et al.; P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity . However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance . The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity . The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site . In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction . A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells . The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction . The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors . The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.

Int J Tuberc Lung Dis, 2001 Mar, 5(3), 272 - 7
Pulmonary resection in the treatment of patients with pulmonary multidrug-resistant tuberculosis in Taiwan; Chiang CY et al.; SETTING: Chronic Disease Control Bureau, Department of Health, Taiwan . OBJECTIVE: To evaluate the role of pulmonary resection in the treatment of pulmonary tuberculosis resistant to isoniazid and rifampin (MDR-TB) . DESIGN: In a retrospective cohort study, 27 MDR-TB patients who underwent pulmonary resection between December 1990 and March 1999 were reviewed . Individually-tailored treatment regimens were selected at a once-weekly staff conference following review of the patient's case history and drug susceptibility results . Surgery was performed for selected patients, essentially those: 1) whose medical treatment had failed, or for whom treatment failure seemed highly likely, or for whom post-treatment relapse seemed likely, 2) with predominantly localised disease, 3) with adequate cardiopulmonary reserve, and 4) whose treatment regimen had been composed of at least two effective drugs to diminish the mycobacterial burden . RESULTS: There was no surgical mortality apart from one peri-operative death (4%) . Three patients (11%) developed complications, and 24 (92%) patients demonstrated sputum conversion and/or remained negative after surgery . Twenty-three patients have already completed treatment, and during a mean of 42 +/- 18 follow-up months (range 15-80 months), one patient relapsed . This patient was disease-free after another course of treatment . CONCLUSION: For selected patients, pulmonary resection may improve the outcome of pulmonary MDR-TB.

Anticancer Res, 2000 Nov-Dec, 20(6D), 5139 - 44
Is there any correlation between MDR1, GST-pi-expression and CEA?
Weissenberger C, Fiebig HH, Lutterbach J, Barke A, Momm F, Muller M, Witucki G, Guttenberger R, Berger DP.
BACKGROUND: The levels of mRNA-expression of multidrug resistance (MDR1) and glutathione-S-transferase pi (GST-pi) were measured and correlated with the immunohistochemical expressions of tumour markers . MATERIALS AND METHODS: Analysis of total mRNA was performed by Northern and slot blots . The expression of carcinoembryonic antigen (CEA) and other tumour markers was assessed by immunohistochemistry . The tumour panel comprised tumours of different histologies . RESULTS: CEA-positive tumours showed a significantly higher ex-pression of MDR1 and GST-pi than CEA-negative tumours . Wilcoxon-Test: mean rank of the MDR1 expression (14.3 vs . 7.8; p < 0.05) and GST-pi expression (15.3 vs . 5.9; p < 0.001) . No other correlation could be found . CONCLUSIONS: The relationship of MDR1 and GST-pi with the tumour marker CEA implies that evaluation of CEA can help in discriminating between tumours with high or low expression of drug resistance . Furthermore, correlation between MDR1, GST-pi and CEA indicates that there might be a common mechanism, regulating drug resistance and expression of CEA.

J Biol Chem, 2001 Jun 29, 276(26), 23674 - 80 Epub 2001 Apr 25.
Coordinate control of sphingolipid biosynthesis and multidrug resistance in Saccharomyces cerevisiae; Hallstrom TC et al.; Multiple or pleiotropic drug resistance often occurs in the yeast Saccharomyces cerevisiae through genetic activation of the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p . Hyperactive alleles of these proteins cause overproduction of target genes that include drug efflux pumps, which in turn confer high level drug resistance . Here we provide evidence that both Pdr1p and Pdr3p act to regulate production of an enzyme involved in sphingolipid biosynthesis in S . cerevisiae . The last step in formation of the major sphingolipid in the yeast plasma membrane, mannosyldiinositol phosphorylceramide, is catalyzed by the product of the IPT1 gene, inositol phosphotransferase (Ipt1p) . Transcription of the IPT1 gene is responsive to changes in activity of Pdr1p and Pdr3p . A single Pdr1p/Pdr3p response element is present in the IPT1 promoter and is required for regulation by these factors . Loss of IPT1 has complex effects on drug resistance of the resulting strain, consistent with an important role for mannosyldiinositol phosphorylceramide in normal plasma membrane function . Direct assay for lipid contents of cells demonstrates that changes in sphingolipid composition correlate with changes in the activity of Pdr3p . These data suggest that Pdr1p and Pdr3p may act to modulate the lipid composition of membranes in S . cerevisiae through activation of sphingolipid biosynthesis along with other target genes.

Toxicol Lett, 2001 Mar 31, 120(1-3), 51 - 7
Up-regulation of transporters of the MRP family by drugs and toxins; Schrenk D et al.; Expression of a variety of ABC efflux pumps including certain conjugate transporters of the multidrug resistance protein (MRP) subfamily is inducible in primate and rodent tissues, and in a variety of cell lines and primary cells in culture . In human cell lines (HepG2, MCF-7), we studied the inducibility of MRPs 1-5 . Similar to the rat mrp2 gene, human mrp2 is inducible by the chemical carcinogen 2-AAF, the chemotherapeutic drug cisplatin and the barbiturate phenobarbital, as demonstrated in Northern and Western Blots . Furthermore, the antibiotic rifampicin was identified as MRP2 inducer in HepG2 cells . MRP1 and 4 mRNAs being expressed in human liver at a very low level could not be detected in HepG2 cells after treatment with various agents . However, MRP3 and 5 mRNAs were detected in addition to MRP2 and their expression was found to be increased by 2-AAF, cisplatin and rifampicin . MRP1 expression was studied in MCF-7 cells where the chemotherapeutic drug vinblastine and tert-butyl hydroquinone but not the MRP2 inducing agents described above acted as inducers.

Toxicol Lett, 2001 Mar 31, 120(1-3), 43 - 9
Characterisation of glucuronidation and transport in V79 cells co-expressing UGT1A1 and MRP1; Cuff RL et al.; The co-ordinated glucuronidation and export of compounds from cells is an important determinant in the detoxification of many compounds in vivo . Many UDP-glucuronosyltransferases (UGTs) and several multidrug resistance proteins (MRPs) have been cloned and individually expressed to assess specificity of glucuronidation and transport . However, to further understand the interplay between glucuronidation and transport we are developing stable cell lines that express different combinations of UGT and MRP isoforms . V79 cells expressing both UGT1A1 and MRP1 have been established . The ability of these cell lines to both glucuronidate and transport compounds was assessed ex vivo using estradiol and bilirubin as substrates.

Toxicol Lett, 2001 Mar 31, 120(1-3), 31 - 41
The importance of drug-transporting P-glycoproteins in toxicology; van Tellingen O; The importance of specific transport in toxicology is becoming increasingly clear and the work on P-glycoprotein has certainly been a major contribution to these growing insights . P-Glycoproteins were discovered by their ability to confer multidrug resistance in mammalian tumour cells . They are localised in the cell membrane where they actively extrude a wide range of compounds including many anti-cancer drugs from the cell . Besides in tumour cells, drug-transporting P-glycoproteins are also expressed in a polarised fashion in normal tissues that perform an excretory or barrier function, such as the liver, kidneys, intestines, brain endothelial cells . Based on this expression profile, it has been proposed that P-glycoproteins are important in protecting the host by reducing exposure to xenobiotics . Further studies with P-glycoprotein knockout mice have clearly established this protective function . In general, the clearance of substrate drugs is lower in knockout mice due to a diminished hepatobiliary excretion, direct intestinal excretion and/or increased enterohepatic cycling . Moreover, their uptake in sanctuary sites, such as the brain or the foetus, was profoundly higher in P-glycoprotein knockout mice, as was the uptake of drugs from the gastro-intestinal tract into the systemic circulation following oral ingestion . These results clearly highlight the impact that transport proteins can play in toxicology.

Neurosci Lett, 2001 May 11, 303(3), 189 - 92
Expression of p-glycoprotein is associated with that of multidrug resistance protein 1 (MRP1) in the vestibular labyrinth and endolymphatic sac of the guinea pig; Saito T et al.; Expression of p-glycoprotein (p-gp) and multidrug resistance protein 1 (MRP1) was detected in the vestibular labyrinth and endolymphatic sac (ES) of the guinea pig by immunohistochemical staining using anti-p-gp monoclonal antibody (mAb) C219 and anti-MRP mAb MRPr1 . P-gp was detected in capillary endothelial cells of the crista ampullaris, utricle, saccule and ES . MRP1 was detected in the epithelial lining of the crista ampullaris, utricle, saccule, and epithelial cells of the ES . Since p-gp and MRP1 act as extrusion pumps, they may coordinate with each other in vestibular organs and ES and play an important role in the blood-labyrinth barrier.

FEBS Lett, 2001 Apr 20, 495(1-2), 94 - 9
Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells; Pascolo L et al.; To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of {3H}UCB was studied in the human trophoblastic, BeWo cell line . When plotted against the unbound UCB concentration {Bf}, uptake exhibited saturative kinetics with a similar apparent Km ( approximately 30 nM) for BeWo cells grown either in polarized (Transwell) or non-polarized fashion (dish) . UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance-associated proteins (MRPs) . MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells . These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.

Eur J Biochem, 2001 May, 268(9), 2629 - 34
Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P-glycoprotein; Isenberg B et al.; P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane . Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy . The most commonly used photoreactive substrate is iodoarylazidoprazosin . Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping . In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides . P-glycoprotein-containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin . After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I-labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing . The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248-312 {transmembrane segment (TM)4 to TM5}, 758-800 (beyond TM7 to beyond TM8) and 1160-1218 (after the Walker A motif of the second nucleotide-binding domain) . Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.

Biochem Biophys Res Commun, 2001 Apr 27, 283(1), 64 - 71
Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid; Kim HS et al.; The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells . Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen . This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels . AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100 . AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100 . Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner . Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells . The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT . However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects . Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator . Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro .

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 201 - 6
Multidrug resistance and ABC transporters in parasitic protozoa; Ouellette M et al.; Drug resistance is an important problem in parasitic protozoa . We review here the role of ABC transporters in drug resistance in parasites . We have concentrated on gene and gene products for which there is a strong evidence for their role in resistance.

J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 171 - 7
MdfA, an interesting model protein for studying multidrug transport; Bibi E et al.; The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each can recognize and expel a broad spectrum of chemically unrelated drugs from the cell . Despite extensive research for many years, the actual mechanism of multidrug transport is still largely unknown . In addition to general questions dealing with energy coupling, the molecular view of substrate recognition by Mdrs is generally obscure . This mini-review describes structural and functional properties of the Escherichia coli Mdr, MdfA, and discusses the possibility that this transporter may serve as a model for studying the multidrug recognition phenomenon and the mechanism of multidrug transport.

J Physiol Biochem, 2000 Dec, 56(4), 307 - 12
P-glycoprotein, glutathione and glutathione S-transferase increase in a colon carcinoma cell line by colchicine; Ruiz-Gomez MJ et al.; The acquisition of resistance to anticancer agents used in chemotherapy is the main cause of treatment failure in malignant disorders, provoking tumours to become resistant during treatment, although they initially respond to it . The main multidrug resistance (MDR) mechanism in tumour cells is the expression of P-gly-coprotein (P-gly), that acts as an ATP-dependent active efflux pump of chemotherapeutic agents . Furthermore, an increased detoxification of compounds mediated by high levels of glutathione (GSH) and glutathione S-transferase (GST), has been found in resistant cells . We developed a study aiming to evaluate the evolution of the main drug resistance markers in tumour cells: P-gly, GSH and GST, during the acquisition of resistance to colchicine, for the purpose of studying the adaptation process and its contribution to the MDR phenomenon . A human colon adenocarcinoma cell line was exposed to colchicine during 82 days, being P-gly, GSH levels and GST activity evaluated by flow cytometry, spectrofluorimetry and spectrophotometry, during exposure time . P-gly and GSH levels increased gradually during the exposure to colchicine, reaching 2.35 and 3.21 fold each . On day 82, GST activity increased 1.84 fold at the end of the exposure period . Moreover, an increment in drug cross-resistance was obtained that ranges from 2.62 to 5.22 fold for colchicine, vinblastine, vincristine and mitomycin C . The increments obtained in P-gly, GSH and GST could probably contribute to the MDR phenomenon in this human colon adenocarcinoma cell line.

Cancer Chemother Pharmacol, 2001 Mar, 47(3), 187 - 98
Novel mechanism of cellular DNA topoisomerase II inhibition by the pyranonaphthoquinone derivatives alpha-lapachone and beta-lapachone; Krishnan P et al.; PURPOSE: The mechanisms of intracellular topoisomerase II inhibition by the pyranonaphthoquinone derivatives alpha-lapachone and beta-lapachone were studied . METHODS: Cell-based mechanistic studies were designed based on the in vitro mechanisms {17} and primarily involved the use of cultured KB (nasopharyngeal tumor cells) cells and the etoposide-resistant sub-line KB-7d . RESULTS: The KB-7d cells exhibited collateral sensitivity to alpha-lapachone; this supports the possibility of catalytic inhibition of topoisomerase II in the cells . Interestingly, both compounds induced an increase (two- to threefold) in reversible double-stranded DNA breaks in cell lines with a reduced expression of topoisomerase II . However, these drug-induced DNA breaks became irreversible at treatment times greater than 1 h . Studies showed that DNA breaks in KB-7d cells were not caused by endonucleases . Use of antioxidants abolished the appearance of cellular DNA breaks; this suggests involvement of the oxidation-reduction cycle of pyranonaphthoquinones in topoisomerase II inhibition; however, irreversible DNA breaks were not a result of drug-induced oxidative stress . CONCLUSIONS: On the basis of the findings, it is proposed that the compounds, on longer incubation with cells, induce abortive dissociation of topoisomerase II from the DNA, leading to an irreversible accumulation of high molecular weight DNA fragments . In addition to establishing topoisomerase II as an intracellular target of alpha-lapachone, the results suggest that both compounds can be classified as neither typical poisons nor as typical catalytic inhibitors of the enzyme . In summary, both compounds are members of a new inhibitor class, and alpha-lapachone, in particular, can be considered a potential lead for the development of drugs to treat multidrug-resistant cell lines with lower expression of topoisomerase II.

N Engl J Med, 2001 Apr 26, 344(17), 1294 - 303
Global trends in resistance to antituberculosis drugs . World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance; Espinal MA et al.; BACKGROUND: Data on global trends in resistance to antituberculosis drugs are lacking . METHODS: We expanded the survey conducted by the World Health Organization and the International Union against Tuberculosis and Lung Disease to assess trends in resistance to antituberculosis drugs in countries on six continents . We obtained data using standard protocols from ongoing surveillance or from surveys of representative samples of all patients with tuberculosis . The standard sampling techniques distinguished between new and previously treated patients, and laboratory performance was checked by means of an international program of quality assurance . RESULTS: Between 1996 and 1999, patients in 58 geographic sites were surveyed; 28 sites provided data for at least two years . For patients with newly diagnosed tuberculosis, the frequency of resistance to at least one antituberculosis drug ranged from 1.7 percent in Uruguay to 36.9 percent in Estonia (median, 10.7 percent) . The prevalence increased in Estonia, from 28.2 percent in 1994 to 36.9 percent in 1998 (P=0.01), and in Denmark, from 9.9 percent in 1995 to 13.1 percent in 1998 (P=0.04) . The median prevalence of multidrug resistance among new cases of tuberculosis was only 1.0 percent, but the prevalence was much higherin Estonia (14.1 percent), Henan Province in China (10.8 percent), Latvia (9.0 percent), the Russian oblasts of Ivanovo (9.0 percent) and Tomsk (6.5 percent), Iran (5.0 percent), and Zhejiang Province in China (4.5 percent) . There were significant decreases in multidrug resistance in France and the United States . In Estonia, the prevalence in all cases increased from 11.7 percent in 1994 to 18.1 percent in 1998 (P<0.001) . CONCLUSIONS: Multidrug-resistant tuberculosis continues to be a serious problem, particularly among some countries of eastern Europe . Our survey also identified areas with a high prevalence of multidrug-resistant tuberculosis in such countries as China and Iran.

Ann Pharm Fr, 2001 Apr, 59(2), 85 - 92
{Molecular aspects of chloroquine and antifols resistance in P . falciparum}; Le Bras J et al.; Drug resistant malaria is mostly due to Plasmodium falciparum, a species highly prevalent in tropical Africa, Amazon and Southeast Asia . P . falciparum is responsible for severe involvement of fever or anaemia prompting more than a million deaths per year . The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality in some human populations from endemic regions . Rationale for chemoprophylaxis is becoming week as multiple drug resistance against well tolerated drugs develops . Plasmodium falciparum drug resistant malaria originate from chromosomal mutations . Analysis using molecular, genetic and biochemical approaches has shown that Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among parasites isolates populations while resistance to antifolinics is highly prevalent in most malarial endemic countries . An established and strong drug pressure and a low antiparasitic immunity probably explains the multidrug-resistance encountered in forests of Southeast Asia and South America . In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilise at an equal level as chloroquine-sensitive malaria . Nevertheless, resistance levels may differs according to places and time . In vivo and in vitro tests are insufficient to give an accurate map of resistance . Biochemical tools at a low cost are urgently needed for a prospective monitoring of resistance.

J Infect Dis, 2001 May 15, 183(10), 1535 - 8 Epub 2001 Apr 13.
High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance Gene pfmdr1; Babiker HA et al.; Polymorphisms were examined in 2 Plasmodium falciparum genes, as were chloroquine responses of clones and isolates from a village in eastern Sudan . There was a significant association between an allele of the P . falciparum chloroquine resistance transporter gene (pfcrt-T76) and both in vitro and in vivo resistance . There was a less significant association with the multidrug resistance gene pfmdr1-Y86 allele . A significant association between pfmdr1-Y86 and pfcrt-T76 was apparent among resistant isolates, which suggests a joint action of the 2 genes in high-level chloroquine resistance.

In Vivo, 2001 Mar-Apr, 15(2), 151 - 6
Effects of naturally occurring glucosides, solasodine glucosides, ginsenosides and parishin derivatives on multidrug resistance of lymphoma cells and leukocyte functions; Berek L et al.; Solamargine, solasonine, ginsenosides and parishin-related compounds were investigated for their effects on mdr efflux pump of lymphoma cells, and their effects on T cell proliferative assays and cell mediated immune functions, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity of human peripheral mononuclear cells . Solamargine and solasonine were the only drugs which inhibited all of the tested immune functions; however, ginsenoside Rc and Rd enhanced T cell proliferative assays and marginally increased the NK cell activity . The majority of the compounds were not able to reverse the multidrug resistance of mouse lymphoma cells . However, ginsenosides Rc, Rd and parishin C were able to moderately reduce the activity of the efflux pump . Parishin, parishin C and crude extract significantly enhanced the ADCC reaction.

Clin Infect Dis, 2001 May 15, 32(10), 1463 - 9 Epub 2001 Apr 20.
Carriage of antibiotic-resistant pneumococci among Asian children: a multinational surveillance by the Asian Network for Surveillance of Resistant Pathogens (ANSORP); Lee NY et al.; To investigate the nasal carriage of antibiotic-resistant pneumococci by children, anterior nasal swabs were done for 4963 children <5 years old in 11 countries in Asia and the Middle East . In total, 1105 pneumococci isolates (carriage rate, 22.3%) were collected, 35.8% of which were found to be nonsusceptible to penicillin . Prevalence of penicillin nonsusceptibility was highest in Taiwan (91.3%), followed by Korea (85.8%), Sri Lanka (76.5%), and Vietnam (70.4%) . Penicillin resistance was related to residence in urban areas, enrollment in day care, and a history of otitis media . The most common serogroups were 6 (21.5%), 23 (16.5%), and 19 (15.7%) . The most common clone, as assessed by pulsed-field gel electrophoresis, was identical to the Spanish 23F clone and to strains of invasive isolates from adult patients . Data in this study documented the high rate of penicillin or multidrug resistance among isolates of pneumococci carried nasally in children in Asia and the Middle East and showed that this is due to the spread of a few predominant clones in the region.

AIDS, 2001 Mar 30, 15(5), 591 - 9
Timing of antiretroviral therapy . Use of Markov modeling and decision analysis to evaluate the long-term implications of therapy; Tebas P et al.; BACKGROUND: The timing of initiation of antiretroviral therapy is controversial . Current guidelines are heavily based on the principle of 'hit early, hit hard', although the long-term implications of this approach are unknown . METHODS: Using Markov modeling and decision analysis we modeled the virologic outcomes over 10 years in a hypothetical population of 10 000 HIV-infected patients in which therapy (with the possibility of three sequential regimens before the development of multidrug-resistant virus) is started immediately versus starting progressively at rates of 5, 10, 15, 20 or 30% of the original population each year . The model used inputs from available clinical trial data: virologic success rate and durability of the response of the first and subsequent regimens . We performed one-way and two-way sensitivity analysis to evaluate changes in the input variables . RESULTS: If therapy is started immediately in all patients, by 10 years 57% would be undetectable, but 38% would have detectable multidrug-resistant virus . In contrast, the population as a whole would have had better virologic outcomes if one waited before starting treatment at any progression rate; for example, initiating therapy in 10% of the subjects per year results in 64% of patients being undetectable and 24% with multidrug-resistant virus . Two-way sensitivity analysis demonstrates that immediate initiation should be at least 15 to 20% better than delayed antiretroviral therapy to justify immediate initiation of therapy over a wide range of success rates of the delayed start . CONCLUSION: Our analysis, utilizing optimistic outcomes based on short-term clinical trials, provides a theoretical basis for questioning the current aggressive early use of therapy and should help prompt studies that look at when and how to start antiretroviral therapy.

Pharmacol Ther, 2001 Feb, 89(2), 207 - 19
Mode of action and mechanisms of resistance for antimalarial drugs; Olliaro P; Understanding the mode of action of and mechanism of resistance to drugs is central to optimising their use, and discovering new therapeutics with novel targets . We have limited understanding of how antimalarial drugs work and how resistance emerges . With few exceptions, antimalarial drugs in current use belong to a limited collection of chemical structures that act on a small number of partially characterised biochemical targets . Resistance has emerged to many of these compounds . The use of closely related compounds has promoted the spread of multidrug resistant parasites . This review intends to collate contemporary knowledge, and also to highlight conflicting views on unresolved issues.

Chin J Dent Res, 2000 May, 3(1), 23 - 6
P-glycoprotein expression in squamous cell carcinoma of the oral and maxillofacial region; Xie ZJ et al.; OBJECTIVE: To investigate the mechanism of drug resistance in oral and maxillofacial carcinoma to chemotherapy . MATERIALS AND METHODS: Forty cases of squamous carcinoma in the oral and maxillofacial region were examined for the multidrug resistance gene product P-glycoprotein using a monoclonal antibody, MRK16 . RESULT: P-glycoprotein was detected in 62.5% of all samples . P-glycoprotein expression was related to the chemotherapy and the differentiation of carcinoma . P-glycoprotein expression was higher in the post-chemotherapy group than in the non-chemotherapy group (P < 0.05) . Well-differentiated tumors expressed P-glycoprotein more frequently (P < 0.05) . P-glycoprotein expression was compared with clinical response to chemotherapy . The accuracy rate of prediction was 75% . CONCLUSION: P-glycoprotein plays an important role in mechanism of drug resistance of squamous cell carcinoma in the oral and maxillofacial region.

Oncogene, 2001 Mar 15, 20(11), 1300 - 6
Gene expression and amplification in breast carcinoma cells with intrinsic and acquired doxorubicin resistance; Turton NJ et al.; The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure . Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance . A cluster of overexpressed genes related to DOX resistance was observed . Included in this cluster was ABCB1 the P-glycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target . Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity . In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identified which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway . Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK . The findings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention.

Gene Ther, 2001 Feb, 8(3), 239 - 46
Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells; Knipper R et al.; We describe the functional analysis of a novel retroviral vector, SF91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (MDR1), in hematopoietic cells . SF91m3 combines several promising features . The vector backbone lacks viral coding sequences and AUG-start codons 5' of the MDR1 cDNA . A point mutation of a cryptic splice acceptor of the MDR1 cDNA increases the probability of transferring an intact provirus . The titer of a PG13 packaging cell clone containing a single proviral integration is high (>2 x 10(6) particles/ml from frozen stocks of serum-free vector harvests) . Human hematopoietic cells transduced with SF91m3 reliably express MDR1 before and after passage through NOD/SCID mice, as shown by quantitative PCR and efflux assays with rhodamine 123 or Hoechst 33342 . Finally, SF91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 x IC(50)) . Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

J Biol Chem, 2001 Jun 29, 276(26), 23492 - 8 Epub 2001 Apr 19.
A unique multifunctional transporter translocates estradiol-17beta -glucuronide in rat liver microsomal vesicles; Battaglia E et al.; A wide array of drugs, xenobiotics, and endogenous compounds undergo detoxification by conjugation with glucuronic acid in the liver via the action of UDP-glucuronosyltransferases . The mechanism whereby glucuronides, generated by this enzyme system in the lumen of the endoplasmic reticulum (ER), are exported to the cytosol prior to excretion is unknown . We examined this process in purified rat liver microsomes using a rapid filtration technique and {(3)H}estradiol-17beta-d-glucuronide ({(3)H}E(2)17betaG) as model substrate . Time-dependent uptake of intact {(3)H}E(2)17betaG was observed and shrinkage of ER vesicles by raffinose lowered the steady-state level of {(3)H}E(2)17betaG accumulation . In addition, rapid efflux of {(3)H}E(2)17betaG from rat liver microsomal vesicles suggested that the transport process is bidirectional . Microsomal uptake was saturable with an apparent K(m) and V(max) of 3.29 +/- 0.58 microm and 0.19 +/- 0.02 nmol.min(-1).mg protein(-1), respectively . Transport of {(3)H}E(2)17betaG was inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and probenecid . Specificity of the transport process was investigated by studying the cis-inhibitory effect of anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on the uptake of {(3)H}E(2)17betaG . Collectively, these data are indicative of a novel multifunctional and bidirectional protein carrier for E(2)17betaG and other anionic compounds in the hepatic ER . This intracellular membrane transporter may contribute to the phenomenon of multidrug resistance.

Gastroenterology, 2001 May, 120(6), 1448 - 58
The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood; Jacquemin E et al.; BACKGROUND & AIMS: We have specified the features of progressive familial intrahepatic cholestasis type 3 and investigated in 31 patients whether a defect of the multidrug resistance 3 gene (MDR3) underlies this phenotype . METHODS: MDR3 sequencing, liver MDR3 immunohistochemistry, and biliary phospholipid dosage were performed . RESULTS: Liver histology showed a pattern of biliary cirrhosis with patency of the biliary tree . Age at presentation ranged from the neonatal period to early adulthood . Sequence analysis revealed 16 different mutations in 17 patients . Mutations were identified on both alleles in 12 patients and only on 1 allele in 5 . Four mutations lead to a frame shift, 2 are nonsense, and 10 are missense . An additional missense mutation probably representing a polymorphism was found in 5 patients . MDR3 mutations were associated with abnormal MDR3 canalicular staining and a low proportion of biliary phospholipids . Gallstones or episodes of cholestasis of pregnancy were found in patients or parents . Children with missense mutations had a less severe disease and more often a beneficial effect of ursodeoxycholic acid therapy . CONCLUSIONS: At least one third of the patients with a progressive familial intrahepatic cholestasis type 3 phenotype have a proven defect of MDR3 . This gene defect should also be considered in adult liver diseases.

Gen Hosp Psychiatry, 2001 Mar-Apr, 23(2), 77 - 83
Depression, anxiety comorbidity, and disability in tuberculosis and chronic obstructive pulmonary disease patients: applicability of GHQ-12; Aydin IO et al.; Our aim was to study anxiety and/or depression comorbidity and the influence of these comorbid conditions on disability for 3 clinical groups of pulmonary tuberculosis and chronic obstructive pulmonary disease (COPD) . We also investigated the applicability of General Health Questionnaire 12 (GHQ12) for these clinical groups as a simple screening test for psychiatric comorbidity . A total of 157 male inpatients were included in the study: 42 with recently diagnosed (RDtb), 39 with defaulted (Dtb), 39 with multidrug resistant tuberculosis (MDRtb) and 38 with COPD . The presence of depression and anxiety was assessed by Composite International Diagnostic Interview (CIDI) . Disability was evaluated by Brief Disability Questionnaire . The validity of GHQ12 for the study groups was examined in order to determine a functional cut-off point . Depression and/or anxiety comorbidity was 19% for RDtb, 21.6% for Dtb, 25.6% for MDRtb and 47.3% for COPD . Patients with psychiatric comorbidity had higher disability scores than the group without psychiatric comorbidity . For the tuberculosis group a 3/4 cut-off point of GHQ had 80.7% sensitivity and 87.1% specificity while a 5/6 cut-off point with 83.3% sensitivity and 80% specificity was applicable to the COPD group.

Crit Rev Oncol Hematol, 2001 May, 38(2), 139 - 70
Mechanisms of action of flavopiridol; Sedlacek HH; Flavopiridol inhibits phosphokinases . Its activity is strongest on cyclin dependent kinases (cdk-1, -2, -4, -6, -7) and less on receptor tyrosine kinases (EGFR), receptor associates tyrosine kinases (pp60 Src) and on signal transducing kinases (PKC and Erk-1) . Although the inhibiting activity of flavopiridol is strongest for cdk, the cytotoxic activity of flavopiridol is not limited to cycling cells . Resting cells are also killed . This fact suggests that inhibition of cdks involved in the control of cell cycle is not the only mechanism of action . Inhibition of cdk's with additional functions (i.e . involved in the control of transcription or function of proteins that do not control cell cycle) may contribute to the antitumoral effect . Moreover, direct and indirect inhibition of receptor activation (EGFR) and/or a direct inhibition of kinases (pp60 Src, PKC, Erk-1) involved in the signal transduction pathway could play a role in the antiproliferative activity of flavopiridol . From pharmacokinetic data in patients it can be concluded that the inhibitory activity (IC50) of flavopiridol on these kinases is in the range of concentrations that might be achieved intracellularly after systemic application of non-toxic doses of flavopiridol . However, no in situ data from flavopiridol treated cells have been published yet that prove that by inhibition of EGFR, pp60 Src, PKC and/or Erk-1 (in addition to inhibition of cdk's) flavopiridol is able to induce apoptosis . Thus many questions regarding the detailed mechanism of antitumoral action of flavopiridol are still open . For the design of protocols for future clinical studies this review covers the essential information available on the mechanism of antitumoral activity of flavopiridol . The characteristics of this antitumoral activity include: High rate of apoptosis, especially in leukemic cells; synergy with the antitumoral activity of many cytostatics; independence of its efficacy on pRb, p53 and Bcl-2 expression; lack of interference with the most frequent multidrug resistance proteins (P-glycoprotein and MRP-190); and a strong antiangiogenic activity . Based on these pharmacological data it can be concluded that flavopiridol could be therapeutically active in tumor patients: independent on the genetic status of their tumors or leukemias (i.e . mutations of the pRb and/or p53, amplification of bcl-2); in spite of drug resistance of their tumors induced by first line treatment (and caused by enhanced expression of multidrug resistance proteins); in combination with conventional chemotherapeutics preferentially given prior to flavopiridol; and due to a complex mechanism involving cytotoxicity on cycling and on resting tumor cells, apoptosis and antiangiogenic activity . In consequence, flavopiridol is a highly attractive, new antitumoral compound and deserves further elucidation of its clinical potency.

Mech Ageing Dev, 2001 Mar, 122(3), 255 - 70
Multidrug resistance gene expression during the murine ontogeny; Schiengold M et al.; Multidrug resistance (MDR) was described initially for tumor cells which become resistant not only to the specific drug to which they are submitted, but also to a large range of unrelated drugs . The expression of mdr genes, responsible for the phenotype, and their product P glycoprotein (Pgp), is currently under intensive study due to their ample distribution in different organisms and their possible physiological roles which include protection against xenobiotics . In mice, three mdr isoforms expressed in some normal tissues are known . In this work, we analyzed by RT-PCR the expression of mdr1, mdr2 and mdr3 in several organs of BALB/c and C57BL/6 mice during ontogeny . A considerable variation in mdr expression among individuals of the same strain, as well as among different organs in individuals of the same age group and among different age groups, was detected . We also observed a strong tendency for the expression of a greater number of active isoforms in old mice . The large expression range of the mdr isoforms point to an important role as a natural defense system.

Biochem J, 2001 May 1, 355(Pt 3), 617 - 24
Differential effects of mitomycin C and doxorubicin on P-glycoprotein expression; Maitra R et al.; Previous studies have demonstrated that mitomycin C (MMC) and other DNA cross-linking agents can suppress MDR1 (multidrug resistance 1) gene expression and subsequent functional P-glycoprotein (Pgp) expression, whereas doxorubicin and other anthracyclines increase MDR1 gene expression . In the present study, with stably transfected Madin-Darby canine kidney C7 epithelial cells expressing a human Pgp tagged with green fluorescent protein under the proximal human MDR1 gene promoter, we demonstrated that MMC and doxorubicin have differential effects on Pgp expression and function . Doxorubicin caused a progressive increase in the cell-surface expression of Pgp and function . In contrast, MMC initially increased plasma membrane expression and function at a time when total cellular Pgp was constant and Pgp mRNA expression had been shown to be suppressed . This was followed by a rapid and sustained decrease in cell-surface expression at later times, presumably as a consequence of the initial decrease in mRNA expression . These studies imply that there are at least two independent chemosensitive steps that can alter Pgp biogenesis: one at the level of mRNA transcription and the other at the level of Pgp trafficking . Understanding the combined consequences of these two mechanisms might lead to novel chemotherapeutic approaches to overcoming drug resistance in human cancers by altering either Pgp mRNA expression or trafficking to the membrane.

Biochem J, 2001 May 1, 355(Pt 3), 587 - 95
Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM cells to mitochondria-mediated apoptosis; Matarrese P et al.; Multidrug resistance caused by P-glycoprotein (P-170) is a phenomenon by which cells exposed to a single drug acquire resistance to other structurally and functionally unrelated drugs . This is a widespread phenomenon described in vivo in the management of infectious as well as non-infectious diseases . Several in vitro models have been developed in order to evaluate physiopathological properties of P-170 . Among these are P-170-expressing variants of the human T-lymphoblastoid CEM cell line called VBL100 . As a general rule, drug resistance normally results in resistance to apoptosis induction . By contrast, a paradoxical activity is exerted in this cell model by the cytokine tumour necrosis factor-alpha (TNF-alpha), which is capable of inducing apoptosis in P-170-expressing variants better than in wild-type (wt) cells . In the present study we partially address the mechanisms underlying this activity . In fact, the susceptibility of VBL100 cells to TNF-alpha appears to be specifically due to the depolarization of their mitochondrial membrane, a key factor for apoptotic induction . The same was observed with staurosporine, a specific mitochondrion-mediated proapoptotic chemical probe . Conversely, other proapoptotic stimuli, such as Fas/CD95 or the anti-cancer drug etoposide, did induce significant cell death in wild type cells only . Thus, schematically, mitochondrially dependent stimuli appeared to be more effective in VBL100-cell killing, while 'physiological' stimuli showed the opposite behaviour . Importantly, under steady-state conditions, VBL100 cells displayed per se a mitochondrial membrane hyperpolarization that appeared strictly related to their high susceptibility to specific apoptotic stimuli . In conclusion, the study of a well-established cell model such as that represented by the wt/VBL CEM lymphoid cell line seems to suggest that the multidrug resistance phenotype can specifically sensitize cells towards 'unphysiological', mitochondria-associated cell death cascade or, in the same fashion, it could shift cells from type I (mainly plasma membrane-associated) towards type II (mainly mitochondrial membrane-associated) phenotype.

Acta Biochim Pol, 2000, 47(3), 763 - 72
Transport of organic anions by multidrug resistance-associated protein in the erythrocyte; Rychlik B et al.; The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years . Further studies identifled the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP) . Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.

Acta Biochim Pol, 2000, 47(3), 751 - 62
Transport of glutathione-conjugates in human erythrocytes; Sharma R et al.; The last step of detoxification of both endogenous and environmental toxicants is typically a conjugation that produces a bulky hydrophilic molecule . The excretion of such conjugates out of cells is of sufficient biological importance to have led to the evolution of ATP-driven export pumps for this purpose . The substrate specificity of such transporters is broad, and in some cases it has been shown to include not only anionic conjugates but also neutral or weakly cationic drugs . In the present article, we review the molecular identity, functional and structural characteristics of these pumps, mainly on the example of human erythrocytes, and discuss their physiological role in detoxification and in the multidrug resistance phenotype of cancer cells.

Acta Biochim Pol, 2000, 47(3), 553 - 64
Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance; Bandorowicz-Pikula J; Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase . Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase) . Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains . A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range . Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases . The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains . These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins . It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.

Biochem Cell Biol, 2001, 79(2), 165 - 75
Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells; Lord-Fontaine S et al.; Multidrug resistance is a major obstacle for the successful use of chemotherapy . The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump . We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine . The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay . We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products . Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid . CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells . Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity . Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products . Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses . CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time) . Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells . Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.

Cytometry, 2001 Apr 15, 46(2), 105 - 13
Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate; Olson DP et al.; Resistance to anticancer drugs has been attributed to an array of cellular changes . The multidrug resistance-related protein (MRP1) is an efflux pump whose overexpression confers resistance to several classes of drugs, such as the anthracyclines, epipodophyllotoxins, and vinca alkaloids . These drugs are mainstays in cancer therapy . MRP1 overexpression is hypothesized to be a causative agent of clinical treatment failure . Consistently accurate methods for detecting this protein are necessary to further understand its biology and delineate its possible clinical relevance . Flow cytometric analysis of multidrug resistance (MDR) is a valuable method to evaluate both antigen expression and function . Using flow cytometry, we assayed MRP1 functional activity in pediatric leukemic blasts and an array of MDR+ and WT cell lines . We conclude that calcein AM, when used in a retention assay with MRP1-specific modulators, is able to reliably detect MRP functional activity . 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF AM) transport is not indicative of MRP1 overexpression . .

Semin Respir Infect, 2001 Mar, 16(1), 27 - 37
Cytokine immunotherapy during bacterial pneumonia: from benchtop to bedside; Moore TA et al.; Bacterial pneumonia is a leading cause of morbidity and mortality within the United States and abroad . Although broad-spectrum antibiotic regiments have led to advances in the treatment of pneumonia, this approach has resulted in the recent emergence of multidrug-resistant bacteria . This adverse effect of aggressive antibiotic therapy underscores the importance for understanding the host inflammatory response to pulmonary bacterial infections . This knowledge can then lead to the development of therapeutic modalities aimed at augmenting host responses, resulting in enhanced resolution of bacterial pneumonia . Ideally, future treatment would combine immunoadjuvant and conventional antibiotic therapy for the treatment of life-threatening bacterial pneumonia . The detailed study of several distinct animal models of bacterial pneumonia has identified cytokines involved in eradication of bacteria deposited within the pulmonary airspace . Furthermore, these studies have led to promising preliminary findings in animal models using therapeutic cytokine immunotherapy.

Oncologist, 2001, 6(2), 133 - 46
Chemotherapy of metastatic breast cancer: what to expect in 2001 and beyond; Esteva FJ et al.; Chemotherapy plays an important role in the management of metastatic breast cancer . The anthracyclines (doxorubicin, epirubicin) and the taxanes (paclitaxel, docetaxel) are considered the most active agents for patients with advanced breast cancer . Traditionally, the anthracyclines have been used in combination with cyclophosphamide and 5-fluorouracil (FAC, FEC) . The taxanes have single-agent activity similar to older combination chemotherapy treatments . There is great interest in developing anthracycline/taxane combinations . Capecitabine is indicated for patients who progress after anthracycline and taxane therapy . Vinorelbine and gemcitabine have activity in patients with metastatic breast cancer and are commonly used as third- and fourth-line palliative therapy . The role of high-dose chemotherapy is not well-defined and remains experimental . Novel cytotoxic therapy strategies include the development of anthracycline, taxane, and oral fluoropyrimidine analogues; antifolates; topoisomerase I inhibitors, and multidrug resistance inhibitors . A better understanding of the biology of breast cancer is providing novel treatment approaches . Oncogenes and tumor-supressor genes are emerging as important targets for therapy . Trastuzumab, a monoclonal antibody directed against the Her-2/neu protein, has been shown to prolong survival in patients with metastatic breast cancer . Other novel biologic therapies interfere with signal transduction pathways and angiogenesis . The challenge for the next decade will be to integrate these promising agents in the management of metastatic and primary breast cancer.

Mol Pharmacol, 2001 May, 59(5), 1171 - 80
Modulation of multidrug resistance protein 1 (MRP1/ABCC1) transport and atpase activities by interaction with dietary flavonoids; Leslie EM et al.; The 190-kDa phosphoglycoprotein multidrug resistance protein 1 (MRP1) (ABCC1) confers resistance to a broad spectrum of anticancer drugs and also actively transports certain xenobiotics with reduced glutathione (GSH) (cotransport) as well as conjugated organic anions such as leukotriene C(4) (LTC(4)) . In the present study, we have investigated a series of bioflavonoids for their ability to influence different aspects of MRP1 function . Most flavonoids inhibited MRP1-mediated LTC(4) transport in membrane vesicles and inhibition by several flavonoids was enhanced by GSH . Five of the flavonoids were competitive inhibitors of LTC(4) transport (K(i), 2.4-21 microM) in the following rank order of potency: kaempferol > apigenin (+ GSH) > quercetin > myricetin > naringenin (+ GSH) . These flavonoids were less effective inhibitors of 17beta-estradiol 17beta-(D-glucuronide) transport . Moreover, their rank order of inhibitory potency for this substrate differed from that for LTC(4) transport inhibition but correlated with their relative lipophilicity . Several flavonoids, especially naringenin and apigenin, markedly stimulated GSH transport by MRP1, suggesting they may be cotransported with this tripeptide . Quercetin inhibited the ATPase activity of purified reconstituted MRP1 but stimulated vanadate-induced trapping of 8-azido-alpha-{(32)P}ADP by MRP1 . In contrast, kaempferol and naringenin stimulated both MRP1 ATPase activity and trapping of ADP . In intact MRP1-overexpressing cells, quercetin reduced vincristine resistance from 8.9- to 2.2-fold, whereas kaempferol and naringenin had no effect . We conclude that dietary flavonoids may modulate the organic anion and GSH transport, ATPase, and/or drug resistance-conferring properties of MRP1 . However, the activity profile of the flavonoids tested differed from one another, suggesting that at least some of these compounds may interact with different sites on the MRP1 molecule.

Mol Pharmacol, 2001 May, 59(5), 1077 - 85
Charged amino acids in the transmembrane domains are involved in the determination of the substrate specificity of rat Mrp2; Ito K et al.; Multidrug resistance-associated protein 2 (MRP2) transports glutathione conjugates, glucuronide conjugates, and sulfated conjugates of bile acids . In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, using the isolated membrane vesicles from Sf9 cells infected with the recombinant baculoviruses . By normalizing the transport activity for compounds by that for estradiol 17beta-D-glucuronide (E(2)17betaG), it was indicated that the site-directed mutagenesis from Lys to Met at 325 (K325M) and from Arg to Leu at 586 (R586L) results in a marked reduction in the transport for glutathione conjugates {2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene (LT) C(4)} without affecting that for 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl) benzothiazole glucuronide and taurolithocholate sulfate . In contrast to the reduced affinity for DNP-SG, the affinity for E(2)17betaG was increased severalfold in these mutant Mrp2s, suggesting the amino acids at 325 and 586 play an important role in distinguishing between glutathione and glucuronide conjugates . The comparable affinity for LTD(4), LTE(4), and LTF(4) in these mutant Mrp2s with that in wild-type Mrp2 indicates that recognition of LTC(4) metabolites by Mrp2 is different from that of LTC(4) . The transport activity for glutathione conjugate was retained on R586K, whereas no such complementary cationic amino acid effect was observed in K325R . In addition, R1206M and E1208Q exhibited the loss of transport activity for the tested compounds . The results of the present study demonstrate that the charged amino acids in the transmembrane domain of rat Mrp2 may play an important role in the recognition and/or transport of its substrates.

Cancer Res, 2001 Apr 1, 61(7), 2961 - 7
Unusual potency of BN 80915, a novel fluorinated E-ring modified camptothecin, toward human colon carcinoma cells; Larsen AK et al.; BN 80915 is the lead compound from a novel class of E-ring modified camptothecin analogues, the homocamptothecins, which show potent antitumor activities in animal models . Here, we report that BN 80915 induces up to 2-fold more cleavable complexes between plasmid DNA and purified human topoisomerase I than SN-38 and camptothecin . BN 80915 also induces DNA-topoisomerase I complexes in living HT-29 colon carcinoma cells, as shown by the in vivo link assay . BN 80915 is an extremely potent inducer of DNA-protein complexes in these cells starting at a concentration of 5 nM in the media . BN 80915 is clearly more potent than SN-38, because at least 20 times more SN-38 is needed to induce comparable levels of cleavable complexes . Kinetic experiments show that BN 80915 induces cleavable complexes within minutes that remain stable for at least 6 h in the presence of drug . Whereas the majority of the complexes are reversed within 15 min after drug removal, a substantial fraction (30%) persists for at least 4 h, in contrast with SN-38-treated cells, where all complexes have disappeared by this time . BN 80915 shows strong antiproliferative effects toward HT-29 cells with an IC50 of 0.3 nM compared with 20 nM for SN-38 and 40 nM for topotecan . BN 80915 is also potent against other colon carcinoma cells as well as toward cells growing in three dimensions as multicellular spheroids . HL-60 cells expressing functional P-glycoprotein or multidrug resistance protein show no cross-resistance toward BN 80915 . Taken together, our results show that BN 80915 is unusually potent toward human colon carcinoma cells because of the formation of high levels of stable, covalent DNA-topoisomerase complexes.

Urology, 2001 Apr, 57(4), 650 - 4
Tamoxifen and colchicine-modulated vinblastine followed by 5-fluorouracil in advanced renal cell carcinoma: a phase II study; Liu JH et al.; OBJECTIVES: Chemotherapy resistance of renal cell carcinoma (RCC) has been attributed in large part to multidrug resistance (MDR) . Reported MDR-modulated chemotherapy for RCC, however, has resulted in only marginal response benefits . In this study, the MDR-modulated effect of paired tamoxifen and colchicine on vinblastine and the possible additive effect of 5-fluorouracil (5-FU) were investigated in the treatment of advanced RCC . METHODS: Chemotherapy was administered every 4 weeks with biweekly vinblastine (4 mg/m(2)/day, intravenously on days 1 and 15) modulated by oral tamoxifen (100 mg/day) and colchicine (1 mg/day) from days -1 to 2 and from days 13 to 16 . 5-FU (800 mg/m(2)/day from days 2 to 5) was administered after vinblastine administration as a continuous infusion . RESULTS: Of 17 eligible patients with advanced RCC available for evaluation, 1 achieved a complete response (CR) and 3 a partial response (PR), with an overall response (CR plus PR) rate of 23.5% . The median overall survival time of all patients was 10 months (95% confidence interval {CI} 3.5 to 16.5); that of our patients with poor, intermediate, and favorable risks as stratified by Motzer's model was 6 (95% CI 1.7 to 10.3), 10 (95% CI 7.9 to 12.2), and 26 (95% CI 24.4 to 27.6) months, respectively . These results are encouraging in view of the poor efficacy of chemotherapy in RCC observed previously . Additionally, the treatment toxicity was limited: toxicity of grade 3 or greater occurred in only 1 patient with leukopenia, and no treatment-related mortality was found . CONCLUSIONS: The encouraging response rates and overall survival with limited toxicity warrant further investigation of this combination therapy as an integrated part of immunochemotherapy for RCC.

Breast Cancer Res, 2001, 3(3), 183 - 91 Epub 2001 Feb 01.
Expression of LRP and MDR1 in locally advanced breast cancer predicts axillary node invasion at the time of rescue mastectomy after induction chemotherapy; Schneider J et al.; BACKGROUND: Axillary node status after induction chemotherapy for locally advanced breast cancer has been shown on multivariate analysis to be an independent predictor of relapse . However, it has been postulated that responders to induction chemotherapy with a clinically negative axilla could be spared the burden of lymphadenectomy, because most of them will not show histological nodal invasion . P-glycoprotein expression in the rescue mastectomy specimen has finally been identified as a significant predictor of patient survival . MATERIALS AND METHODS: We studied the expression of the genes encoding multidrug resistance associated protein (MDR1) and lung cancer associated resistance protein (LRP) in formalin-fixed, paraffin-embedded tumor samples from 52 patients treated for locally advanced breast cancer by means of induction chemotherapy followed by rescue mastectomy . P-glycoprotein expression was assessed by means of immunohistochemistry before treatment in 23 cases, and by means of reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after treatment in 46 (6 failed) . LRP expression was detected by means of immunohistochemistry, with the LRP-56 monoclonal antibody, in 31 cases before treatment . Immunohistochemistry for detecting the expression of c-erb-B2, p53, Ki67, estrogen receptor and progesterone receptor are routinely performed in our laboratory in every case, and the results obtained were included in the study . All patients had received between two and six cycles of standard 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) chemotherapy, with two exceptions {one patient received four cycles of a docetaxel-adriamycin combination, and the other four cycles of standard cyclophosphamide-methotrexate-5-fluorouracil (CMF) polychemotherapy} . Response was assessed in accordance with the Response Evaluation Criteria In Solid Tumors (RECIST) . By these, 2 patients achieved a complete clinical response, 37 a partial response, and the remaining 13 showed stable disease . This makes a total clinical response rate of 75.0% . None achieved a complete pathological response . RESULTS: MDR1 mRNA expression detected by RT-PCR was associated with the presence of invaded axillary nodes at surgery in 18/22 cases (81.8%), compared with 13/24 (54.2%) in the group with undetectable MDR1 expression . This difference was statistically significant (P < 0.05) . LRP expression in more than 20% of tumor cells before any treatment was associated with axillary nodal metastasis after chemotherapy and rescue mastectomy in 17/23 cases, compared with 3/8 in nonexpressors . Again, this difference was highly significant (P < 0.01) . LRP expression before treatment and MDR1 mRNA expression after treatment were significantly interrelated (P < 0.001), which might reflect the presence of chemoresistant clones liable to metastasize to the regional nodes . Persistence of previously detected MDR1-positivity after treatment (7/9 compared with 0/2 cases) was significantly associated with axillary node metastasis (P < 0.05) . Finally, in a logistic regression multivariate model, histology other than ductal, a Ki67 labeling index of at least 20% and the combination of LRP and MDR1 positivity emerged as independent predictors of axillary node invasion at the time of rescue mastectomy . CONCLUSION: The expression of different genes involved in resistance to chemotherapy, both before and after treatment with neoadjuvant, is associated with the presence of axillary node invasion at rescue surgery in locally advanced breast cancer . This might reflect the presence of intrinsically resistant clones before any form of therapy, which persist after it, and could be helpful both for prognosis and for the choice of individual treatment.

J Clin Oncol, 2001 Apr 15, 19(8), 2179 - 88
Natural killer/natural killer-like T-cell lymphoma, CD56+, presenting in the skin: an increasingly recognized entity with an aggressive course; Mraz-Gernhard S et al.; PURPOSE: To describe and identify the clinical and pathologic features of prognostic significance for natural killer (NK) and NK-like T-cell (NK/T-cell) lymphoma presenting in the skin . PATIENTS AND METHODS: This study was a retrospective review of 30 patients with CD56+ lymphomas initially presenting with cutaneous lesions, with analysis of clinical and histopathologic parameters . RESULTS: The median survival for all patients was 15 months . Those with extracutaneous manifestations at presentation (11 patients) had a shorter median survival of 7.6 months as compared with those without extracutaneous involvement (17 patients), who had a more favorable median survival of 44.9 months (P =.0001) . Age, gender, extent of cutaneous involvement, and initial response to therapy had no statistically significant effect on survival . Seven patients (24%) had detectable Epstein-Barr virus (EBV) within neoplastic cells . The patients with tumor cells that coexpress CD30 (seven patients) have not yet reached a median survival after 35 months of follow-up as compared with those with CD30- tumor cells (20 patients), who had a median survival of 9.6 months (P <.02) . Routine histopathologic characteristics had no prognostic significance nor did the presence of CD3epsilon, EBV, or multidrug resistance . CONCLUSION: NK/T-cell lymphoma is an aggressive neoplasm; however, a subset with a more favorable outcome is identified in this study . The presence of extracutaneous disease at presentation is the most important clinical variable and portends a poor prognosis . The extent of initial skin involvement does not reliably predict outcome . Patients from the United States with NK/T-cell lymphoma presenting in the skin have a low incidence of demonstrable EBV in their tumor cells . Patients with coexpression of CD30 in CD56 lymphomas tend to have a more favorable outcome.

Drug Metab Dispos, 2001 May, 29(5), 634 - 7
Multidrug resistance p-glycoprotein 2 is essential for the biliary excretion of indocyanine green; Huang L et al.; Multidrug resistance P-glycoprotein 2 (Mdr2) is a phospholipid translocator in the canalicular membrane that is essential for the formation of biliary phospholipid vesicles and mixed lipid/bile salt micelles . Incorporation into biliary vesicles and micelles is thought to contribute to the hepatobiliary excretion of certain hydrophobic organic anions, such as indocyanine green (ICG) . The present studies characterized the biliary excretion of two hydrophobic organic anions, ICG and estradiol-17beta(beta-D-glucuronide) (E(2)17G), in the single-pass isolated perfused liver and the biliary excretion of glutathione (GSH) in vivo in wild-type and Mdr2-/- female mice . The biliary excretion of ICG (0.4 micromol) was reduced by 90%, while the biliary excretion of total GSH was decreased by 65% in Mdr2-/- mice relative to wild-type mice . In contrast, the biliary excretion of E(2)17G (0.1 micromol) was increased by 30% in Mdr2-/- mice . These data indicate that the absence of Mdr2 differentially influences the biliary excretion of these organic anions and suggest that phospholipid vesicles and mixed micelles in bile are essential for the biliary excretion of ICG.

Antimicrob Agents Chemother, 2001 May, 45(5), 1539 - 46
4'-Ethynyl nucleoside analogs: potent inhibitors of multidrug-resistant human immunodeficiency virus variants in vitro; Kodama EI et al.; A series of 4'-ethynyl (4'-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates . Among such analogs examined, 4'-E-2'-deoxycytidine (4'-E-dC), 4'-E-2'-deoxyadenosine (4'-E-dA), 4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine, and 4'-E-2'-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 microM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600) . These 4'-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1(M41L/T215Y), HIV-1(K65R), HIV-1(L74V), HIV-1(M41L/T69S-S-G/T215Y), and HIV-1(A62V/V75I/F77L/F116Y/Q151M) . Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed . The 4'-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1(Y181C), and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays . The antiviral activity of 4'-E-thymidine and 4'-E-dC was blocked by the addition of thymidine and 2'-deoxycytidine, respectively, while that of 4'-E-dA was not affected by 2'-deoxyadenosine, similar to the antiviral activity reversion feature of 2',3'-dideoxynucleosides, strongly suggesting that 4'-E analogs belong to the family of nucleoside reverse transcriptase inhibitors . Further development of 4'-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.

Antimicrob Agents Chemother, 2001 May, 45(5), 1528 - 34
Resistance and adaptation to quinidine in Saccharomyces cerevisiae: role of QDR1 (YIL120w), encoding a plasma membrane transporter of the major facilitator superfamily required for multidrug resistance; Nunes PA et al.; As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant . Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1 . Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress . However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine . Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein . We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment . However, we were not able to prove that Qdr1p is directly implicated in this export . Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1.

Antimicrob Agents Chemother, 2001 May, 45(5), 1467 - 72
Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants Selected with a fluoroquinolone; Jellen-Ritter AS et al.; The development of fluoroquinolone resistance in Escherichia coli may be associated with mutations in regulatory gene loci such as marRAB that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump . We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones from marRAB- or acrAB-inactivated E . coli K-12 strains having a single mutation in the quinolone-resistance-determining region of gyrA . Mutant 3-AG100MKX, obtained from a mar knockout strain after two selection steps, showed enhanced expression of acrB in a reverse transcriptase PCR associated with insertion of IS186 into the AcrAB repressor gene acrR . In vitro selection experiments with acrAB knockout strains yielded MDR mutants after a single step . Enhanced efflux in these mutants was due to increased expression of acrEF and associated with insertion of IS2 into the upstream region of acrEF, presumably creating a hybrid promoter . These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in E . coli.

J Biol Chem, 2001 Jun 22, 276(25), 23197 - 206 Epub 2001 Apr 11.
Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein; Ren XQ et al.; MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells . MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)) . It has been demonstrated that GSH plays an important role in MRP1-mediated MDR . However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known . We synthesized {(125)I}11-azidophenyl agosterol A ({(125)I}azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent . The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4) . Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH . This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1 . The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.

Leuk Res, 2001 May, 25(5), 423 - 31
8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFalpha-induced apoptosis is via inactivation of NF-kappaB; Yin Y et al.; These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects . The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity . CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis . 8-Cl-adenosine increased the cytosolic levels of IkappaBalpha which prevented NF-kappaB nuclear translocation . 8-Cl-adenosine also prevented TNFalpha-induced IkB decay and NF-kappaB activation in CEM and CEM/VLB100 cells.

Anticancer Res, 2001 Jan-Feb, 21(1A), 445 - 50
Reversal of multidrug resistance to epirubicin by cyclosporin A in liposomes or intralipid; Lo YL et al.; Clinical applications of the first-generation multidrug resistance (MDR) modulators, such as cyclosporin A (CsA) have been hampered because of their severe side effects in vivo . In this study, we utilized liposomes and Intralipid to provide selective delivery of CsA to tumor cells as well as to circumvent toxicities associated with CsA by altering the pharmacodistribution properties of encapsulated CsA . The MDR reversing effect of CsA in free, liposomal or Intralipid formulations on the uptake and transport of epirubicin in Caco-2 cells and rat intestines was evaluated . The results showed that CsA in free or liposomal formulations significantly enhanced the intracellular accumulation of epirubicin in a dose-related fashion in Caco-2 cells, with the highest enhancement at 2 microM: These formulations substantially ameliorated the apical to basolateral absorption of epirubicin in Caco-2 cells and markedly increased mucosal to serosal absorption of epirubicin in rat jejunum and ileum . CsA in free, liposomal or Intralipid formulations all significantly reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers . CsA encapsulated in liposomes showed greater enhancement than other formulations . In conclusion, liposomal preparations of CsA may circumvent MDR and have the advantage of diminishing side effects, thus providing a useful alternative dosage form for intravenous administration of CsA to be combined with cytotoxic agents for the treatment of resistant tumors.

Anticancer Res, 2001 Jan-Feb, 21(1A), 317 - 20
Drug resistance modification using pulsing electromagnetic field stimulation for multidrug resistant mouse osteosarcoma cell line; Hirata M et al.; Multidrug resistance (MDR) is one of the major problems in osteosarcoma chemotherapy . Therefore, methods of overcoming MDR are urgently needed . In this study, we investigated the effects of pulsing electromagnetic field stimulation (PEMFs) on a MDR murine osteosarcoma cell line which strongly expresses P-glycoprotein (P-gp) . To assess the reversal effects of PEMFs on doxorubicin (DOX) resistance, MTT assay was applied . Viable cells were assessed by the trypan blue exclusion test . Fluorescence intensity of DOX binding to nuclear DNA of each cell was measured using a cytofluorometer . Changes in P-gp expression in each cell were detected by the indirect immunofluorescence method using an antibody to Pgp . PEMFs increased DOX binding ability to nuclear DNA and inhibited cell growth, although it had no significant effect on P-gp expression . These findings indicated that PEMFs reversed the DOX resistance of the MOS/ADR1 cells by inhibiting P-gp function . The results suggested that PEMFs may be useful as a local treatment for MDR osteosarcoma.

Neurogastroenterol Motil, 2001 Apr, 13(2), 163 - 8
Differential gene expression in the small intestines of wildtype and W/W(V) mice; Takayama I et al.; Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/W(V) mice . These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein . We examined gene expression in the small intestines of wildtype and W/W(V) mice . RNA expression in the jejunums of wildtype and W/W(V) mutants was studied using a differential gene expression METHOD: Seven known genes were differentially expressed in wildtype and W/W(V) mice . COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug-resistance complex, class 4) were suppressed in both fed and fasted W/W(V) mice . Expression of another five genes was increased in W/W(V) mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6-5 (encoding phosphorylcholine {PC} T-cell suppressor factor {TsF}) . Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state . We conclude that several genes are differentially expressed in the small intestines of W/W(V) mice where the major lesion is loss of pacemaker ICC . Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.

Br J Dermatol, 2001 Apr, 144(4), 745 - 50
Chemotherapy induces or increases expression of multidrug resistance-associated protein in malignant melanoma cells; Ichihashi N et al.; BACKGROUND: Human malignant melanoma is notoriously resistant to chemotherapeutic agents . Melanoma-derived cell lines are often markedly chemoresistant, suggesting that cellular mechanisms mediate generation of the multidrug resistance (MDR) phenotype . This phenotype is often due to P-glycoprotein (Pgp) and the MDR-associated protein (MRP), which are drug transporter proteins associated with resistance to a broad spectrum of lipophilic drugs . OBJECTIVES: To determine the relationships between the expression of the MDR gene MDR-1 (the product of which is Pgp) or the MRP gene, and clinical chemoresistance of malignant melanoma . METHODS: We examined changes in the expression of MDR-1 and MRP genes at the mRNA level before and after chemotherapy by reverse transcription-polymerase chain reaction (RT-PCR) analysis using formalin-fixed, paraffin-embedded sections of 18 specimens taken from eight melanoma patients . mRNA expression of the MDR-1 and MRP gene-specific PCR products was quantitatively determined by densitometry and compared with that of an internal standard (beta-actin) . RESULTS: Five of seven primary melanomas were found to express the MRP gene to a certain extent even before chemotherapy . After first and second courses of chemotherapy, six patients had an increased ratio of MRP mRNA to beta-actin mRNA compared with the prechemotherapy levels in the same patients . None of the cases of melanoma expressed MDR-1 . CONCLUSIONS: These results suggest that a significant mRNA level of MRP gene was intrinsically present in malignant melanoma even before exposure to chemotherapeutic drugs and increased in its expression after chemotherapy, suggesting that MRP plays a part in increasing the chemoresistance of malignant melanoma during chemotherapy.

FEBS Lett, 2001 Apr 6, 494(1-2), 99 - 104
A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant; Holzmann K et al.; Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype . We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5'-leader . Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis . L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types . MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts . Our results suggest alternative splicing as one mechanism for regulation of MVP expression.

Clin Cancer Res, 2001 Mar, 7(3), 562 - 9
The prognostic value of molecular marker analysis in patients treated with trimodality therapy for esophageal cancer; Harpole DH Jr et al.; The purpose of this study was to define the prognostic value of a group of molecular tumor markers in a well-staged population of patients treated with trimodality therapy for esophageal cancer . The original pretreatment paraffin-embedded endoscopic esophageal tumor biopsy material was obtained from 118 patients treated with concurrent cisplatin + 5-fluorouracil (5-FU) + 45 Gy radiation followed by resection from 1986 until 1997 at the Duke University Comprehensive Cancer Center . Three markers of possible platinum chemotherapy association {metallothionein (MT), glutathione S-transferase-pi (GST-pi), P-glycoprotein (P-gp or multidrug resistance)} and one marker of possible 5-FU association {thymidylate synthase (TS)} were measured using immunohistochemistry . The median cancer-free survival was 25.0 months, with a significantly improved survival for the 38 patients who had a complete response (P < 0.001) . High-level expression of GST-pi, P-gp, and TS were associated with a decreased survival . MT was not significant in this population . Multivariate analysis identified high-level expression in two of the platinum markers (GST-pi and P-gp) and the 5-FU marker TS as independent predictors of early recurrence and death . In conclusion, this investigation measured three possible markers associated with platinum and one possible marker associated with 5-FU in a cohort of esophageal cancer patients . Independent prognostic significance was observed, which suggests that it may be possible to predict which patients may benefit most from trimodality therapy . These data need to be reproduced in a prospective investigation.

Oncol Rep, 2001 May-Jun, 8(3), 597 - 603
A phase II trial of mitomycin C, 5'-deoxy-5-fluorouridine, etoposide and medroxyprogesterone acetate (McVD-MPA) as a salvage chemotherapy to anthracycline-resistant tumor in relapsed breast cancer and its mechanism(s) of antitumor action; Kim R et al.; To assess the therapeutic efficacy in the combination of mitomycin C (MMC), 5'-deoxy-5-fluorouridine (5'-DFUR), etoposide (VP-16) and medroxyprogesterone acetate (MPA) (McVD-MPA) to anthracycline-resistant tumor as a salvage chemotherapy, a phase II trial was conducted in patients with relapsed breast cancer . Fifty-five patients were enrolled in this trial and 54 were assessable, who had all previously been treated with an anthracycline regimen . The treatment schedule was designed with the intravenous administration of MMC (6 mg/m2) on day 1 followed by peroral administration of VP-16 (75 mg/m2) on day 2, 4, 6 and the peroral administration of 5'-DFUR (600 mg/m2) and MPA (400 mg/m2) on day 1 through 21 in one cycle . The overall tumor response rate was 40.7% (22/54) including 16.6% (9 cases) in complete response and 24.0% (13 cases) in partial response, and the long no change (NC) was observed in 18.5% (10/54) out of 44.4% (24/54) in NC . Of the patients with primary resistance to anthracycline 30.0% responded to McVD-MPA therapy . Bone and liver metastases responded in 50.0% and 50.0%, whereas soft tissue and lung metastases responded in 36.8% and 35.2%, respectively . The mean time to response and response duration were 2.7 and 15.6 months, respectively . The overall survival of the patient treated with the McVD-MPA was superior to the non-treatment of second line therapy, and the median survival between McVD-MPA and non-treatment was 86 days and 50 days, respectively . The major adverse effect was observed in hematological toxicity (31.7%) such as leukopenia and thrombocytopenia and non-hematological toxicity of gastrointestinal events (31.7%), the toxicity was less than grade 2, and was tolerable during the treatment . In the experiment of MDA-MB-231 breast cancer cell line that was overexpressed with P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP), the mechanism(s) by which McVD-MPA induces the antitumor effect to anthracycline-resistant tumor may be explained at least in part as follows: i) The treatment of MMC suppressed the expression of P-gp and MRP in a dose-and time-dependent manner, connecting the increase of the intracellular concentration of VP-16; ii) The treatment of MMC enhanced the expression of thymidine phosphorylase to increase the production of 5-FU from 5'-DFUR in the antiangiogenic effect of MPA . These results indicate that the combination chemotherapy of the McVD-MPA may be an effective regimen to anthracycline-resistant tumor as a salvage chemotherapy to prolong the survival in the patient with relapsed breast cancer.

Emerg Infect Dis, 2001 Mar-Apr, 7(2), 306 - 11
New technology for detecting multidrug-resistant pathogens in the clinical microbiology laboratory; Peterson LR et al.; Northwestern Memorial Hospital instituted in-house molecular typing to rapidly assess microbial clonality and integrated this typing into an infection control program . We compared data on nosocomial infections collected during 24 months before and 60 months after implementing the new program . During the intervention period, infections per 1,000 patient-days fell 13% (p=0.002) and the percentage of hospitalized patients with nosocomial infections decreased 23% (p=0.000006) . In our hospital, the percentage of patients with nosocomial infections is 43% below the U.S . rate . Our typing laboratory costs approximately $400,000 per year, a savings of $5.00 for each dollar spent.

Int Orthop, 2001, 24(6), 307 - 10
The co-expression of p53 protein and P-glycoprotein is correlated to a poor prognosis in osteosarcoma; Park YB et al.; Multidrug resistance (MDR), mediated by P-glycoprotein (P-gp), is an in vitro phenomenon observed within tumour cells, suggesting cross-resistance to unrelated drugs, and expression of P-gp may therefore affect the prognosis and incidence of recurrence after treatment . The mutant p53 protein causes reduced tumour suppression . Co-expression of p53 and P-gp is related to short survival, increased tumour activity and drug resistance . The purpose of this study was to measure the expression of p53 protein and P-gp in osteosarcoma tissue and assess its prognostic significance . Fifty-two tumour specimens were evaluated . The correlation between p53 and P-gp expression was significant (P=0.0008) . In univariate analysis of survival, p53 protein was not significant (P=0.2) but P-gp was significant (P=0.0001) . The co-expression of p53 and P-gp was the strongest indicator of a short survival according to multivariate analysis (P=0.0004).

Ugeskr Laeger, 2001 Mar 26, 163(13), 1842 - 6
{Resistant tuberculosis in Denmark}; Thomsen VO et al.; INTRODUCTION: Increased rates of multidrug-resistant (MDR) tuberculosis (TB) has been reported from countries close to Denmark . We evaluated the incidence of drug resistance in Denmark in order to determine the magnitude of the problem . MATERIALS AND METHODS: Susceptibility testing was performed in isolates from 85.4% of all notified patients during 1991-1998 . Epidemiological information was retrieved from the mandatory notification forms . RESULTS: Total drug resistance remained largely constant, although a minor increase was observed in 1997-1998 . Monoresistance was observed in 7.3% of the isolates . Among 3.6% polyresistant isolates, resistance to isoniazid and streptomycin accounted for 2.8%, whereas MDR accounted for 0.5% . The MDR strains displayed different restriction fragment length polymorphism (RFLP) patterns, and no matches were identified in the international MDR database . Drug resistance in untreated Danes and foreigners were 5.9% and 14.6%, respectively . Among Danes and foreigners with previous TB, 6.2% and 22.7% had drug resistance, respectively . Increased drug resistance was found among untreated Danes aged 25-54 years mainly due to a single isoniazid- and streptomycin-resistant RFLP-cluster . Among all patients with isoniazid- and streptomycin-resistance, 77.0% had clustered strains . DISCUSSION: In conclusion, although drug resistance among untreated Danes was close to the rate estimated in good national programmes, close monitoring is needed in future years, as active transmission of isoniazid- and streptomycin-resistant Mycobacterium tuberculosis was demonstrated.

FASEB J, 2001 Apr, 15(6), 995 - 1005
Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells; Wartenberg M et al.; Tumor vascularization is the rate-limiting step for the progression of cancer . Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells . Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) . In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O(2)-sensitive microelectrodes . After 24--48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days . Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure . Furthermore, tumor vascularization resulted in elevated levels of HIF-1 alpha, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein . Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.

Invest New Drugs, 2001, 19(1), 93 - 100
Excellent response to gemcitabine in a massively pre-treated woman with extensive cutaneous involvement after recurrence of breast cancer; Arends J et al.; A 50-year-old woman presented with local relapse of breast cancer 6 years after partial mastectomy . Relapse was accompanied by extended skin induration due to tumor cell embolization of dermal lymphatics . During the following years the patient was exposed to 11 different anti-tumor regimens including 13 cytotoxic drugs (including alkylating agents, antitumor antibiotics, vinca alcaloids, epipodophyllotoxins, and taxanes), 4 anti-hormonal, and 2 immunologic attempts . Paclitaxel achieved a prolonged local improvement for some 7 months, but further various treatments were ineffective . At that time gemcitabine therapy was initiated and tumor infiltration of the skin was visibly diminished only 2 weeks later . After that tumor regressed further for 5 months and remained stable with continued doses of gemcitabine during much of the woman's last year . The patient died of acute myeloid leukemia (AML) 4 years after the local recurrence of breast cancer . Since multiple treatments using a plethora of aggressive cytotoxic drugs may render several classes of chemotherapy agents ineffective due to cross-resistance, it seems advisable to select mild agents that are not subject to multidrug resistance mechanisms and display a unique mode of action as demonstrated in this case by gemcitabine.

Int J Cancer, 2001 Apr 15, 92(2), 195 - 202
Up-regulation of vaults may be necessary but not sufficient for multidrug resistance; Siva AC et al.; Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w . vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA) . Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein-mediated multidrug resistance (MDR) . To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines . Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells . Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line . Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines . Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype . This implies a requirement of an additional factor(s) for vault-mediated MDR .

Int J Cancer, 2001 Apr 15, 92(2), 187 - 94
Multidrug resistance gene 1 expression in salivary gland adenocarcinomas and oral squamous-cell carcinomas; Uematsu T et al.; In combined chemotherapy for head-and-neck cancer (HNC), salivary gland-cell adenocarcinoma (SGA) shows insufficient clinical outcome, and it has been suggested that the sensitivity and/or the mechanism of resistance to anti-cancer drugs are different between SGA and oral squamous-cell carcinoma (SCC) . The aim of our study was to clarify whether P-glycoprotein (P-gp) expression is associated with multidrug resistance (MDR) in HNC and the difference in the process of its development between SGA and SCC . In immunohistochemical analysis, P-gp expression was found in the ductal cells of salivary glands but not in oral mucosal epithelium . In cancer tissues, a few SCC cells in 12 of 37 and most cells in all SGAs expressed P-gp . The intensive P-gp expression was significantly found in SGA compared with SCC . In an in vivo chemotherapeutic model using tumor-bearing nude mice, P-gp expression in counterparts was observed in only a few cells of the HSY line, while no P-gp expression was observed in Hepd cells . However, P-gp expression was developed in both HSY and Hepd cell lines after vincristine (VCR) treatment . RT-PCR showed that the mean ratios of mdr1 mRNA expression levels in HSY clones were 3.7-fold higher than those in Hepd clones after VCR treatment, while each cell line exhibited both induction and activated production of P-gp . These results suggest that P-gp-related MDR in SGA is an inherent phenotype caused by both high levels of P-gp induction and activated P-gp production during VCR treatment, while that in SCC is an acquired phenotype chiefly caused by induction of P-gp .

Anticancer Drugs, 2001 Mar, 12(3), 267 - 73
Effect of hydroxyzine on the transport of etoposide in rat small intestine; Kan WM et al.; Etoposide, an anti-neoplastic agent and a substrate of P-glycoprotein (P-gp), exhibits variable oral bioavailability . P-gp, the multidrug resistance gene (mdr1) product, has been considered as an absorption barrier against intestinal drug absorption . Terfenadine, an antihistamine, has been shown to be a P-gp inhibitor . The current study was designed to assess the effect of hydroxyzine, an antihistamine, on the transport of etoposide in the small intestine . Everted rat gut sacs were used to determine the absorption and exsorption of etoposide under different conditions, as rhodamine 123 was chosen to evaluate the role of P-gp in the drug interaction . The results showed that the transport of etoposide was significantly increased from the luminal site to the serosal site in the jejunum by 2- and 4-fold after 90 min in the presence of hydroxyzine and quinidine, respectively . A similar trend was observed in the ileal sacs . This in vitro exsorption study also demonstrated that hydroxyzine could reduce the efflux of etoposide to the luminal site in either jejunum or ileum . The effect of hydroxyzine on the pharmacokinetics of etoposide differed by the in vivo route of administration, thus assuming clinical importance for chemotherapeutic treatment.

Anticancer Drugs, 2001 Mar, 12(3), 247 - 58
Sequential gene expression of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and lung resistance protein: functional activity of P-gp and MRP present in the doxorubicin-resistant human K562 cell lines; Grandjean F et al.; Previous studies have reported that P-glycoprotein (P-gp), a transmembrane efflux pump involved in multidrug resistance (MDR), was overexpressed in the doxorubicin (Dox)-resistant human erythroleukemia cell line K562 . Nevertheless, several results suggested that P-gp was not the only mechanism involved in these resistant cells . Sequential co-expression of other MDR-associated proteins was sometimes reported, as MDR-associated protein (MRP) and lung resistance protein (LRP), in different MDR cell lines . Thus, mRNA expression and stability of P-gp, MRP and LRP were analyzed, while their corresponding protein levels were quantified in correlation with functional assay, in the K562 cell line and two Dox-resistant variants (K562/R) . Their P-gp content was in accordance with their degree of resistance, but not as much in the level of mRNA expression, suggesting a post-transcriptional regulation . On the other hand, MRP could play a minor role in MDR because of an unchanged expression in K562/R sublines . A surprising progressive disappearance of LRP in both resistant cells suggested that the original mechanism of drug redistribution may be operative, involving a negative role for LRP.

Anticancer Drugs, 2001 Mar, 12(3), 213 - 20
Preclinical antitumor activity of the azonafide series of anthracene-based DNA intercalators; Dorr RT et al.; The azonafides are a series of anthracene-based DNA intercalators which inhibit tumor cell growth in vitro at low nanomolar concentrations and are not affected by the multidrug resistance phenomenon (MDR) . Prior studies have described antitumor efficacy in murine tumor models including L-1210 and P-388 leukemias, and B-16 melanoma . The current results extend these cell line observations to human tumors tested in the NCI panel of 56 cell lines, in freshly isolated tumors tested in colony-forming assays in soft agar and in several animal models . In the NCI panel, the overall mean 50% cell kill (LC50) for the unsubstituted azonafide, AMP-1, was 10(-5.53) M, with some selectivity noted in melanomas (10(-6.22) M) . The mean LC50 for the 6-ethoxy substituted analog, AMP-53, was 10(-5.53) M, with some selectivity found in non-small cell lung cancer (10(-5.91)) and renal cell carcinoma (10(-5.84)) . In freshly isolated human tumors tested in soft agar, there was marked activity (mean IC50 in microg/ml) for AMP-53 in four cell types: breast cancer (0.09), lung cancer (0.06), renal cell carcinomas (0.06) and multiple myeloma (0.03) . These effects were superior to doxorubicin and to several other azonafides, including AMP-1, AMP-104 and the 6-hydroxyethoxy derivative, AMP-115 . Compound AMP-1 was shown to be superior to amonafide in the mammary 16C breast cancer model in B6CF31 mice, but it had little activity in Colon-38 nor in M5076 ovarian sarcomas in vivo . Nine azonafides were evaluated in the Lewis lung cancer model in C57/bl mice, but only AMP-53 demonstrated significant efficacy with a treated/control x 100% (T/C) value of 30% . Because AMP-53 demonstrated the greatest breadth of activity, it was then evaluated in several human tumor cell lines growing in mice with severe combined immunodeficiency disease (SCID) . Only three tumors were sensitive (T/C<42%), including HL-60 leukemia (T/C=39%), MCF-7 breast cancer (T/C=39%) and A549 non-small cell lung cancer (T/C=37%) . Overall, these results demonstrate that the 6-ethoxy substituted azonafide, AMP-53, has consistent (in vitro and in vivo) experimental antitumor activity in human breast and lung cancer, and could be considered for clinical testing in patients with MDR tumors.

Eur J Cancer, 2001 Mar, 37(5), 660 - 7
Reversal of multidrug resistance and increase in plasma membrane fluidity in CHO cells with R-verapamil and bile salts; Schuldes H et al.; Studies with multidrug resistance modifiers indicate that perturbations of the cell membrane structure may influence P-glycoprotein (P-gp)-mediated drug transport . We describe studies of plasma membrane order using electron-paramagnetic resonance (EPR) in resistant (CH(R)C5) and sensitive (AUXB1) chinese hamster ovary cells treated with R-verapamil and bile salts . Cell growth rates were determined in presence of doxorubicin mitomycin and cisplatin . The plasma membrane order in untreated resistant cells was higher than in the sensitive cells . Both the bile salt taurochenodeoxycholate (TCDC; 0.2-1.6 mM) and R-verapamil (1-3 microM) lowered the membrane order in the CH(R)C5 cells to that in the sensitive cells and reversed the resistance to doxorubicin and mitomycin . The bile salt tauroursodeoxycholate (TUDC; 0.2-3 mM) did not lower membrane order and did not sensitise CH(R)C5 cells . Neither R-verapamil, TCDC nor TUDC reduced the membrane order of the sensitive cells AUXB1 cells . These results support the view that changes in multidrug resistance in Chinese hamster ovary cells and P-gp function are associated with alterations in the fluidity of the plasma membrane.

Cancer Res, 2001 Mar 15, 61(6), 2552 - 7
Peptide transport by the multidrug resistance protein MRP1; de Jong MC et al.; Small hydrophobic peptides were studied as possible substrates of the multidrug resistance protein (MRP)-1 (ABCC1) transmembrane transporter molecule . As observed earlier for P-glycoprotein- (Pgp; ABCB1) overexpressing cells, MRP1-overexpressing cells, including cells stably transfected with the MRP1 cDNA, showed distinct resistance to the cytotoxic peptide N-acetyl-Leu-Leu-norleucinal (ALLN) . Resistance to this peptide and another toxic peptide derivative, which is based on a Thr-His-Thr-Nle-Glu-Gly backbone conjugated to butyl and benzyl groups (4A6), could be reversed by MRP1 inhibitors . The reduced toxicity of 4A6 in MRP1-overexpressing cells was found to be associated with lower accumulation of a fluorescein-labeled derivative of this peptide . Glutathione (GSH) depletion had a clear effect on resistance to ALLN but hardly affected 4A6 resistance . In a limited structure-activity study using peptides that are analogous to 4A6, MRP1-overexpressing cells were found to be resistant to these peptides as well . Remarkably, when selecting A2780 ovarian cancer cells for resistance to ALLN, even in the absence of Pgp blockers, resulting cell lines had up-regulated MRP1, rather than any of the other currently known multidrug resistance transporter molecules including Pgp, MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCCS), and the breast cancer resistance protein ABCG2 . ALLN-resistant, MRP1-overexpressing cells were found to be cross-resistant to 4A6 and the classical multidrug resistance drugs doxorubicin, vincristine, and etoposide . This establishes MRP1 as a transporter for small hydrophobic peptides . More extensive structure-activity relationship studies should allow the identification of clinically useful peptide antagonists of MRP1.

J Pharm Sci, 2001 May, 90(5), 638 - 46
Cytokines influence mRNA expression of cytochrome P450 3A4 and MDRI in intestinal cells; Bertilsson PM et al.; Inflammation and infection may have the potential to increase the bioavailability of drugs . This effect could be because of a reduced metabolism of xenobiotics in the liver and/or the intestines, or because of alterations in small intestinal permeability, mucosal flow, and expression of drug efflux transporters such as P-glycoprotein (Pgp) . To assess the impact on intestinal epithelium of some proinflammatory cytokines and macrophages on permeability and mRNA expression of CYP3A4 and MDRI (multidrug resistance, coding for Pgp), we used the Caco-2 cell line as a model . Exposure to proinflammatory cytokines and macrophages decreased the mRNA expression of CYP3A4 and increased the expression of MDR1 mRNA in the Caco-2 cells . In parallel, the cell layer permeability, as measured by sodium fluorescein flux, increased for all cytokine and macrophage treatments, whereas the effect on transepithelial electrical resistance (TEER) varied . Our findings suggest that inflammation and infection trigger several different cellular responses that may affect drug bioavailability; that is hampered CYP3A4 expression, increased permeability of the epithelial cell layer, and enhanced Pgp-mediated counteractive transport .

J Biol Chem, 2001 Jun 15, 276(24), 21199 - 208 Epub 2001 Apr 03.
Functionally similar vanadate-induced 8-azidoadenosine 5'-{alpha-(32)P}Diphosphate-trapped transition state intermediates of human P-glycoprotin are generated in the absence and presence of ATP hydrolysis; Sauna ZE et al.; P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells . Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation . In this study we compare Vi-induced {alpha-(32)P}8-azido-ADP trapping into Pgp in the presence of {alpha-(32)P}8-azido-ATP (with ATP hydrolysis) or {alpha-(32)P}8-azido-ADP (without ATP hydrolysis) . Vi mimics P(i) to trap the nucleotide tenaciously in the Pgp.{alpha-(32)P}8-azido-ADP.Vi conformation in either condition . Thus, by using {alpha-(32)P}8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis . Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of {alpha-(32)P}8-azido-ATP or {alpha-(32)P}8-azido-ADP . The trapped {alpha-(32)P}8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions . Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of {alpha-(32)P}8-azido-ADP trapping into Pgp in the absence of hydrolysis . These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP) . Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ({(125)I}iodoarylazidoprazosin) as well as nucleotide (2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate) . Analyses of Arrhenius plots show that trapping of Pgp with {alpha-(32)P}8-azido-ADP (in the absence of hydrolysis) displays an approximately 2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of {alpha-(32)P}8-azido-ATP (62 kJ/mol) . In aggregate, these results demonstrate that the Pgp.{alpha-(32)P}8-azido-ADP (or ADP).Vi transition state complexes generated either in the absence of or accompanying {alpha-(32)P}8-azido-ATP hydrolysis are functionally indistinguishable.

Trends Parasitol, 2001 Mar, 17(3), 122 - 6
Possible modes of action of the artemisinin-type compounds; Olliaro PL et al.; Artemisinin-type compounds are used for the treatment of uncomplicated and severe forms of malaria . They reduce parasitaemia more rapidly than any other antimalarial compound known, and are effective against multidrug-resistant parasites . However, uncertainties remain as to how they act on the parasite and cause toxicity . In this review, we summarize current ideas.

J Invest Dermatol, 2001 Apr, 116(4), 541 - 8
Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes; Baron JM et al.; Cytochrome P450 enzymes metabolize various endogenous and exogenous small molecular weight compounds . Transport-associated proteins, such as P-glycoprotein, multidrug resistance-associated protein and lung resistance protein are overexpressed in drug-resistant cell lines, as well as in human tumors from various histologic origins, including malignant melanoma . Little is known about the expression and function of cytochrome enzymes and multidrug resistance-associated transport proteins in human skin; therefore, the aim of this study was to analyze the expression pattern of cytochrome enzymes and multidrug resistance-associated transport proteins in proliferating human epidermal keratinocytes under constitutive conditions and after induction with various inducers . Reverse transcription-polymerase chain reaction revealed constitutive expression of cytochromes 1A1, 1B1, 2B6, 2E1, and 3A5 in keratinocytes and showed expression of cytochrome 3A4 after incubation with dexamethasone . The expression of cytochrome 1A1 was enhanced on the mRNA level after induction with benzanthracene . Reverse transcription-polymerase chain reaction analysis of the multidrug resistance-associated transport proteins revealed constitutive expression of multidrug resistance-associated proteins 1 and 3-6, and lung resistance protein in human epithelial keratinocytes and was negative for multidrug resistance 1 and 2 . Expression of 1 was seen after induction with dexamethasone . Reverse transcription-polymerase chain reaction results were confirmed by immunoblots which showed expression of cytochromes 1A1, 2B6, 2E1, and 3A, multidrug resistance-associated proteins 1, 3, and 5 as well as multidrug resistance 1 after induction with dexamethasone . Immunohistology showed positive immunofluorescence in skin specimens for cytochromes 1A1, 2B6, 2E1, and 3A and multidrug resistance-associated protein 1 and multidrug resistance 1 . Constitutive activity of cytochrome 1A1, 2B, 2E1, and 3A enzymes was measured by catalytic assays . These results show that keratinocytes of the human skin express various transport-associated enzymes and detoxifying metabolic enzymes . Previous studies have revealed that cytochrome enzymes and transport-associated proteins play complementary parts in drug disposition by biotransformation (phase I) and anti-transport (phase III) and act synergistically as a drug bioavailability barrier.

Br J Cancer, 2001 Apr 6, 84(7), 959 - 64
Dexrazoxane significantly impairs the induction of doxorubicin resistance in the human leukaemia line, K562; Sargent JM et al.; Dexrazoxane combined with doxorubicin (+ 5-fluorouracil + cyclophosphamide - the FAC regime) leads to a significant decrease in doxorubicin cardiotoxicity and a significant increase in median survival time for patients with advanced breast cancer responsive to FAC . The reason for this increase in survival may be due to interference with the mechanism involved in the emergence of multidrug resistance (MDR) . In order to test this hypothesis, we induced resistance to doxorubicin in the K562 cell line by growing cells in increasing concentrations of doxorubicin (10-30 nM) in the presence and absence of dexrazoxane (20 nM) . The doxorubicin sensitivity of all resultant sublines was measured using the MTT assay . Flow cytometry was used to assess the MDR1 phenotype, measuring P-glycoprotein expression with MRK 16 antibody and drug accumulation in the presence and absence of PSC 833 for functional P-glycoprotein . Long-term growth in doxorubicin increased the cellular resistance (IC(50)) of K562 cells in a concentration-dependent manner (r(2 )= 0.908) . Doxorubicin resistance was not induced in the presence of dexrazoxane (P< 0.0001) for several months . In parallel, the expression of functional P-glycoprotein was delayed after concomitant addition of dexrazoxane to the selecting medium (P< 0.001) . Dexrazoxane did not act as a conventional modulator of P-glycoprotein . These results suggest that dexrazoxane may delay the development of MDR1, thus allowing responders to the FAC regime to continue to respond .

Biochemistry, 2001 Apr 10, 40(14), 4332 - 9
Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein; Ruth A et al.; The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs . The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates . MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide . This selection yielded the I186N substitution, adjacent to G185V . Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells . In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance . The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2 . Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites . Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs . Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs . In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number . These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.

Hepatology, 2001 Apr, 33(4), 783 - 91
Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver; Soroka CJ et al.; The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis . In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat . Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression . In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein . Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region . By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished . Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary . After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes . Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation . This was in contrast to the progressive decrease in Mrp2 protein . Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.

Hepatology, 2001 Apr, 33(4), 776 - 82
Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes; Rippin SJ et al.; To maintain ongoing vectorial bile secretion, hepatocytes localize distinct transport systems at their basolateral and canalicular membrane domains . Here we compare the expression of the basolateral Na(+)-taurocholate cotransporter (Ntcp) and organic anion transporting polypeptides 1 and 2 (Oatp1, Oatp2) and the canalicular bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) in primary cultured rat hepatocytes . During 72 hours of culturing time the messenger RNA (mRNA) and protein levels of Ntcp and Oatp1 decreased in parallel to 2% to 7% of initial values at 3 hours . Although Oatp2 mRNA exhibited a similar down-regulation to 4%, Oatp2 protein and function were maintained at 25% to 47% of initial values . Furthermore, Bsep and Mrp2 protein levels were maintained at about 50%, while the Mrp2 mRNA showed a transient up-regulation to 154% at 24 and 48 hours . Also, induction of Mrp1 mRNA and protein was observed starting after 24 hours . These results indicate transcriptional down-regulation or decreased mRNA stability of Ntcp and Oatp1, transcriptional and posttranslational regulation of Oatp2, Bsep, and Mrp2 and transcriptional up-regulation of Mrp1 in primary cultured hepatocytes . Furthermore, and most importantly, the observed changes in transporter expression closely resemble the altered transporter phenotypes of cholestatic and proliferating hepatocytes in vivo, thus indicating that primary cultured hepatocytes acquire a cholestatic phenotype, and that the transporter expression might be a suitable differentiation marker for maintenance of hepatocytes in vitro.

Nat Cell Biol, 2001 Apr, 3(4), 384 - 91
Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly; Seol JH et al.; SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs) . Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2) . Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase) . V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme . Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here . Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.

Eur J Pharmacol, 2001 Mar 23, 416(1-2), 19 - 24
Steroids affect collateral sensitivity to gemcitabine of multidrug-resistant human lung cancer cells; Bergman AM et al.; Gemcitabine is phosphorylated by deoxycytidine kinase and thymidine kinase 2 and during S-phase incorporated into DNA . The steroids cortisol and dexamethasone, which regulate cell proliferation and gene expression, are pumped out of the cell by the membrane efflux pumps P-glycoprotein and multidrug resistance-associated protein (MRP), which are blocked by verapamil . In parental non-small cell lung cancer (NSCLC) cells (SW1573), 5 microM cortisol and 100 nM dexamethasone decreased sensitivity to gemcitabine . However, both cortisol and dexamethasone only decreased sensitivity with verapamil in MRP (2R120) and P-glycoprotein (2R160) overexpressing variants . Cortisol decreased deoxycytidine kinase activity in SW1573 cells and cortisol with verapamil in 2R120 and 2R160 cells . Dexamethasone with verapamil decreased deoxycytidine kinase activity in 2R160 . Cortisol decreased thymidine kinase 2 activity in 2R120 and 2R160 cells . Dexamethasone decreased thymidine kinase 2 activity in SW1573, 2R120 and 2R160 cells . In conclusion, since dexamethasone is frequently used to treat side effects of oncolytic therapy, a decrease of sensitivity to gemcitabine by steroids might be clinically relevant.

AIDS Res Hum Retroviruses, 2001 Mar 20, 17(5), 401 - 7
Anti-HIV type 1 activity of 3'-fluoro-3'-deoxythymidine for several different multidrug-resistant mutants; Kim EY et al.; The objective of this work was to test the antiviral activity of a potent nucleoside reverse transcriptase inhibitor, 3'-fluoro-3'-deoxythymidine (FLT), on both a wild-type human immunodeficiency virus (HIV-1) isolate and multidrug-resistant HIV-1 patient isolates . Drug-resistant viral isolates were selected on the basis of four different categories of well-characterized and representative multidrug-resistant mutants . The isolates included three variants containing 151M alone or in combination; three variants containing 215Y and 41L, 67N, 184V, 210W, and 219N in combination; two insertion mutant viruses (69 + EA and 69 + SA); and two deletion mutant viruses (del67NG and del67GS), the latter two groups both also containing other significant mutations . The activity of FLT and AZT against these isolates was determined by drug susceptibility assays and by measuring viral antigen p24 by ELISA . The cytotoxicity of FLT and AZT was assessed in PHA-stimulated PBMCs . Development of resistant mutants under FLT pressure was attempted by passaging HIV-1 isolates in SupT1 cells and stepwise increasing the concentration of FLT . The multidrug-resistant mutant HIV-1 isolates exhibited 7-fold to >100-fold increased resistance to AZT, but showed IC(50) values for FLT of 0.0014-0.0168 microM, which were lower than or similar to that of wild type (0.0075 microM) . The cellular cytotoxicities of FLT and AZT fell into a similar range in PBMCs . The development of HIV mutants resistant to FLT appeared to be slower than for other RT inhibitors . HIV isolates with mutations resulting in multidrug resistance had no evidence of resistance to FLT . FLT may be useful in salvage therapies for patients harboring resistant strains and a reassessment of its therapeutic potential seems required.

Curr Med Chem, 2001 May, 8(6), 685 - 713
Structure-activity relationships of multidrug resistance reversers; Wiese M et al.; Multidrug resistance, MDR, is a major obstacle in the chemotherapeutic treatment of cancer . MDR can be reversed by drugs that vary widely in their chemical structure and main biological action . Many efforts are directed to find out the relationships between the structure and MDR reversal effect of these drugs . In this review we try to summarize the results of a variety of studies on identification of structure-activity relationships, SARs, and quantitative SARs, QSARs, of different MDR reversing drugs . As any reasonable (Q)SAR study relies on a real or putative presentation about the mechanism of action of the studied compounds, the most significant MDR mechanisms revealed till now are shortly discussed . Special attention is paid to P-glycoprotein, P-gp, related MDR as the most experimentally and clinically tested form of drug resistance . The currently proposed models of P-gp functioning and mechanisms of MDR modulation are presented . Problems that can arise in (Q)SARs studies are discussed in advance to allow the reader to judge on possible pitfalls . The physicochemical and structural properties of MDR modulators as found by different research groups are commented and summarized . From the discussed studies it can be concluded that the careful selection of relevant structural and biological data processed with appropriate QSAR and especially 3D-QSAR methods, is a promising approach to structure-activity studies of MDR reversers.

Int J Cancer, 2001 Apr 1, 92(1), 115 - 22
Overcoming multi-drug resistance using an intracellular anti-MDR1 sFv; Heike Y et al.; We made an intracellular single-chain variable fragment (sFv) from the C219 monoclonal antibody that recognized the intracellular domain of the multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) . Immuno-cytochemistry using the FITC conjugated anti-C-myc tag antibody showed that the sFv protein was expressed in the cytoplasm of the cells . Although transfection of the sFv did not result in the down-regulation of P-gp expression in P-gp positive MDR cells as determined by flow cytometry analysis, Adriamycin (ADM) uptake and Rhodamine123 (Rh123) retention were increased by the C219 intra-cellular sFv transfection . The transfected cells exhibited a higher sensitivity to ADM using a 10-day colony formation assay . The conventional 3-day MTT assay showed the drug resistant tendency in C219 sFv transfected cell we tested . The growth rate of C219 sFv transfected cells was delayed in all non-MDR and MDR cells that might be the reason why C219 transfected cells exhibited the drug resistant tendency in the MTT assay . Despite this unexpected effect of C219 sFv on growth rate, our data suggest that the intra-cellular sFv technique could knockout MDR functionally and may offer a means of increasing the effectiveness of tumor chemotherapy .

J Pediatr Oncol Nurs, 2001 Mar-Apr, 18(2), 50 - 4
The treatment of retinoblastoma: a case study; Tomlinson D; Research in the treatment of retinoblastoma and multidrug resistance has led to new treatment protocols for children . This case study introduces information regarding a clinical trial for the treatment of intraocular retinoblastoma . It also highlights important nursing issues in the care of these children and their families .

Cancer Res, 2001 Mar 1, 61(5), 1991 - 5
4-Demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a novel anticancer agent active against tumor cell lines with different resistance mechanisms; Marchini S et al.; The activity of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a new alkycycline with high antitumor activity against a broad range of cancer cells, was evaluated in vitro and in vivo in cells selected for resistance to different anticancer agents . Both in vitro and in vivo, PNU-159548 did retain its activity in cells expressing the multidrug resistance (MDR) phenotype, associated to MIDR-1 gene overexpression or with an alteration in the topoisomerase II gene (altered MDR), independently on the drug used for the selection of the resistant cell line . According to these data, the intracellular uptake of PNU-159548 is not influenced by the presence of MDR-1 . PNU-159548 was also active, both in vitro and in vivo, against cells showing resistance to various alkylating agents iincluding cisplatin, cyclophosphamide, and melphalan) and topoisomerase I-inhibitors . Cells defective in nucleotide excision repair, which did show hypersensitivity to treatment with UV irradiation and alkylating agents, showed only a marginally increased sensitivity to PNU-159548 . Similarly, the activity of the drug was not influenced by the mismatch repair system, as assessed in two different cellular systems deficient in hMLH1 expression and in which hMLH1 activity was restored by chromosome 3 transfer . The results obtained clearly indicate that the new anticancer agent PNU-159548 is able to overcome the classical mechanisms of resistance emerging after treatment with the most clinically used anticancer agents, and it could represent an alternate choice in the treatment of those tumors refractory to conventional therapy.

Cancer Biother Radiopharm, 2001 Feb, 16(1), 17 - 23
Technetium-99m-tetrofosmin would be a substrate for multidrug resistance-associated protein (MRP): comparison between a leukemia cell line with high MRP gene expression and its parental cell line; Li XF et al.; The kinetics of cellular accumulation and retention of technetium-99m-tetrofosmin (99mTc-TF) were investigated in wild type HL60/WT cell line and in its doxorubicin-resistant HL60/DOX cell line with multidrug resistance-associated protein (MRP), but without P-gp overexpression, to determine whether 99mTc-TF is a substrate for MRP . METHODS: The accumulation and washout of 99mTc-TF were observed in both cell lines at 37 degrees C . The effect of verapamil on the kinetics was also assessed . RESULTS: 99mTc-TF net accumulation was significantly lower in HL60/DOX (1.35 +/- 0.23%) than in HL60/WT (12.79 +/- 0.47%) at 60 min (P < 0.001) . Three minutes after exchanging the incubation solution to the tracer-free medium, only 18.20 +/- 0.34% of 99mTc-TF remained in HL60/DOX, whereas 84.74 +/- 0.65% did in HL60/WT (P < 0.001) . In the presence of 10 microM verapamil, 99mTc-TF net accumulation in HL60/DOX was 302% of the control and the washout was significantly delayed . CONCLUSION: 99mTc-TF would be a substrate for MRP and 99mTc-TF may be used as a functional imaging agent of MRP in vivo.

Acta Haematol, 2000, 104(4), 174 - 80
Expression of functional markers in acute nonlymphoblastic leukemia; Han K et al.; Multidrug resistance parameters, tissue infiltration parameters, receptors for colony-stimulating factors (CSFr) and cell cycle parameters were analyzed using flow cytometry in 145, 109 initial and 36 relapsed or refractory, acute nonlymphoblastic leukemia (ANLL) patients to find out clinically more reliable functional parameters . Lung resistance-associated protein (LRP) was most frequently expressed in ANLL (44.1%) followed by P-glycoprotein (PGP) (35.9%) and multidrug resistance-associated protein (MRP) (8.3%) . LRP and PGP were expressed more frequently in relapsed or refractory ANLL than initial ANLL cases . Complete remission rate after standard chemotherapy falls in PGP-positive cases (p = 0.001) . CD44-positive ANLL cases relapsed more frequently . The organ tropism is different depending on the infiltration parameters, vascular cell adhesion molecule to splenomegaly, matrix metalloprotease-2 to hepatomegaly and to extramedullary infiltration other than spleen, liver or lymph node . The percentage of the granulocyte-macrophage-CSFr expression was high in M4 and M5, and granulocyte-CSFr-positive ANLL showed less extramedullary infiltration (p = 0.007) and more PGP expression . Ki-67 was expressed significantly less in refractory ANLL than initial ANLL and DNA topisomerase IIalpha was expressed significantly more in the surviving patients group . In conclusion, analysis of these new functional parameters could help to predict and overcome the clinical behavior of each ANLL at the time of diagnosis .

J Biol Chem, 2001 May 4, 276(18), 14972 - 9 Epub 2001 Feb 14.
Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil; Loo TW et al.; Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell . Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp . MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K(m) value (25 microM) that was similar to that of verapamil . 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil . The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil . Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil . Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11) . These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil.

J Biol Chem, 2001 May 11, 276(19), 16076 - 82 Epub 2001 Jan 22.
The structure of the multidrug resistance protein 1 (MRP1/ABCC1) . crystallization and single-particle analysis; Rosenberg MF et al.; Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux . MRP1 is also an efficient transporter of conjugated organic anions . Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD) . However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH(2) terminus . Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein . In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids . The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell . The overall dimensions of the MRP1 monomer are approximately 80 x 100 A . The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates . These data represent the first structural information of this transporter to approximately 22-A resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.

J Biol Chem, 2001 May 18, 276(20), 17420 - 8 Epub 2001 Feb 20.
Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species; Wartenberg M et al.; Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS) . A transient expression of Pgp was observed during the growth of multicellular tumor spheroids . Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK . Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated . Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression . The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e . the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation . ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

J Biol Chem, 2001 May 18, 276(20), 16833 - 9 Epub 2001 Feb 23.
The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis; Sirakova TD et al.; Multidrug-resistant tuberculosis is a major global health emergency . Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis . The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen . A variety of multiple methyl-branched fatty acids are among such unique lipids . We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids . One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct . Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis . Thin-layer and radio gas-chromatographic analyses of lipids derived from {1-14C}propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta- and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids . Consequently, pks2 mutant does not produce sulfolipids . Sulfolipid deficiency in pks2 mutant was confirmed by two-dimensional thin-layer chromatographic analysis of lipids derived from {1-14C}propionic acid and 35SO4(-2) . With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M . tuberculosis.

J Biol Chem, 2001 May 11, 276(19), 15616 - 24 Epub 2001 Feb 21.
Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport; Ito K et al.; Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions . In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact . In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide . A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr . Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane . In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of {(3)H}vincristine comparable to those in vector control-transfected cells . Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16 . In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1 . This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents . Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.

J Biol Chem, 2001 May 18, 276(20), 16667 - 73 Epub 2001 Feb 20.
P-glycoprotein does not protect cells against cytolysis induced by pore-forming proteins; Johnstone RW et al.; P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR) . In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function . We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response . There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation . Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different pore-forming proteins . Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp(+ve) or P-gp(-ve) cells to the pore-forming proteins complement, perforin, or pneumolysin . Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.

J Biol Chem, 2001 Apr 20, 276(16), 13231 - 9 Epub 2001 Jan 23.
Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines; Zhang DW et al.; Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines . Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs . We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1 . These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1 . This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) . Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance . To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein . The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport . Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate . These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.

Biochim Biophys Acta, 2001 Mar 30, 1531(1-2), 111 - 31
The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo); Santini MT et al.; The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated . In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared . The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts . However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells . In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types . The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts . From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals . The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.

FEBS Lett, 2001 Mar 23, 493(1), 31 - 5
Nucleotide-induced conformational changes in the human multidrug resistance protein MRP1 are related to the capacity of chemotherapeutic drugs to accumulate or not in resistant cells; Manciu L et al.; Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface . Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells . On the contrary, three compounds (4'-deoxy-4'-iodo-doxorubicin, 4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells . Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport . MRP1 was reconstituted in lipid vesicles as previously described {Manciu, L., Chang, X.B., Riordan, J.R . and Ruysschaert, J.-M . (2000) Biochemistry 39, 13026-13033} . The reconstituted protein was shown to conserve its ATPase and drug transport activity . Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes . Binding of drugs (4-demethoxy-daunorubicin and 3'-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.

Gynecol Oncol, 2001 Apr, 81(1), 18 - 24
Evaluation of chemoresistance markers in women with epithelial ovarian carcinoma; Goff BA et al.; OBJECTIVE: Preliminary studies have suggested that lung resistance protein (LRP), multidrug resistance protein (MRP), and p27 may be useful markers of chemoresistance . Our goal was to evaluate the expression of LRP, MRP, and p27 in normal ovaries and chemosensitive and chemoresistant ovarian carcinomas . METHODS: Fourteen women with normal ovaries and fifty women with epithelial ovarian carcinoma who underwent cytoreductive surgery from 1996 through 1998 had specimens stained with immunocytochemistry for LRP, MRP, and p27 . All women received paclitaxel/platinum-based chemotherapy . Twenty-nine women had a disease-free survival (DFS) of at least 12 months after completion of chemotherapy (sensitive) and 21 women had persistent disease during treatment (resistant) . RESULTS: Evaluation of LRP expression revealed significant differences between the normal ovaries and cancers in both the epithelial (57% vs 90%, P = 0.03) and stromal (86% vs 32%, P = 0.001) components . Evaluation of MRP expression revealed significant differences between normal ovaries and cancers in the epithelial component (7% vs 66%, P = 0.001) but not in the stromal component (14% vs 4%, P = 0.1) . Evaluation of p27 revealed significant reductions in expression in cancers compared with normal ovaries for both the epithelial (90% vs 55%, P = 0.02) and stromal (88% vs 5%, P = 0.001) components . Comparison between the chemosensitive and chemoresistant groups revealed no significant differences in expression of LRP and MRP, in either the epithelial or stromal component, but significantly lower levels of p27 were expressed in the epithelial component of the chemoresistant group (P = 0.01) . CONCLUSIONS: The expression of LRP, MRP, and p27 is significantly different in ovarian cancers compared with normal ovaries . Low levels of p27 expression are associated with chemoresistance; however, LRP and MRP expression are not prognostic for chemosensitivity .

J Biol Chem, 2001 Jun 15, 276(24), 20876 - 81 Epub 2001 Mar 27.
Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT); Harris MJ et al.; The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules . In these tissues MRP2 specifically localizes to the apical membrane . The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells . The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1 . Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells . Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization . In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells . Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.

J Assoc Physicians India, 1998 Feb, 46(2), 194 - 8
Acquired drug resistance in tuberculosis in Harayana, India; Janmeja AK et al.; Sputa of 200 treatment failure cases of pulmonary tuberculosis over a period of 1991-1995 were subjected to culture and sensitivity testing against commonly used anti-tuberculosis drugs . Out of 200 cases, 75% revealed resistance to one or more anti-TB drugs, resistance to isoniazid was observed in 72% cases, it was 49% for rifampicin, and 37% for streptomycin, while the resistance rate for other drugs was low . Majority of patients had resistance to two or three drugs concomitantly while resistance to 4, 5 and 6 drugs was of very low order and resistance to reserved drugs like kanamycin, ethionamide and cycloserine was encountered infrequently (1%) . Out of 200 treatment failure patients multidrug resistance (MDR) was seen in 59% cases as 16% revealed resistance to isoniazid alone and strains in 22% cases were sensitive to all drugs . The study concludes that acquired MDR against first line antituberculosis drugs had increased as significant resistance against 3 drug combinations was observed although resistance against 2 drugs concomitantly was insignificant . Most ominous acquired drug resistance was seen against rifampicin in our region . The trends of drug resistance in the country and Haryana State are compared and their implications on outcome of chemotherapy are discussed.

Med Mycol, 2001 Feb, 39(1), 109 - 16
Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei; Katiyar SK et al.; Infections with Candida krusei have increased in recent years as a consequence of its intrinsic resistance to fluconazole, an antifungal azole widely used in immunocompromised individuals to suppress infections due to azole-susceptible C . albicans . One established mechanism for azole resistance is drug efflux by ATP binding cassette (ABC) transporters . Since these transporters recognize structurally diverse drugs, their overexpression can lead to multidrug resistance (MDR) . To identify C . krusei genes potentially involved in azole resistance, PCR was performed with primers corresponding to conserved sequences of MDR-related ABC transporters from other fungi . Two genes, ABC1 and ABC2, were identified; Southern blots suggested that both have one or two related gene copies in the C . krusei genome . ABC1 RNA was constitutively expressed at low levels in log phase cells while ABC2 RNA was undetectable . However, both genes were upregulated as cultures approached stationary phase, and this upregulation was correlated with decreased susceptibility to the lethal activity of the azole derivative miconazole . Furthermore, ABC1 was upregulated following brief treatment of C . krusei with miconazole and clotrimazole (but not other azoles), and the unrelated compounds albendazole and cycloheximide . The latter two compounds antagonized fluconazole activity versus C . krusei, supporting a role for the ABC1 transporter in azole efflux . Finally, miconazole-resistant mutants selected in vitro demonstrated increased constitutive expression of ABC1 . Based on these expression data, genetic and functional characterization of the ABC1 transporter to directly test its role in C . krusei azole resistance would appear to be warranted.

J Antibiot (Tokyo), 2001 Jan, 54(1), 44 - 55
Design of a novel mammalian screening system for the detection of bioavailable, non-cytotoxic streptogramin antibiotics; Aubel D et al.; Screening and development of new antibiotic activities to counteract the increasing prevalence of multidrug-resistant (MDR) human pathogenic bacteria has once again become a priority in human chemotherapy . Here we describe a novel mammalian cell culture-based screening platform for the detection of streptogramin antibiotics . Quinupristin-dalfopristin (Synercid), a synthetically modified streptogramin, is presently the sole effective agent in the treatment of some MDR nosocomial infections . A Streptomyces coelicolor transcriptional regulator (Pip) has been adapted to modulate reporter gene expression (SEAP, secreted alkaline phosphatase) in Chinese hamster ovary cells (CHO) in response to streptogramin antibiotics . This CHO cell-based technology was more sensitive in detecting the production of the model streptogramin pristinamycin, from Streptomyces pristinaespiralis, than antibiogram tests using a variety of human pathogenic bacteria as indicator strains . The reporter system was able to detect pristinamycin compound produced by a single S . pristinaespiralis colony . The assay was rapid (17 hours) and could be carried out in a high-throughput 96-well plate assay format or a 24-well transwell set-up . This novel mammalian cell-based antibiotic screening concept enables detection of bioavailable and non-cytotoxic representatives of a particular class of antibiotics in a single assay and represents a promising alternative to traditional antibiogram-based screening programs.

Anticancer Res, 2000 Sep-Oct, 20(5C), 4019 - 22
MRP expression of testicular cancers and its clinical relevance; Eid H et al.; BACKGROUND: The expression of a multidrug resistance associated protein (MRP) has been investigated in a variety of human tumors . However, there is a lack of data regarding its expression in germ cell testicular tumors (GCTTs) . PATIENTS AND METHODS: MRP expression was examined by immunohistochemistry (IHC) using mouse monoclonal antibody (MRPm6) against human MRP in 56 testis cancer specimens . This antigen was also correlated with the histology, metastatic behavior, clinical stage and tumor suppressor protein p53 immunostaining of GCTTs . RESULTS: All testis tumors, regardless of their histology, metastatic status and clinical stage gave positive signals . MRP was positive not only in the cytoplasm but, very interestingly, in the nuclei . CONCLUSION: Our results suggested that ala GCTTs express high levels of MRP protein with no relation to any of clinicopathological variables investigated here . Since germ cell tumors are very sensitive to chemotherapy, the role of MRP as mediator of drug resistance seems unconvincing in this malignancy . MRP is located in the cytoplasm and the nuclei of tumor cells and may be involved in transportation and/or redistribution certain substrates from the nucleus to the cytoplasm.

Anticancer Res, 2000 Sep-Oct, 20(5C), 3759 - 65
Expression of apoptosis, cell proliferation, and drug resistance genes in pediatric Wilms' tumors; Ramachandran C et al.; The expression of genes associated with apoptosis, cell proliferation and drug resistance in tumor cells was investigated in two pediatric Wilms' tumor patients (MCH-WT-1 and MCH-WT-3) for their association with cell cycle, daunorubicin accumulation and clinical data . DNA content, cell cycle and drug accumulation were analyzed immediately after surgery by flow cytometry and mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) assay . Primary cell cultures were established from tumor specimens and tumor cells in both cases showed epithelial morphology . Although cell proliferation markers (Ki67 and PCNA) were expressed in both cases, MCH-WT-3 showed higher levels of mRNA expression, which corresponded, with metastatic behavior of the tumor in the patient . While p53 and p21 mRNAs were expressed at low levels in MCH-WT1, MCH-WT-3 showed high levels of p21 mRNA only . The increased expression of cyclin kinase inhibitor (p21) in MCH-WT-3 compared to MCH-WT-1 correlated with a higher percentage of G0/G1 cell population in the tumor specimen . Despite the expression of multidrug resistance markers (MDR1 and LRP) in MCH-WT-1, flow cytometric analysis showed tumor cell populations with very low and high daunorubicin accumulation and with the absence of any effect for verapamil and dipyridamole on daunorubicin accumulation of tumor cells.

Emerg Infect Dis, 2001 Jan-Feb, 7(1), 123 - 7
Hospital control and multidrug-resistant pulmonary tuberculosis in female patients, Lima, Peru; Willingham FF et al.; We examined the prevalence of tuberculosis (TB), rate of multidrug-resistant (MDR) TB, and characteristics of TB on a female general medicine ward in Peru . Of 250 patients, 40 (16%) were positive by sputum culture and 27 (11%) by smear, and 8 (3%) had MDRTB . Thirteen (33%) of 40 culture-positive patients had not been suspected of having TB on admission . Six (46%) of 13 patients whose TB was unsuspected on admission had MDRTB, compared with 2 (7%) of 27 suspected cases (p = 0.009) . Five (63%) of 8 MDRTB patients were smear positive and therefore highly infective . In developing countries, hospital control, a simple method of reducing the spread of MDRTB, is neglected.

J Virol, 2001 Apr, 75(8), 3988 - 92
Amino acid deletion at codon 67 and Thr-to-Gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistance profiles; Imamichi T et al.; The potential roles of an amino acid deletion at codon 67 (Delta67) and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase of human immunodeficiency virus (HIV) type 1 in drug sensitivity and relative replication fitness were studied . Our results suggest that the Delta67 and T69G changes can be categorized as mutations associated with multidrug resistance . The combination of both mutations with an L74I change (Delta67+T69G/L74I) leads to a novel 3'-azido-3'-deoxythymidine resistance motif and compensates for impaired HIV replication.

Biochem Biophys Res Commun, 2001 Mar 23, 282(1), 257 - 63
Reactive oxygen species-related induction of multidrug resistance-associated protein 2 expression in primary hepatocytes exposed to sulforaphane; Payen L et al.; Expression of multidrug resistance-associated protein 2 (MRP2), an efflux pump contributing to biliary secretion of xenobiotics, was investigated in primary rat and human hepatocytes exposed to sulforaphane, a naturally-occurring chemopreventive agent . Northern blot indicated that sulforaphane increased MRP2 mRNA levels in primary rat hepatocytes; it also induced expression of drug metabolizing enzymes such as glutathione S-transferase A1/2 isoforms and NAD(P)H:quinone oxidoreductase in a dose-response and time-course manner similar to that observed for the upregulation of MRP2 transcripts . This sulforaphane-related increase of MRP2 mRNAs paralleled increased expression of 190 kD MRP2 protein as assessed by Western blotting; it was fully abolished by the transcription inhibitor actinomycin D . MRP2 induction was associated with increased cellular production of reactive oxygen species (ROS) and addition of dimethyl sulfoxide, that reduced sulforaphane-related formation of ROS, and also decreased MRP2 mRNA levels in sulforaphane-treated primary rat hepatocytes; this suggests that sulforaphane-mediated production of ROS may contribute to MRP2 induction . This link between ROS and MRP2 regulation was further supported by the increase of MRP2 expression occurring in response to t-butylhydroquinone, known to regulate drug metabolizing enzymes through ROS formation . In addition to rat cells, primary human hepatocytes exposed to sulforaphane also displayed induced MRP2 expression evidenced at both mRNA and protein levels . All these observations strongly support the conclusion that the export pump MRP2 can be classified among the detoxifying proteins that are regulated by sulforaphane and that are thought to contribute, at least in part, to its anticarcinogenic properties .

Int J Tuberc Lung Dis, 2001 Jan, 5(1), 59 - 64
Outbreak of multidrug-resistant tuberculosis at a methadone treatment program; Conover C et al.; SETTING: An out-patient methadone treatment program MTP) . OBJECTIVE: To investigate transmission of multidrug-resistant tuberculosis (MDR-TB) in the MTP . DESIGN: Cases were defined as MTP clients or staff who developed TB between 1 January 1994 and 1 January 1996, with at least one positive culture for Mycobacterium tuberculosis resistant to isoniazid and rifampin . Contacts were identified, located and evaluated . RESULTS: Thirteen cases of MDR-TB occurred among 462 clients and staff . One fifth (6/30) of the members of a counseling group for human immunodeficiency virus (HIV) infected clients developed MDR-TB . Individuals known to be HIV positive were at greater risk for TB than those who were HIV negative (RR 5.2, 95%CI 1.2-22.7) . Of 449 clients and staff identified as contacts, 393 (87.5%) were located and screened . Among those with a negative baseline tuberculin skin test, 18.5% (56/303) were skin test converters . Attendance at the MTP during a period when the index case was infectious was associated with an increased risk of conversion (RR 2.5, 95%CI 1.1-6.0) . CONCLUSION: Extensive transmission of MDR-TB occurred at an out-patient MTP serving numerous clients with HIV infection . This outbreak underscores the importance of developing effective strategies to prevent TB transmission in this setting.

Int J Tuberc Lung Dis, 2001 Jan, 5(1), 46 - 52
Resistance of Mycobacterium tuberculosis to four first-line anti-tuberculosis drugs in Japan, 1997; Abe C et al.; SETTING: Five years after the last survey of drug-resistant tuberculosis in Japan, a nationwide survey was conducted by the Tuberculosis Research Committee . OBJECTIVE: To determine the prevalence of and risk factors for resistance to four first-line anti-tuberculosis drugs . DESIGN: Cultures were obtained from patients hospitalized at 78 hospitals in different districts of Japan throughout a 6-month period, 1 June-30 November 1997 . Drug susceptibility testing was carried out at the Research Institute of Tuberculosis, Tokyo, one of the supranational reference laboratories of the WHO/IUATLD global project . RESULTS AND CONCLUSION: Among patients with no prior treatment, resistance to any of the four drugs was found in 10.3%, and the prevalence of primary multidrug resistance (MDR) was 0.8% . The prevalence of acquired resistance was 42.4% for any of the four drugs and 19.7% for MDR, indicating a high prevalence rate compared with those reported in the WHO/IUATLD global project . About 73% of resistant isolates from new cases were resistant to one drug, while 64.3% of resistant isolates from the re-treatment cases were resistant to two or more drugs (P < 0.0001) . No significant differences in resistance rates by sex, age group, nationality, district, and/or accompanying diseases were observed in any of the new or re-treatment cases . Other factors associated with the high prevalence in re-treatment cases remain to be determined.

Int J Tuberc Lung Dis, 2001 Jan, 5(1), 32 - 9
Prevalence of drug-resistant tuberculosis in an HIV endemic area in northern Thailand; Yoshiyama T et al.; SETTING: Chiang Rai Province in Northern Thailand, where human immunodeficiency virus (HIV) infection has been prevalent since the 1990s . OBJECTIVE: To observe the prevalence of drug-resistant tuberculosis (TB) and investigate the factors related to the level of drug resistance in an HIV endemic area . DESIGN: Population-based surveillance study covering the whole province . METHOD: Drug susceptibility testing was performed at the Thai Ministry of Public Health laboratory for all sputum smear-positive TB patients diagnosed in hospitals in Chiang Rai Province over a 25-month period in 1996-1998 . Patient characteristics were obtained through interview by trained personnel . HIV testing was performed with informed consent . RESULTS: Among the 1077 incident patients without previous history of treatment, the proportion of patients with resistance to isoniazid was 13.2%, 10.8% to rifampicin, 15.6% to streptomycin, and 5.8% to ethambutol . Multidrug resistance (MDR), i.e., resistance to at least both isoniazid and rifampicin, was observed in 6.3% . Factors associated with primary MDR-TB were HIV positivity (OR 2.2, 95%CI 1.3-3.9), age <50 years (OR 2.0), and treatment in the provincial hospital (OR 2.3), compared to patients treated in the community and private hospitals . Stratified analysis shows a significantly high prevalence of primary MDR-TB among HIV-positive patients treated in the provincial hospital against HIV-negative patients or HIV-positive patients in other hospitals . CONCLUSION: The prevalence of primary MDR-TB in this area was high . It is necessary to strengthen TB control activities in order to reduce the burden of MDR-TB.

J Neurooncol, 2000 Dec, 50(3), 227 - 37
Drug resistance-associated factors in primary and secondary glioblastomas and their precursor tumors; Tews DS et al.; Malignant gliomas are largely resistant to current chemotherapeutic strategies often displaying a multidrug-resistant phenotype . Mechanisms involved in drug resistance are reduced cellular drug accumulation through membrane efflux pumps, drug detoxification as well as alterations in drug target specificity . In 27 primary and 17 secondary glioblastomas and their astrocytic precursor tumors, we studied the immunohistochemical expression profile of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), metallothionein, and topoisomerase II alpha . Glial tumor cells in all glioblastomas showed constant up-regulation of LRP, MRP, and topoisomerase II alpha . P-gp was found in 90% of the primary and 60% of the secondary glioblastomas . In precursor tumors, these drug resistance-related factors were expressed in varying proportions . Metallothionein, also found in normal and activated astrocytes, was retained in all neoplastic phenotypes . Furthermore, metallothionein, P-gp, LRP, and topoisomerase II alpha were strongly expressed by normal and neoplastic vessels which may confer to impaired penetration of therapeutic agents through the blood-brain and blood-tumor barrier . However, the expression profiles of drug resistance-related proteins neither differed between primary and secondary glioblastomas nor revealed any correlation to precursor or recurrent tumors . Nevertheless, inhibition of these factors may be promising approaches to the management of malignant gliomas.

Zhonghua Xue Ye Xue Za Zhi, 1998 Dec, 19(12), 619 - 22
{Efficient expression of multidrug resistance gene mdr1 in retroviral vector under control of an internal ribosome entry site}; Fu J et al.; OBJECTIVE: To test the efficiency of human mdr1 gene expression under the control of an internal ribosome entry site (IRES) . METHODS: The expression retroviral vector Halmdr1 was constructed from pSXLC/pHa system which contains an IRES from encephalomyocarditis virus . The vector was introduced into ecotropic packaging cells GP + E86 by liposome-mediated transfection . The retrovirus producing cells were obtained by 50 micrograms/L vincristine selection . The success of transfer and expression of mdr1 gene were determined by polymerase chain reaction (PCR) and flow cytometry (FCM) . RESULTS: Virus in the supernatant of the producer cells, in which the integration of mdr1 gene was confirmed by PCR, was titrated to 2.0 x 10(5) cfu/ml . The selected producer cells exhibited a 24-52-fold increase of resistance to vincristine, daunorubicin and taxol in comparison with control cells GP + E86 . The expression of mdr1 gene was confirmed by both reverse transcription PCR at RNA level and FCM at protein level, although the HaImdr1 transfectants showed somewhat less expression of P-gp when compared to that with cap-dependent translation of Ha mdr1 . CONCLUSION: mdr1 gene can be effectively translated and expressed under the control of IRES in cap-independent manner, it may be used as a dominant selectable marker in bicistronic vectors for gene therapy.

Anticancer Drugs, 2001 Feb, 12(2), 107 - 16
Doxorubicin-peptide conjugates overcome multidrug resistance; Mazel M et al.; A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein (P-gp), which is capable of lowering intracellular drug concentrations . To overcome this problem, we tested the capability of two peptide vectors that are able to cross cellular membranes to deliver doxorubicin in P-gp-expressing cells . The antitumor effect of peptide-conjugated doxorubicin was tested in human erythroleukemic (K562/ ADR) resistant cells . The conjugate showed potent dose-dependent inhibition of cell growth against K562/ADR cells as compared with doxorubicin alone . Doxorubicin exhibited IC50 concentrations of 65 microM in the resistant cells, whereas vectorized doxorubicin was more effective with IC50 concentrations of 3 microM . After treatment of the resistant cells with verapamil, the intracellular levels of doxorubicin were markedly increased and consequent cytotoxicity was improved . In contrast, treatment of resistant cells with verapamil did not cause any further enhancement in the cell uptake nor in the cytotoxic effect of the conjugated doxorubicin, indicating that the conjugate bypasses the P-gp . Finally, we show by the in situ brain perfusion method in P-gp-deficient and competent mice that vectorized doxorubicin bypasses the P-gp present at the luminal site of the blood-brain barrier . These results indicate that vectorization of doxorubicin with peptide vectors is effective in overcoming multidrug resistance.

Histopathology, 2001 Mar, 38(3), 209 - 16
High expression of MDR-1 gene and P-glycoprotein in initial and re-biopsy specimens of relapsed B-cell lymphoma; Liu Q et al.; AIMS: Multidrug resistance (MDR) is a major obstacle in the treatment of lymphoma . The expression of MDR-1 mRNA and P-glycoprotein (MDR-1/P-gp) has been linked to MDR . We aimed to investigate the expression of MDR-1/P-gp in B-cell lymphoma . METHODS AND RESULTS: Samples at diagnosis and relapse from 10 patients with B-cell lymphoma were obtained . We also obtained 14 unselected control cases of B-cell lymphoma at diagnosis . The expression of mRNA and protein were determined semiquantitatively by RT-PCR and immunohistochemistry . High MDR-1 and P-gp expressions were found in seven and seven of 10 samples obtained at diagnosis, eight and eight of 10 samples obtained at relapse, and three and four of 14 control cases at diagnosis, respectively . The results of RT-PCR paralleled those of immunohistochemistry . Concordance of high MDR-1/P-gp expression was noted in 27 of 34 samples (r = 0.73, P = 0.001) . There were no significant changes in MDR-1/P-gp expression in all cases at relapse and during the clinical course following chemotherapy . In the 14 control cases, the average survival time was 12.7 months in MDR-1/P-gp positive cases and 29.0 months in the MDR-1/P-gp negative cases (P = 0.20) . CONCLUSIONS: Our results showed that at least some B-cell lymphomas express MDR-1/P-gp, which could be detected by different methods, and suggested that high MDR-1/P-gp expression in tumour cells may be associated with a high probability of relapse and poor prognosis.

Brain Res, 2001 Mar 23, 895(1-2), 253 - 7
Expression of multidrug resistance protein 1 (MRP1) in the rat cochlea with special reference to the blood-inner ear barrier; Saito T et al.; Expression of multidrug resistance protein 1 (MRP1) was detected in the rat cochlea by RT-PCR and immunohistochemistry using anti-MRP monoclonal antibody MRPr1 . Use of primers specific for rat mrp1 gene resulted in the amplification of an expected 394 bp fragment prepared from brain and cochlear tissues . Immunohistochemically, MRP was found in the choroid plexus, stria vascularis, spiral ligament, spiral prominence and cochlear nerve in the modiolus . From these results, it was suggested that MRP in the rat cochlea might function as an extrusion pump and play an important role in the blood-inner ear barrier.

FASEB J, 2001 Mar, 15(3), 719 - 30
Ceramide glycosylation potentiates cellular multidrug resistance; Liu YY et al.; Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide-induced programmed cell death . This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents {Liu, Y . Y., Han, T . Y., Giuliano, A . E., and M . C . Cabot . (1999) J . Biol . Chem . 274, 1140-1146} . We previously found that glucosylceramide, the glycosylated form of ceramide, accumulates in adriamycin-resistant breast carcinoma cells, in vinblastine-resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy . Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting . In adriamycin-resistant MCF-7-AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs . Sensitivity to the various drugs by GCS antisense transfection increased 7- to 240-fold and was consistent with the resumption of ceramide-caspase-apoptotic signaling . GCS targeting had little influence on cellular sensitivity to either 5-FU or cisplatin, nor did it modify P-glycoprotein expression or rhodamine-123 efflux . GCS antisense transfection did enhance rhodamine-123 uptake compared with parent MCF-7-AdrR cells . This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.

Int J Tuberc Lung Dis, 2001 Feb, 5(2), 170 - 6
Tuberculosis as an occupational hazard for health care workers in Estonia; Kruuner A et al.; SETTING: Tuberculosis incidence has been increasing in the Baltic states since the 1990s, accompanied by the emergence of drug resistance, including multidrug resistance (MDR) . In this changing situation, the potential threat of nosocomial spread of tuberculosis to other patients and health care workers (HCW) has remained unrecognised . OBJECTIVE: To investigate the risk of tuberculosis in health care workers in Estonia . DESIGN: Cases of tuberculosis registered among HCWs from 1994 to 1998 were evaluated . The case records were analysed retrospectively and combined with bacteriological data including data on drug resistance . RESULTS: Sixty-seven HCWs (23 physicians, 23 nurses and seven laboratory technicians, 12 assistant nurses and two cleaners), all of whom tested negative for human immunodeficiency virus, were diagnosed as having active tuberculosis . The incidence of tuberculosis among HCWs (mean 91/100,000/year) was 1.5 to three times higher than in the general population . In a chest hospital in charge of regional tuberculosis care, the incidence was 30 to 90 times higher, and was highest among physicians . In 49 HCWs tuberculosis was confirmed by culture . Among these, drug resistance was detected in 23 (49%), 18 (38%) of whom had MDR tuberculosis . CONCLUSIONS: Health care workers, especially those working in a chest hospital where tuberculosis patients were treated, were found to be at an elevated risk of tuberculosis . MDR tuberculosis poses a particular threat which is difficult to combat.

Int J Tuberc Lung Dis, 2001 Feb, 5(2), 129 - 36
Low failure rate in standardised retreatment of tuberculosis in Nicaragua: patient category, drug resistance and survival of 'chronic' patients; Heldal E et al.; SETTING: IUATLD collaborative programme, Nicaragua . OBJECTIVE: To analyse reported trends in the retreatment failure rate (2SRHZE/1RHZE/5R3H3E3), and assess demographic characteristics, drug resistance and survival in patients who fail retreatment . DESIGN: A retrospective, descriptive study . Reports from 1988-1996 were analysed and records of 69 patients who failed retreatment were reviewed . RESULTS: The treatment success rate in new cases improved from 71% in 1988-1991 to 79% in 1992-1996, the default rate decreased from 16% to 10%, and the failure rate remained stable at 2-3% . The proportion of previously treated patients among all smear-positives decreased from 20% to 15% . In retreatment patients the failure rate declined from 6.6% to 4.3% and the average annual number of failures from 24 to 13 . In 1992-1996, 64 patients, 0.8% of new smear-positive cases treated during this period, failed retreatment; the corresponding figures for 1988-1991 are 95 and 1.6% . Among 69 retreatment failure cases reviewed, there was male predominance and increasing age during the study period . Drug susceptibility results were available for 38, of whom 89% were resistant to isoniazid and rifampicin . The median survival of patients after failure was 3.9 years . CONCLUSION: Treatment results improved over the study period . The proportion of patients on retreatment out of all smear positives treated decreased, as did the absolute number of failures and the retreatment failure rate . Development of multidrug resistance has been largely prevented in Nicaragua; the low failure rate justifies the continued use of the standardised retreatment regimen.

Jpn J Thorac Cardiovasc Surg, 2001 Feb, 49(2), 141 - 4
Surgical management of multidrug-resistant tuberculosis; Sasano S et al.; We report surgical resections in 3 patients with active multidrug-resistant tuberculosis . All cases involved strains of Mycobacterium tuberculosis resistant to at least isoniazid and rifampin and patients who were poor candidates for medical therapy alone . We conducted pulmonary resections (partial resection in case 1, lobectomy in case 2, and segmentectomy in case 3) . The optimum multiple-drug regimen, based on drug susceptibility studies, was used preoperatively and postoperatively . In all cases, sputum smears and cultures yielded negative results postoperatively, and continue to be negative for Mycobacterium tuberculosis to date . It is recommended that, if localized disease is present and medical treatment is likely to fail, pulmonary resection be conducted for multidrug-resistant Mycobacterium tuberculosis.

Antimicrob Agents Chemother, 2001 Apr, 45(4), 1263 - 70
Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins; Partridge SR et al.; The first 13.6 kb of the mercury and multidrug resistance transposon Tn1696, which includes the class 1 integron In4, has been sequenced . In4 is 8.33 kb long and contains the 5'-conserved segment (5'-CS) and 2.24 kb of the 3'-conserved segment (3'-CS) flanking four integrated cassettes . The 3'-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS6100 flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons . This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn21, and other related class 1 integrons . In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism . In4 lies between the resII and resI sites of a backbone mercury resistance transposon which is >99.5% identical to Tn5036 . Although Tn21 and Tn1696 are both classified as members of the Tn21 subfamily of the Tn3 transposon family, the backbone mercury resistance transposons are only 79 to 96% identical . Tn21 also contains a region of about 0.7 kb not found in Tn1696 . The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance transposons . Thus, Tn21 and Tn1696 have independent histories and origins . Other transposons (Tn1403 and Tn1412) that include a class 1 integron also have independent origins . In all except Tn21, the integron is located within the res region of the backbone transposon.

Int J Mol Med, 2001 Apr, 7(4), 397 - 400
Expression of multidrug resistance associated transporters (MDR1, MRP1, LRP and BCRP) in porcine oocyte; Takebayashi Y et al.; Transporters such as P-glycoprotein (MDR1), multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP) are associated with multidrug resistance in various carcinoma cell lines . The expression of these molecules has been also characterized in human normal tissues . However, the expression of these molecules in oocyte is still unclear . In order to obtain more insight into the physiological role of these transporters, their expression in porcine oocyte were examined by reverse transcriptase-polymerase chain reaction . MDR1, MRP1 and LRP genes, but not BCRP gene were found to be expressed in porcine oocyte . After the subcloning and sequence analysis of MDR1, MRP1 and LRP genes, the high homology of these transporters were observed between porcine and human gene . These findings suggest that MDR1, MRP1 and LRP play an important physiological role(s) in an oocyte.

Am J Respir Crit Care Med, 2001 Mar, 163(3 Pt 1), 717 - 20
Exogenous reinfection with tuberculosis on a European island with a moderate incidence of disease; Caminero JA et al.; The frequency and determinants of exogenous reinfection and of endogenous reactivation of tuberculosis in patients previously treated are poorly understood . In Gran Canaria Island, Spain, between 1991 and 1996, 962 tuberculosis cases were confirmed by culture . Drug susceptibility testing was performed on available bacterial isolates and IS6110-based RFLP genotyping was carried out . Twenty-three patients (2.4%) had two positive cultures separated by at least 12 mo, 18 of whom had bacterial DNA available for genotypic analysis . The initial and final isolates from eight (44%) were different genotypes, indicating exogenous reinfection . Six of them were retreated after cure and two retreated after default . Six were HIV seronegative and two were HIV seropositive . Endogenous reactivation was seen in the remaining 10 patients of whom eight were retreated after default and two after cure . Three of the eight (38%) being retreated after default developed multidrug resistance . One genotype was responsible for a second episode of tuberculosis in five cases, three exogenous reinfections and two endogenous reactivations . In the context of a moderate incidence of tuberculosis, exogenous reinfection is an important cause of TB recurrence, even in HIV-seronegative patients.

Pharmacotherapy, 2001 Mar, 21(3), 351 - 4
Periorbital cellulitis secondary to Conidiobolus incongruus; Temple ME et al.; A previously healthy, 18-month-old girl developed edema and erythema around her left eye 1 week after getting sand in that eye . The patient did not respond to oral or intravenous antibiotics . A mass developed around the eye, and biopsy revealed Conidiobolus incongruus . The patient failed to respond to amphotericin B 1 mg/kg, and susceptibility tests indicated multiantifungal resistance . A combination of antifungal therapy, hyperbaric oxygen, and surgery was required for successful treatment . Three months after treatment the child was disease free . There is no definitive therapy for Conidiobolus incongruus infections, although various drugs have been administered with some success . When susceptibility tests determine multidrug resistance, radical resection with antifungal chemotherapy and hyperbaric oxygen may be necessary as well as lifesaving.

Int J Oncol, 2001 Apr, 18(4), 767 - 73
The anti-malarial artesunate is also active against cancer; Efferth T et al.; Artesunate (ART) is a semi-synthetic derivative of artemisinin, the active principle of the Chinese herb Artemisia annua . ART reveals remarkable activity against otherwise multidrug-resistant Plasmodium falciparum and P . vivax malaria . ART has now been analyzed for its anti-cancer activity against 55 cell lines of the Developmental Therapeutics Program of the National Cancer Institute, USA . ART was most active against leukemia and colon cancer cell lines (mean GI50 values: 1.11+/-0.56 microM and 2.13+/-0.74 microM , respectively) . Non-small cell lung cancer cell lines showed the highest mean GI50 value (25.62+/-14.95 microM) indicating the lowest sensitivity towards ART in this test panel . Intermediate GI50 values were obtained for melanomas, breast, ovarian, prostate, CNS, and renal cancer cell lines . Importantly, a comparison of ART's cytotoxicity with those of other standard cytostatic drugs showed that ART was active in molar ranges comparable to those of established anti-tumor drugs . Furthermore, we tested CEM leukemia sub-lines resistant to either doxorubicin, vincristine, methotrexate, or hydroxyurea which do not belong to the N.C.I . screening panel . None of these drug-resistant cell lines showed cross resistance to ART . To gain insight into the molecular mechanisms of ART's cytotoxicity, we used a panel of isogenic Saccaromyces cerevisiae strains with defined genetic mutations in DNA repair, DNA checkpoint and cell proliferation genes . A yeast strain with a defective mitosis regulating BUB3 gene showed increased ART sensitivity and another strain with a defective proliferation-regulating CLN2 gene showed increased ART resistance over the wild-type strain, wt644 . None of the other DNA repair or DNA check-point deficient isogenic strains were different from the wild-type . These results and the known low toxicity of ART are clues that ART may be a promising novel candidate for cancer chemotherapy.

Am J Physiol Renal Physiol, 2001 Apr, 280(4), F636 - 45
Two apical multidrug transporters, P-gp and MRP2, are differently altered in chronic renal failure; Laouari D et al.; Tubular function is altered in chronic renal failure (CRF) . Whether drug secretion by renal tubules is modified in CRF is questioned because of frequent accumulation of various toxins in CRF . This function mainly involves ATP-dependent drug transporters, particularly P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) 2, both present in apical membrane of epithelial cells . The present study was aimed at determining the changes in P-gp and MRP2 expression induced by experimental CRF in kidney and liver . The relationship between MRP2 and glutathione metabolism changes was examined because MRP2 transports GSSG and glutathione conjugates . Rats underwent either 80% subtotal nephrectomy (Nx) or sham operation, and determinations were performed 3 and 6 wk later . CRF induced a 70--200% rise in protein and mRNA expression of MRP2 after 3 and 6 wk post-Nx in remnant kidney and after 6 wk in liver . However, P-gp expression was unchanged by CRF . Relative to whole kidney mass, total MRP2 levels decreased by only 27% in Nx rats whereas total P-gp levels were reduced by 60% . Renal GSSG and total glutathione levels were increased by 30% in Nx rats, but glutathione-S-transferase (GST) activity was normal; liver GSSG levels and GST activity were reduced in Nx rats . In conclusion, CRF resulted in specific overexpression of MRP2 in kidney and liver . This could be an adaptative response to some elevated circulating toxins . The later MRP2 induction and different glutathione changes in liver compared with kidney suggest different mechanisms for MRP2 induction and/or action in these two tissues.

Curr Opin Mol Ther, 2000 Aug, 2(4), 459 - 67
Technology evaluation: Valspodar, Novartis AG; Tai HL; Valspodar (PSC-833) is a derivative of cyclosporin but devoid of the immunosuppressive and nephrotoxic properties seen in cyclosporin A . It exhibited high affinity binding to Mdr1 P-glycoprotein (P-gp) and demonstrated multidrug resistance-reversing activity superior to cyclosporin A and verapamil both in vitro and in vivo . Preclinical and phase I/II clinical data have indicated that plasma levels of PSC-833 with multidrug resistance-reversing activities are achievable . Potent inhibition of intestinal, hepatobiliary and blood-brain barrier P-gp function has been demonstrated . Since valspodar is also a substrate of cytochrome P450 3A (CYP3A), dual interactions of this compound with P-gp and CYP3A are the basis for the pharmacokinetic interactions seen in preclinical and clinical studies . A new formulation of the drug has recently been developed with better oral bioavailability (60%) and less interindividual variability . The toxicity profiles of valspodar are acceptable and dose-limited by transient and reversible cerebellar ataxia . It has shown multidrug resistance-modulating activities towards acute myeloid leukemia, multiple myeloma and ovarian cancer in phase I/II clinical trials . Phase III studies with respect to these three diseases are ongoing.

Eur J Biochem, 2001 Mar, 268(6), 1561 - 7
P-glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine; Frezard F et al.; The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP-dependent extrusions of drugs from cells by P-glycoprotein (P-gp) and/or by multidrug resistance protein (MRP1) . Despite the huge amount of research that has been performed on the mechanisms of P-gp-mediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P-gp . Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form . The aim of the work reported here was to determine which molecular form is actively pumped out by P-gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping . We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels . Using 3'-deamino, 3'-hydroxyl doxorubicin (OH-DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P-gp-mediated efflux of OH-DOX would not depend on the pH being in the range 6.6--8.4 . The P-gp-mediated efflux of daunorubicin (DNR) and 3'-hydroxy-4-amino (WP608) was determined at different pH values . These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P-gp-mediated efflux cannot be assigned to lipohilicity variation, and (b) their pKa values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms . Our data show that both forms are recognized by P-gp but the neutral form is pumped about three times more efficiently than the charged form . This is corroborated by results showing the active efflux (checked at pH(i) 7.3 only) of five other anthracycline containing a basic center . We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P-gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.

Biochim Biophys Acta, 2001 Mar 9, 1511(1), 7 - 16
Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump; Akita H et al.; Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep) . In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep . ATP-dependent uptake of {(3)H}taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and {(3)H}taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR . In addition, ATP-dependent uptake of {(3)H}TLC-S (K(m)=3.9 microM) and {(3)H}taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively . TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively . In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively) . By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM) . Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport . These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

An Acad Bras Cienc, 2001 Mar, 73(1), 57 - 69
Multidrug resistance in tumour cells: characterization of the multidrug resistant cell line K562-Lucena 1; Rumjanek VM et al.; Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients . The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein . However, the resistance process is multifactorial . Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents . This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562 . This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G . Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line . On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1 . In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used . Changes in the cytoskeleton of this cell line were also observed.

Cancer Res, 2001 Feb 15, 61(4), 1693 - 8
Drug resistance induced by ouabain via the stimulation of MDR1 gene expression in human carcinomatous pulmonary cells; Brouillard F et al.; The inhibition of the Na+/K+-ATPase by cardiotonic drugs like ouabain deeply perturbs both the properties of the cell membrane and the ionic composition of the cytoplasm and hence alters fundamental cell reactions . These three types of reactions may be involved in the stimulation of multidrug resistance 1 (MDR-1) gene expression and the synthesis of permeability glycoprotein {P-glycoprotein (P-gp)} . We have determined whether ouabain, which binds to an extracellular motif of the Na+/K+-ATPase, stimulates MDR-1 gene expression by measuring both mRNA and protein and whether the resulting P-gp extrudes hydrophobic compounds and causes resistance to antimitotic agents . The experiments were performed on Calu-3 cells, a human cell line from a pulmonary carcinoma . Northern blotting showed that treating the cells with submicromolar concentrations of ouabain stimulated MDR-1 gene expression within 24 h . The ouabain-induced stimulation of MDR-1 expression was not restricted to Calu-3 cells but also occurred in human carcinomatous colon (T-84 and HT-29) and hepatic (H7V3) cells . However, it is not ubiquitous because it was not found in HeLa cells . The stimulation was reproduced by other Na+/K+-ATPase inhibitors and occurred via enhanced gene transcription, apparently due to the increased cytosolic calcium concentration . Ouabain also increased the membrane content of P-gp, as detected by immunoblotting and immunohistology . We have developed a microvideo assay based on the properties of acetoxymethyl ester calcein and calcein to show that this P-gp extruded the hydrophobic acetoxymethyl ester calcein . Ouabain also caused the Calu-3 cells to become resistant to doxorubicin and vinblastine . Thus, although ouabain acts extracellularly, it may stimulate MDR-1 gene expression and P-gp synthesis and make cells resistant to hydrophobic cytotoxic compounds.

Cancer Res, 2001 Feb 15, 61(4), 1486 - 92
Identification and characterization of A-105972, an antineoplastic agent; Wu-Wong JR et al.; A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells . After screening more than 60,000 compounds, A-105972 was identified and selected for further testing . A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells . The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type . The potency of A-105972 is similar in cells expressing wild-type or mutant p53 . A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle . Using {3H}A-105972, we found that A-105972 bound to purified tubulin . Unlabeled A-105972 competed with {3H}A-105972 binding with an IC50 value of 3.6 microL . Colchicine partially inhibited {3H}A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect . Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies . DNA ladder studies also indicated that A-105972 induced apoptosis . A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation . A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388 . These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells . A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.

Cancer Res, 2001 Feb 15, 61(4), 1469 - 76
The pharmacological phenotype of combined multidrug-resistance mdr1a/1b- and mrp1-deficient mice; Johnson DR et al.; Two major classes of plasma membrane proteins that actively extrude a wide range of structurally diverse hydrophobic amphipathic antineoplastic agents from cells, with different mechanisms of action, lead to multidrug resistance . To study the importance of these ATP-binding cassette transporters to the toxicity of cancer chemotherapy agents, we have used mice genetically deficient in both the mdr1a and mdr1b genes {mdr1a/1b(-/-) mice}, the mrp1 gene {mrp1(-/-) mice}, and the combined genes mdr1a/1b and mrp1 {mdr1a/1b(-/-), mrp1(-/-) mice} and embryonic fibroblasts derived from wild-type mice and from the three gene knockout animals . The consequences of export pump deficiencies were evaluated primarily using vincristine and etoposide . Mice deficient in the three genes, mdr1a/1b and mrp1, exhibited a 128-fold increase in toxicity to vincristine and a 3-5-fold increase in toxicity to etoposide; increased toxicity to embryonic fibroblast cells from triple knockout mice also occurred with vincristine and etoposide . Vincristine, which normally does not express toxicity to the bone marrow and to the gastrointestinal mucosa when used at therapeutic doses, caused extensive damage to these tissues in mdr1a/1b(-/-), mrp1(-/-) mice . The findings indicate that the P-glycoprotein and mrpl are compensatory transporters for vincristine and etoposide in the bone marrow and the gastrointestinal mucosa and emphasize the potential for increased toxicities by the combined inhibition of these efflux pumps.

Cancer Res, 2001 Feb 15, 61(4), 1412 - 4
P-Glycoprotein and multidrug resistance-related protein expressions in relation to technetium-99m methoxyisobutylisonitrile scintimammography findings; Kao CH et al.; The purpose of this study was to retrospectively study 48 patients with infiltrating ductal breast cancer to evaluate the relationship between the degree of accumulation of technetium-99m methoxyisobutylisonitrile (Tc-MIBI) and P-glycoprotein (Pgp) or multidrug resistance-related protein (MRP) expression in breast cancer tissues . Before surgery or biopsy, all 48 patients underwent scintimammography started 10 min after the injection of Tc-MIBI . Tumor:background (T:B) ratios were calculated from the Tc-MIBI scintimammography . Immunohistochemical analysis was performed on the pathological specimens of the 48 breast tumors to determine Pgp and MRP expression . According to the results of immunohistochemical analysis, the 48 breast cancers were separated into four groups: (a) group 1, 12 cancers with both positive Pgp expression and positive MRP expression; (b) group 2, 12 cancers with positive Pgp expression and negative MRP expression; (c) group 3, 12 cancers with negative Pgp expression and positive MRP expression; and (d) group 4, 12 cancers with both negative Pgp expression and negative MRP expression . Among the four groups, the T:B ratio was lowest in group 1 (1.13+/-0.10) and highest in group 4 (2.17+/-0.14), respectively (P < 0.05) . The T:B ratios of groups 2 (1.30+/-0.25) and 3 (1.32+/-0.26) were between those of groups 1 and 4 . Our data confirmed that Tc-MIBI scintimammography is useful for determining Pgp and MRP expression in patients with breast cancers.

Leukemia, 2001 Jan, 15(1), 80 - 8
Cyclosporin increases cellular idarubicin and idarubicinol concentrations in relapsed or refractory AML mainly due to reduced systemic clearance; Smeets M et al.; The feasibility of adding both the multidrug resistance modulator cyclosporin (CsA) and granulocyte colony-stimulating factor (G-CSF) to a standard salvage regimen of idarubicin (IDA) and cytarabine was evaluated in patients with resistant or relapsed acute myeloid leukemia and myelodysplastic syndrome . Three patients received IDA 12 mg/m2/day, the next four patients 9 mg/m2/day . The dose of CsA was 16 mg/kg/day . Six patients showed Pgp expression and none MRP1 expression . Grade III or IV toxicity (CTC-NCIC criteria) was registered in six patients for gastrointestinal, two patients for cardiovascular and one patient for neurological complications . Three patients died in hypoplasia and three patients showed leukemic regrowth . Three control patients were treated with IDA 12 mg/m2/day and cytarabine, but no CsA and G-CSF . The plasma IDA and idarubicinol (ida-ol) area under the curve's of patients treated with IDA 12 mg/m2 plus CsA were higher (P< 0.05) than in controls . Cellular IDA concentrations were almost similar, but cellular ida-ol concentrations were significantly higher (P < 0.05) in the presence of CsA than in controls . We conclude that the toxicity either with IDA 12 or 9 mg/m2/day was too high . The modulating effect of CsA was mainly based on changes in plasma kinetics of IDA and ida-ol, although ida-ol cellular clearance was delayed in the presence of CsA.

Zhonghua Xue Ye Xue Za Zhi, 1998 May, 19(5), 247 - 9
{A study of the GSH contents, GST activity and the expression of GST isoenzyme and MRP in drug-resistant leukemic cell lines}; Xia R et al.; OBJECTIVE: To understand the non-mdr1 mechanism in multidrug-resistant leukemic cell lines . METHODS: GSH contents and GST activity were measured by biochemical methods, the expression of GST isoenzyme alpha, pi, mu and MRP mRNAs were examined by Northern blot, and the expression of GST alpha, pi, mu proteins were examined by Western blot in sensitive HL-60, K562 and resistant K562/H20, HL-60/Adr, K562/VCR leukemic cell lines, respectively . RESULTS: Compared with K562/S, GSH contents and GST activities were increased in K562/H20 and K562/VCR, and GST alpha, pi or GST mu, pi were overexpressed in K562/H20 or K562/VCR, while there was no difference of GSH contents, GST activities or GST isoenzyme expressions between HL-60/Adr and HL-60/S . The increased expressions of MRP mRNA were revealed in the three resistant cell lines . CONCLUSION: GSH, GST and MRP involved in the drug resistance of K562/H20 and K562/VCR, while only MRP associated with drug resistance of HL-60/Adr.

J Acquir Immune Defic Syndr, 2001 Mar 1, 26(3), 266 - 73
Frequency of genotypic and phenotypic drug-resistant HIV-1 among therapy-naive patients of the German Seroconverter Study; Duwe S et al.; Genotypic and phenotypic resistance of viral reverse transcriptase (RT) and protease (PR) was determined for 64 therapy-naive, HIV-1-infected seroconverters of the German Seroconverter Study coordinated by the Robert Koch-Institut, Berlin . The date of seroconversion of patients and the laboratory, clinical, and therapeutic follow-up data were documented . Samples were collected between 1996 and 1999 . Phenotypic resistant HIV-1 were found in 8 (13%) seroconverters; in most cases resistance was weak and mainly directed against RT inhibitors (4 nucleoside reverse transcriptase inhibitors {NRTIs}, 2 nonnucleoside reverse transcriptase inhibitors {NNRTIs}, 1 combination NRTI/NNRTI) . Only one infection with a weak PR inhibitor resistance was identified . Transmission of multidrug-resistant HIV-1 has not yet been observed . Frequently at least one or more amino acid mutations associated with antiretroviral drug resistance were detected by genotypic analysis . The mean number of resistance-associated mutations in the RT of the transmitted virus has increased significantly since 1996 . Studies have shown the improved benefit of initial antiretroviral therapy if based on genotypic resistance data . In view of the considerably high level of transmission of resistant HIV-1 in Germany, which is also seen in other studies in Europe and the United States, we suggest determining the genotypic resistance pattern before starting therapy of newly HIV-1-infected patients.

J Acquir Immune Defic Syndr, 2001 Feb 1, 26(2), 145 - 50
Primary genotypic and phenotypic HIV-1 drug resistance in recent seroconverters in Madrid; Briones C et al.; OBJECTIVE: Transmission of drug-resistant HIV-1 strains is increasing with widespread use of antiretroviral drugs in developed countries . This study examined the prevalence of resistant viruses in recent seroconverters in Madrid, Spain . DESIGN: HIV isolates from 30 consecutive participants with positive or indeterminate HIV antibody test results and a negative test result at a mean of 6.6 months earlier were examined for HIV drug resistance . All study subjects admitted to having very recently engaged in high-risk practices . All were therapeutically naive and were recruited between 1997 and 1999 in a referring health care facility for sexually transmitted diseases . METHODS: Population-based sequencing of the viral reverse transcriptase (RT) and protease (PR) regions derived from plasma viral RNA was performed . Phenotypic resistance was assessed by a recombinant virus assay . RESULTS: Overall prevalence of genotypes associated with reduced susceptibility was 26.7% (8 of 30 participants) . Resistance mutations were seen against nucleoside analogues in 7 (23.3%), nonnucleoside reverse transcriptase inhibitors in 1 (3.3%), and protease inhibitors in 2 (6.7%) . Zidovudine-resistance mutations M41L and/or T215Y were the commonest, found in 20% (6 of 30 participants) . Resistance mutations to at least two antiretroviral families (multidrug-resistance) were detected in 2 (6.7%) study subjects . A median infectious dose (IC50) increase of fourfold for any drug was found in 7 patients, and in 2 was > tenfold for zidovudine (genotype M41L + T215Y) and lamivudine (genotype M184V), respectively . CONCLUSIONS: Drug-resistant HIV variants were present in over one quarter of individuals recently diagnosed as infected in Madrid, Spain . Therefore, resistance testing at baseline should be considered for the optimal design of first-line antiretroviral combinations.

AIDS, 2001 Mar 9, 15(4), 483 - 91
Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood--brain barrier; van der Sandt IC et al.; OBJECTIVE: To investigate the involvement of P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) on the active transport of the HIV protease inhibitors amprenavir, ritonavir and indinavir . METHODS: The transport behaviour of ritonavir, indinavir and amprenavir in the presence and absence of Pgp modulators and probenecid was investigated in an in vitro blood--brain barrier (BBB) co-culture model and in monolayers of LLC-PK1, LLC-PK1:MDR1, LLC-PK1:MRP1 and Caco-2 cells . RESULTS: All three HIV protease inhibitors showed polarized transport in the BBB model, LLC-PK1:MDR1 and Caco-2 cell line . The Pgp modulators SDZ-PSC 833, verapamil and LY 335979 inhibited polarized transport, although their potency was dependent on both the cell model and the HIV protease inhibitor used . Ritonavir and indinavir also showed polarized transport in the LLC-PK1 and LLC-PK1:MRP1 cell line, which could be inhibited by probenecid . HIV protease inhibitors were not able to inhibit competitively polarized transport of other HIV protease inhibitors in the LLC-PK1:MDR1 cell line . CONCLUSIONS: Amprenavir, ritonavir and indinavir are mainly actively transported by Pgp, while MRP also plays a role in the transport of ritonavir and indinavir . This indicates that inhibition of Pgp could be useful therapeutically to increase HIV protease inhibitor concentrations in the brain and in other tissues and cells expressing Pgp . The HIV protease inhibitors were not able to inhibit Pgp-mediated efflux when given simultaneously, suggesting that simultaneous administration of these drugs will not increase the concentration of antiretroviral drugs in the brain.

Clin Pharmacol Ther, 2001 Mar, 69(3), 169 - 74
Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects; Cascorbi I et al.; BACKGROUND: P-glycoprotein, the gene product of MDR1, confers multidrug resistance against antineoplastic agents but also plays an important role in the bioavailability of common drugs in medical treatment . Various polymorphisms in the MDR1 gene were recently identified . A silent mutation in exon 26 (C3435T) was correlated with intestinal P-glycoprotein expression and oral bioavailability of digoxin . OBJECTIVE: We wanted to establish easy-to-use and cost-effective genotyping assays for the major known MDR1 single nucleotide polymorphisms and study the allelic frequency distribution of the single nucleotide polymorphisms in a large sample of volunteers . METHODS: In this study, the distribution of the major MDR1 alleles was determined in 461 white volunteers with the use of polymerase chain reaction and restriction fragment length polymorphism . RESULTS: Five amino acid exchanges were found with allelic frequencies of 11.2% for Asn21Asp and 5.5% for Ser400Asn . Strikingly, in exon 21 three variants were discovered at the same locus: 2677G (56.4%), 2677T (41.6%), and 2677A (1.9%), coding for 893Ala, Ser, or Thr . A novel missense Gln1107Pro mutation was found in two cases (0.2%) . The highest frequencies were observed for intronic and silent polymorphisms; C3435T occurred in 53.9% of the subjects heterozygously, and 28.6% of individuals were homozygous carriers of 3435T/T with functionally restrained P-glycoprotein . CONCLUSION: This study provides the first analysis of MDR1 variant genotype distribution in a large sample of white subjects . It gives a basis for large-scale clinical investigations on the functional role of MDR1 allelic variants for bioavailability of a substantial number of drugs.

Int J Gynecol Cancer, 2000 Jan, 10(S1), 47 - 52
Options for modulation of drug resistance in ovarian cancer; Arts HJ et al.; The objective of this paper is to present an update of mechanisms responsible for drug resistance in ovarian cancer and the possible therapeutic options to modulate this resistance using literature review with emphasis on data acquired in studies comprising ovarian tumor samples . The classic concepts on resistance in ovarian cancer, namely platinum and multidrug resistance, are briefly discussed, followed by a description of more recent insights concerning the role of apoptosis in the development of chemoresistance . A wide variety of mechanisms may be responsible for drug resistance in ovarian cancer . However, a growing body of evidence indicates that defects in the intra- and extracellular apoptotic pathways are an important cause of resistance to cytotoxic agents which opens several new treatment strategies.

Biochem Pharmacol, 2001 Mar 1, 61(5), 555 - 63
Differential sensitivities of the MRP gene family and gamma-glutamylcysteine synthetase to prooxidants in human colorectal carcinoma cell lines with different p53 status; Lin-Lee YC et al.; 1Recent molecular cloning studies have identified six members in the multidrug-resistance protein (MRP) gene family . However, the regulation of expression of these genes is largely unknown . We previously reported that expression of MRP1, encoding multidrug-resistance associated protein, and gamma-GCSh, which encodes the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS), could be up-regulated by prooxidants {Yamane et al., J Biol Chem 1998;273:31075-85} . In the present study, we investigated whether different members of the MRP family exhibit different responses to induction by prooxidants, and whether p53 status influences the levels of induction . A panel of colorectal cancer cell lines with different p53 status, i.e . HCT116 containing wild-type p53, and HT29, SW480, and Caco2 containing mutant p53, was treated with tert-butylhydroquinone (t-BHQ) and pyrrolidinedithiocarbamate (PDTC) . MRP1 and gamma-GCSh mRNA levels were determined by the RNase protection assay, using gene-specific probes . We report here that induction of MRP1 and gamma-GCSh expression by these prooxidants varied among the different cell lines, and p53 mutations were not always associated with elevated levels of induction . These results suggest that the effects of p53 on the induced expression of MRP1 and gamma-GCSh depend on the environment of the cell and/or nature of p53 mutations . In an isogenic HCT116 cell line containing p53(-/-) alleles, we demonstrated that, as for MRP1, expression of MRP2 and MRP3 was induced by the prooxidants, whereas expression of MRP4 and MRP5 was not . MRP6 mRNA was not detectable . Induction of MRP2 expression by prooxidants seemed to be independent of p53 status . Our results demonstrated the differential regulation of the MRP gene family by p53 mutation under oxidative stress.

J Virol, 2001 Apr, 75(7), 3291 - 300
Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1; Bleiber G et al.; Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity . The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as "fitness") in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background . Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs . In peripheral blood mononuclear cells, replication rates (means +/- standard deviations) of RT recombinants were 72.5% +/- 27.3% and replication rates of PR recombinants were 60.5% +/- 33.6% of the rates of NL4-3 . PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% +/- 23.5% of NL4-3 replication rates . Cloning of the cognate RT improved fitness of some PR mutant clones . For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% +/- 10.7%, and integrase content diminished by 23.3% +/- 18.4%), a novel mechanism for a decreased-fitness phenotype . Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT . Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure . These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Blood, 2001 Mar 15, 97(6), 1888 - 91
The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells; Sellers SE et al.; Transduction of murine stem cells with a multidrug-resistance 1 gene (MDR1) retrovirus results in dramatic ex vivo and in vivo expansion of repopulating cells accompanied by a myeloproliferative disorder . Given the use of MDR1-containing vectors in human trials, investigations have been extended to nonhuman primates . Peripheral blood stem cells from 2 rhesus monkeys were collected, CD34-enriched, split into 2 portions, and transduced with either MDR1 vectors or neo vectors and continued in culture for a total of 10 days before reinfusion . At engraftment, the copy number in granulocytes was extremely high from both MDR vectors and neo vectors, but the copy number fell to 0.01 to 0.05 for both . There were no perturbations of the leukocyte count or differential noted . After 3 cycles of stem cell factor/granulocyte colony-stimulating factor, there were no changes in the levels of MDR1 vector- or neo vector-containing cells . There was no evidence for expansion of MDR1 vector-transduced cells . Long-term engraftment with MDR1 vector- and neo vector-transduced cells occurred despite prolonged culture.

Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 827 - 34
Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms; Demeule M et al.; Endothelial cells (EC) were isolated from brain, lung, and renal cortex using magnetic microbeads cross-linked to an antibody directed against the platelet-endothelial cell adhesion molecule-1 (PECAM-1) . Levels of endothelial nitric oxide synthase (eNOS) and PECAM-1 were measured by Western blots and both were enriched in the positively selected EC fractions . The multidrug resistance P-glycoprotein (P-gp) was strongly enriched (59-fold) in the EC fraction from brain and was absent in the negative fraction, in which the glial fibrillary acidic protein (GFAP), an astrocyte marker, was present . Lower P-gp levels were detected in EC from renal cortex and lung . Reverse transcription-polymerase chain reaction analysis showed that the mdr1a gene was preferentially expressed in EC fraction from the brain . The mdr1b gene was found in EC from renal cortex whereas both mdr1 genes were detected in EC from lung . Our results indicate that EC can be isolated using microbeads and that the isoform of P-gp found in brain is mostly mdr1a, associated with EC .

Br J Cancer, 2001 Mar 2, 84(5), 680 - 5
Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML; Sargent JM et al.; Treatment failure in AML is often attributed to P-glycoprotein-associated multidrug resistance . However, the importance of increased DNA repair in resistant cells is becoming more apparent . In order to investigate the ability of the DNA repair inhibitor aphidicolin to modulate drug resistance, we continually exposed blasts cells, isolated from 22 patients with AML, to a variety of agents +/- 15 microM aphidicolin for 48 hours . Cell survival was measured using the MTT assay . Overall, there was no significant effect of aphidicolin on sensitivity to daunorubicin, doxorubicin, etoposide or fludarabine . However, there was a marked increase in sensitivity to ara-C with a median 4.75-fold increase overall (range 0.8-80-fold;P< 0.005) . The effect of aphidicolin was significantly greater in blast cells found resistant in vitro to ara-C (8.9-fold compared to 2.12-fold, P< 0.01) . This observation was further validated by the correlation between ara-C LC(50)and extent of modulation effect (P< 0.05) . Cells isolated from 10 cord blood samples were also tested in order to establish the haematological toxicity of combining ara-C and aphidicolin . The therapeutic index (LC(50)normal cells/tumour cells) for ara-C + aphidicolin was higher than that for ara-C alone suggesting no increased myelotoxicity for the combination . Increased cytotoxicity without increased haematotoxicity makes the combination of ara-C plus aphidicolin ideal for inclusion in future clinical trials .

Leukemia, 2001 Mar, 15(3), 398 - 405
MDR1 expression in poor-risk acute myeloid leukemia with partial or complete monosomy 7; van den Heuvel-Eibrink MM et al.; Expression of the multidrug resistance (MDR1) phenotype, encoded by the MDR1 gene, is an adverse prognostic factor for CR and survival in acute myeloid leukemia (AML) . Other prognostic factors, such as specific cytogenetic abnormalities, have been identified in AML . We have investigated the expression of the MDR1 gene in untreated AML patients with monosomy 7 (n = 12), and partial deletions (n = 7) of the long arm of chromosome 7 (respectively -7/7q-), because of the extremely bad prognosis associated with these cytogenetic abnormalities and because of the fact that the MDR1 gene is located on chromosome 7q21.1.The findings were compared with the level of MDR1 expression in a group of 42 other AML patients, matched for age with favourable, neutral or complex cytogenetic abberations . MDR1 mRNA expression, as measured by the RNase protection assay was significantly higher in the -7/7q- group vs other AML patients (median 1.3 vs 0.1 arbitrary units, P = 0.02) . Protein expression of MDR1 in the -7/7q- group, as determined with the monoclonal antibody MRK16, was found to be similar to the levels found in the control group . With a functional rhodamine retention assay using the modulator PSC833, increased MDR1 activity was observed in the -7/7q- group as compared to the control group of patients (P = 0.05) . Considering the higher MDR1 mRNA expression and equal or slightly elevated level of protein expression of MDR1, we studied the presence of MDR1 genes in this group of -7/7q- patients . Fluorescence in situ hybridization (FISH) studies, using a specific MDR1 probe revealed no loss of an MDR1 allele in any of the deleted q- arms of the seven patients with 7q-, whereas all monosomy 7 patients lacked one MDR1 gene homologue . To determine whether there was selective loss of the MDR1 gene in the -7/7q- patients, the genetic polymorphism of the MDR1 gene was used . Both allelic variants (G and T) were represented in the -7/7q- and in the control group, showing a predominance for GT at position 2677 of the MDR1 gene in the control group . In the 12 monosomy 7 patients loss of the MDR1 allele was random . Methylation studies of the CpG island of the MDR1 gene revealed no hypermethylation in any of the -7/7q- patients . We conclude that MDR1 expression in -7/7q- AML patients is upregulated at transcriptional, but not at translational level, suggesting that mechanisms other than MDR1 are responsible for the poor prognosis in these patients.

Zhonghua Zhong Liu Za Zhi, 2000 Nov, 22(6), 487 - 9
{Studies of multidrug resistance gene expression and apoptosis in stomach carcinoma}; Chen X et al.; OBJECTIVE: To investigate the influence of multidrug resistance gene (MDR) expression and apoptosis on stomach cancer . METHODS: A total of 80 cases of paraffin embedded stomach cancer tissues was stained immunohistochemically for P-gp, GST-pi and TOPO II using monoclonal antibodies and apoptosis (in terms of apoptotic index, AI) in tissues was musured by the TUNEL method . The results were correlated with tumor type, clinical stage and prognosis . RESULTS: Normal stomach tissue expression of P-gp was associated with better survival, but higher level of P-gp expression in tumor tissues was associated with poor prognosis . The expression of GST-pi didnot show a relationship with cell differentiation of stomach cancer and clinical stage of the disease, but positive correlation did exist between intensity of expression and survival time . Although the expression of TOPO II was associated with tumor cell differentiation, it did not affect prognosis . Apoptosis was demonstrated in 73% of the cases and was related to clinical stage of stomach carcinoma . CONCLUSION: While P-gp expression in tumor cells is indicative of poor prognosis, its expression in normal tissue has the opposite effect . Positive correlation exits between GST-pi expression intensity in tumor cells and survival . MDR and apoptosis are related to the biologic behavior of the tumor.

Zhonghua Zhong Liu Za Zhi, 2000 Nov, 22(6), 456 - 9
{Protection of hematopoietic cells against toxicity of anticancer agents by transfecting mdr1 gene into bone marrow cells}; Chen L et al.; OBJECTIVE: To investigate whether the hematopoietic cells can be protected from the toxic effect of anticancer agents by transfecting mdr1 gene into bone marrow cells . METHODS: The mdr1 gene was transfected into murine and human bone marrow cells by Lipofectin . Expression of mdr1 was assessed by RT-PCR and flow cytometry . The functional activity of mdr1 in transfected cells was examined by rhodamine retention assay . Resistance to chemotherapeutic drugs of human and murine mdr1 gene transfected bone marrow cells was ascertained in vivo and that of murine cells was assessed in vivo in a bone marrow transplantation model . RESULTS: Multidrug resistance 1 gene was successfully transfected into and expressed in murine and human bone marrow cells . The transfected cells were resistant to doxorubicin; Vp-16, vincristine and colchicine . Bone marrow cells transfected with mdr1 gene reconstituted the hematopoietic function and offered resistance to doxorubicin in recipient mice . CONCLUSION: To transfect mdr1 gene into bone marrow cells is a promissing approach to protect bone marrow cells from the myelosuppressive effect of chemotherapeutic agents.

Life Sci, 2001 Feb 2, 68(11), 1323 - 31
Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from peripheral blood; Laupeze B et al.; Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions . In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay . Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells . Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes . Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels . Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.

Infect Control Hosp Epidemiol, 2001 Feb, 22(2), 109 - 11
Multidrug-resistant bacteria infection control: study of compliance with isolation precautions in a Paris university hospital; Vidal-Trecan GM et al.; Isolation practices in a university hospital were analyzed for 137 patients with multidrug-resistant bacteria . Isolation was ordered in writing by physicians for 40% and instituted by nurses for 60%; 74% were isolated . Compliance depended on physician ordering in writing (odds ratio, 36.3; 95% confidence interval, 4.8-274.9) . Nurses complied best with hand washing.

J Contin Educ Health Prof, 2000 Summer, 20(3), 146 - 55
Evaluating provider prescribing practices for the treatment of tuberculosis in Virginia, 1995 to 1998: an assessment of educational need; Richardson NL; BACKGROUND: Although the use of epidemiologic studies to demonstrate learning needs appears to be infrequent, this study addressed the discrepancies in the prescribing practices for the initial treatment of tuberculosis in Virginia between public and private clinicians, comparing them with the treatment regimens recommended by the Centers for Disease Control and Prevention and the American Thoracic Society (CDC-ATS) . METHODS: Data examined were the 1995 to 1998 reported cases of tuberculosis within the Commonwealth of Virginia . The study population consisted of 770 laboratory-confirmed tuberculosis cases, living in Virginia, whose isolates were tested for isoniazid susceptibility and were prescribed an initial drug regimen . Prevalence rates, prevalence odds ratios, and logistic regression were used to determine the estimated risk for receipt of the CDC-ATS treatment regimen . RESULTS: Of the 770 cases, 28.7% did not receive the CDC-ATS recommended drug regimen . Prevalence rates and odds for not receiving the recommended regimen were highest among persons of United States origin, Caucasians, females, persons under age 15, and persons from the southwest region of Virginia . Logistic regression indicated a slight increase in the estimated risk of not receiving the CDC-ATS regimen from a private physician (OR: 1.40; CI: 0.97, 2.04) when compared to a public physician . FINDINGS: Private tuberculosis care providers were less compliant with CDC-ATS guidelines than public tuberculosis care providers . Because providers did not follow the recommended treatment guidelines universally, it is advised that all tuberculosis care providers in Virginia would benefit from increased education regarding adequate treatment regimens for tuberculosis and the prevention of multidrug-resistant tuberculosis.

Biochem Pharmacol, 1999 May 15, 57(10), 1147 - 52
HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833; Drewe J et al.; The effect of P-glycoprotein inhibition on the uptake of the HIV type 1 protease inhibitor saquinavir into brain capillary endothelial cells was studied using porcine primary brain capillary endothelial cell monolayers as an in vitro test system . As confirmed by polymerase chain reaction and Western blot analysis, this system functionally expressed class I P-glycoprotein (pgp1A) . P-Glycoprotein isoforms pgp1B or pgp1D could not be detected . The uptake of saquinavir into endothelial cells could be described as the result of a diffusional term of uptake and an oppositely directed saturable extrusion process . Net uptake of saquinavir into cultured brain endothelial cells could be increased significantly up to 2-fold by SDZ PSC 833 in a dose-dependent manner, with an IC(50) of 1.13 microM . In addition, the HIV protease inhibitor ritonavir inhibited p-glycoprotein-mediated extrusion of saquinavir with an IC(50) of 0.2 microM, indicating a high affinity of ritonavir for p-glycoprotein . In conclusion, we showed that the HIV protease inhibitor ritonavir is a more potent inhibitor of P-glycoprotein than the multidrug resistance (MDR)-reversing agent SDZ PSC 833 . The inclusion of this drug in combination regimens may greatly facilitate brain uptake of HIV protease inhibitors, which is especially important in patients suffering from AIDS dementia complex.

Hepatology, 2001 Mar, 33(3), 509 - 18
Regulation of the dynamic localization of the rat Bsep gene-encoded bile salt export pump by anisoosmolarity; Schmitt M et al.; Canalicular transport via the bile salt export pump (Bsep) represents the rate-controlling step in taurocholate excretion, whose capacity is under osmotic control . The short-term effects of anisoosmolarity and Ca(2+)-withdrawal on the localization of Bsep and the tight junction proteins Zo-1 and occludin were studied in perfused rat liver by immunohistochemistry, confocal microscopy, and densitometry . Under normoosmotic conditions, Bsep was found in the canalicular membrane and showed a punctate intracellular localization . Hypoosmolarity resulted in the translocation of intracellular Bsep to the canalicular membrane, whereas hyperosmolarity induced a retrieval of Bsep . Following hyperosmolar retrieval of Bsep and multidrug resistance protein 2 (Mrp2) from the canalicular membrane, in the putative intracellular vesicles Bsep and Mrp2 colocalized in 15% of these vesicles, whereas 85% stained either positive for Bsep (61%) or Mrp2 (24%) . Anisotonicity had no effect on the linear staining patterns of occludin and Zo-1, indicating no increase in paracellular permeability . Omission of calcium produced cholestasis characterized by a disruption of occludin, whereas the localization of Zo-1, Bsep, and Mrp2 remained unaffected . It is concluded (1) that hyperosmolarity induces retrieval of Bsep from the canalicular membrane, which correlates to cholestasis . Hypoosmolarity leads to choleresis accompanied by a rapid recruitment of intracellular Bsep to the canalicular membrane . (2) Bsep- and Mrp2-specific vesicles participate in the short-term osmoregulation of canalicular secretion, however, a cause-effect relationship between bile salt excretion and transporter localization remains to be established . (3) Ca(2+)-depletion induces cholestasis by disruption of occludin-determined tight junctional permeability, whereas internalization of canalicular transporters play a minor role.

J Clin Microbiol, 2001 Mar, 39(3), 871 - 8
Reverse dot blot assay (insertion site typing) for precise detection of sites of IS6110 insertion in the Mycobacterium tuberculosis genome; Steinlein LM et al.; We have developed an amplification-based reverse dot blot assay for the detection of specific sites of insertion of the Mycobacterium tuberculosis insertion sequence IS6110 . In this assay, a set of biotin-labeled amplicons representing the various copies of IS6110 and their flanking sequences is generated by linker-mediated PCR . The amplicons are then hybridized to immobilized oligonucleotide probes that are specific for known IS6110 insertion sites . The method was evaluated using an array of oligonucleotide probes corresponding to IS6110 insertion sites from M . tuberculosis strains CDC1551, Erdman, and H37Rv, and multidrug-resistant strain W . A set of 72 DNA samples from 60 M . tuberculosis clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly reproducible . This method of identifying insertion sites has been named "insite" and can be used for the genotyping of M . tuberculosis complex strains based on IS6110 insertion site profiles.

Eur J Pharmacol, 2001 Feb 16, 413(2-3), 131 - 41
Transport of new non-cross-resistant antitumor compounds of the benzoperimidine family in multidrug resistant cells; Tkaczyk-Gobis K et al.; Multidrug resistance (MDR) phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or multidrug resistance-associated protein (MRP1) . Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins . The influx and efflux of drugs across the cell membrane are in large part responsible for their intracellular concentrations, and in the search for new compounds able to overcome MDR, it is of prime importance to determine the molecular parameters whose modification would lead to an increase in the kinetics of uptake and/or to a decrease in the pump-mediated efflux . Here, we studied three members of a new family of benzoperimidine antitumor compounds which exhibit comparable cytotoxicity towards resistant cells expressing P-glycoprotein, or MRP1, and sensitive cells . We used spectrofluorometric methods to determine the kinetics of the uptake and release of these three drugs in different cell lines: the erythroleukemia cell line K562 and the resistant K562/Adr expressing P-glycoprotein, the small-cell lung cancer cell line GLC4 and resistant GLC4/Adr expressing MRP1 . We also studied, using confocal microscopy, the intracellular distribution of these drugs in NIH/3T3 cells . Our data show that (i) the kinetics for the uptake of these drugs is very rapid, higher than 2 x 10(-17) mole cell(-1) s(-1), (ii) the drugs are strongly accumulated in the nucleus and lysosomes, (iii) the three drugs are recognized and pumped out by both transporters, as shown by the inhibition of P-glycoprotein- and MRP1-mediated efflux of pirarubicin by benzoperimidine, with inhibitory constants of 1.5 and 2.1 microM for P-glycoprotein and MRP1, respectively, suggesting that benzoperimidine is transported by the two transporters with K(m) approximately 2 microM . In conclusion, the fast uptake kinetics of the benzoperimidines counterbalance their efflux by P-glycoprotein and MRP1.

Semin Oncol, 2000 Dec, 27(6 Suppl 12), 30 - 6
Chemotherapy sensitization by rituximab: experimental and clinical evidence; Wilson WH; For most lymphomas, chemotherapy is palliative because of an inability to overcome drug resistance within the lymphoma cells and attempts at overcoming specific drug resistance mechanisms, such as multidrug resistance, have had limited success . However, accumulating evidence suggests that the ability to activate apoptotic pathways may be an important determinant of chemotherapy sensitivity and presents a potentially important new therapeutic strategy . Studies have shown that distinct cellular thresholds exist for apoptosis, and it is likely that multiple developmental and environmental factors converge in a dynamic process to regulate this set point . If the threshold for inducing apoptosis is limiting, then strategies to modulate these pathways may profoundly enhance the efficacy of cytotoxic therapy . Monoclonal antibodies against the CD20 receptor have been shown to directly induce apoptosis and may serve to modulate the threshold for chemotherapy-induced apoptosis . Recent clinical studies of the monoclonal antibody, rituximab (Rituxan; Genentech, Inc, South San Francisco, CA and IDEC Pharmaceutical Corporation, San Diego, CA), and combination chemotherapy have produced unexpectedly high rates of response and progression-free survival, suggesting rituximab improves the efficacy of chemotherapy . Taken together, the results from in vitro and clinical studies suggest that rituximab may modulate the sensitivity of B-cell lymphomas to chemotherapy.

Jpn J Cancer Res, 2001 Feb, 92(2), 220 - 30
Phase I study of intravenous PSC-833 and doxorubicin: reversal of multidrug resistance; Minami H et al.; PSC-833 reverses multidrug resistance by P-glycoprotein at concentrations < or = 1000 ng / ml . A phase I study of PSC-833 and doxorubicin was conducted to determine the maximum tolerated dose and to investigate pharmacokinetics . PSC-833 was intravenously infused as a 2-h loading dose (LD) and a subsequent 24-h continuous dose (CD) . Doxorubicin was infused over 5 min, 1 h after the LD . The starting dose was 1 mg / kg for both LD and CD with 30 mg / m(2) doxorubicin; these dosages were increased to 2 and 10 mg / kg and 50 mg / m(2), respectively . Thirty-one patients were treated . Nausea / vomiting was controllable with granisetron and dexamethasone . Neutropenia and ataxia were dose limiting . Steady-state concentrations of PSC-833 > 1000 ng / ml were achieved at a 2 mg / kg LD and a 10 mg / kg CD . Ex-vivo bioassay revealed that activity in serum for reversing multidrug resistance was achieved in all patients; IC(50) of P-glycoprotein expressing 8226 / Dox(6) in patients' serum was decreased from 5.9 to 1.3 microg / ml (P < 0.0001) by PSC-833 administration . Doxorubicin clearance was 24.3 +/- 13.7 (mean +/- SD) liter / h/m(2), which was lower than the 49.0 +/- 16.9 liter / h/m(2) without PSC-833 (P < 0.0001) . The relationship between doxorubicin exposure and neutropenia did not differ between patients treated and not treated with PSC-833 . The recommended phase II dose of PSC-833 was 2 and 10 mg / kg for LD and CD, respectively, which achieved a sufficient concentration in serum to reverse drug resistance, as confirmed by bioassay . The dose of doxorubicin should be reduced to 40 mg / m(2), not because of the pharmacodynamic interaction between PSC-833 and doxorubicin affecting hematopoiesis, but because of pharmacokinetic interaction.

Jpn J Cancer Res, 2001 Feb, 92(2), 211 - 9
Involvement of the multidrug resistance protein 3 in drug sensitivity and its expression in human glioma; Haga S et al.; The multidrug resistance protein (MRP) family belongs to the ATP-binding cassette superfamily (ABC) of transporters, which are involved in ATP-dependent transport of hydrophobic compounds . One of the MRP family, MRP1, is partially associated with the multidrug resistance phenotype in brain tumors . In this study, we asked whether another MRP family gene, MRP3, could affect drug sensitivity to anticancer agents in human glioma cell lines and clinical glioma specimens . We first produced two antisense transfectants by introduction of antisense MRP3 cDNA into the glioma cell line NHG2, which endogenously expresses MRP3 . The two MRP3 antisense transfectants showed 2- to 5-fold increases in drug sensitivity to etoposide and cisplatin compared with NHG2 cells, but their sensitivity to vincristine or nitrosourea was not changed . Two MRP3 cDNA sense transfectants of pig kidney cell lines showed 4- to 6-fold drug resistance to etoposide, but only 1.4- to 1.5-fold to cisplatin . We next compared the mRNA levels of four ABC transporters, multidrug resistance 1 (MDR1), MRP1, MRP2 and MRP3 in clinical samples, including 34 patients with gliomas, by quantitative RT-PCR analysis . In some of the clinical samples, increased expression of MRP1 and MRP3 was apparent in malignant gliomas . In situ hybridization revealed that glioma cells were stained with MRP3 probe . MRP3 may modulate drug sensitivity to certain anticancer agents in human gliomas.

Phytochemistry, 2001 Jan, 56(2), 153 - 9
The ABC-like vacuolar transporter for rye mesophyll flavone glucuronides is not species-specific; Klein M et al.; In many cases, the vacuolar uptake of secondary metabolites has been demonstrated to be strictly specific for a given compound and plant species . While most plants contain glycosylated secondary substances, few cases are known where flavonoids may also carry negative charges, e.g . as glucuronide conjugates . Vacuolar transport of glucosylated phenylpropanoid derivatives has been shown to occur by proton substrate antiport mechanisms (Klein, M., Weissenbock . G., Dufaud, A., Gaillard, C., Kreuz, K., Martinoia, E., 1996 . Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside . J . Biol . Chem . 271, 29,666-29,671) . In contrast, flavone glucuronides appearing specifically in rye mesophyll vacuoles are taken up by direct energisation utilising MgATP, strongly arguing for the presence of an ATP-binding cassette (ABC) transporter belonging to the subfamily of multidrug resistance-associated proteins (MRP) on the rye vacuolar membrane (Klein, M., Martinoia, E., Hoffmann-Thoma, G., Weissenbock, G., 2000 . A membrane-potential dependent, ubiquitous ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides regulation of glucturonide uptake by glutathione and its conjugates . Plant Journal 21, 289-304) . MRPs are known to transport negatively charged organic anions . Results presented here suggest that the vacuolar directly energised MRP-like glucuronate pump for plant-specific flavone glucuronides is ubiquitously present in diverse plant species since rye flavone glucuronides are taken up into vacuoles isolated from the barley mesophyll or from the broccoli stalk parenchyma representing two species which do not synthesise glucuronidated secondary compounds . According to the transport characteristics and inhibition profile observed we propose the existence of a high-capacity, uncoupler-insensitive vacuolar ABC transporter for flavone glucuronides and possibly other negatively charged organic compounds -- plant-born or xenobiotic -- irrespective of the plant's capability to endogenously produce glucuronidated compounds.

Zhonghua Fu Chan Ke Za Zhi, 2000 Nov, 35(11), 677 - 9
{Study of mdr1 antisense oligodeoxynucleotides on reversal of multidrug resistance in ovarian carcinoma cell line SKOV3}; Tong Y et al.; OBJECTIVE: To investigate the effect of mdr1 antisense oligodeoxynucleotides (ASON) on reversal of multidrug resistance in ovarian carcinoma cells . METHODS: Drug resistance ovarian carcinoma cells SKOV3/mdr1 transducted with human multidrug resistance gene (mdr1) were served as models . The positive rate and function of the mdr1 gene product P-glycoprotein (P-gp) in SKOV3/mdr1 cells after mdr1-ASON (250 micrograms/ml) treatment were determined by flow cytometry and rhodamine 123 efflux trial . Drug resistance of SKOV3/mdr1 cells was also observed by cell colony culture . RESULTS: P-gp positive rate of SKOV3/mdr1 cells after mdr1-ASON treatment was decreased from 38.9% to 21.3% (P < 0.01) . Intracellular rhodamine retension in SKOV3/mdr1 cells after mdr1-ASON treatment was increased from 32.1% to 50.7% (P < 0.01) . Under effect of Taxol 5 ng/ml, the relative percents of drug-resistant colony in mdr1-ASON treated SKOV3/mdr1 cells and in SKOV3/mdr1 cells was 8% and 63%, respectively, (P < 0.01) . Under effect of Doxorubicin 100 ng/ml, the relative percents of drug-resistant colony in mdr1-ASON treated SKOV3/mdr1 cells and in SKOV3/mdr1 cells was 34% and 79%, respectively, (P < 0.01) . CONCLUSION: mdr1-ASON can reverse multidrug resistance of ovarian carcinoma cell in a certain extent so as to increase chemotherapeutic sensitivity of ovarian carcinoma cells.

Biol Pharm Bull, 2001 Feb, 24(2), 111 - 8
Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions; Terasaki T et al.; Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen . These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen . Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake . These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport . TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity . They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity . TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc . TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2 . The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side . TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo . These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.

Mol Cell Biochem, 2001 Jan, 216(1-2), 103 - 10
Induction of P-glycoprotein mRNA transcripts by cycloheximide in animal tissues: evidence that class I Pgp is transcriptionally regulated whereas class II Pgp is post-transcriptionally regulated; Lee CH; P-glycoprotein (Pgp) are a small family of plasma membrane proteins capable of transporting substrates across cell membranes . Class I and class II Pgp are able to transport drugs and have been shown to mediate multidrug resistance (MDR) . Class III Pgp is a long chain phospholipid transporter and does not mediate MDR . The expression and regulation of Pgp genes in animal tissues are not well understood . In this study, the protein synthesis inhibitor cycloheximide was used as a tool to understand Pgp gene expression and regulation in animal tissues . The sensitive RNase protection assay was used to detect changes in Pgp mRNA levels and nuclear run-on assay was used to determine whether transcription or post-transcription is important . The results showed that cycloheximide significantly induced class II Pgp expression in all tissues examined . This was predominantly through post-transcriptional effect . In contrast, the relatively modest increase in class I Pgp expression by cycloheximide was found to be mainly due to increased transcriptional activity . On the other hand, cycloheximide induced class III Pgp expression in some tissues while caused decay of class III Pgp mRNA in other tissues . The transcriptional and post-transcriptional mechanisms exerted by cycloheximide on Pgp genes are discussed . These findings have implications for our understanding of gene regulation in animal tissues and MDR reversal strategies in vivo.

J Tongji Med Univ, 2000, 20(3), 231 - 4
The effect of different interventional treatment on P-Glycoprotein in different histopathological types and grades of primary hepatocellular carcinoma; Xiao E et al.; To study the effect of the different interventional treatment on P-Glycoprotein (Pgp) in different histopathological types of primary hepatocellular carcinoma (PHC), 98 surgically and histologically verified PHC specimens were obtained . The patients included 57 patients treated by surgical resection alone and 41 patients receiving second-stage surgical resection after four kinds of interventional treatment . SABC immunohistochemical staining with a monoclonal antibody against human Pgp was used to observe the Pgp in all specimens . The positive rate of Pgp was 100% in group of chemotherapy alone (P < 0.05), 62.5% in group of chemotherapy combined with iodized oil (P > 0.05), 46.6% in group of chemotherapy combined with iodized oil and spongia gelatini absorbens (Sga) (P > 0.05), 18.18% in group of chemotherapy combined with Ethanol-iodized-oil and Sga (P < 0.05) and 52.63% in group of surgical resection alone . The positive rate of Pgp varied with different histopathological types, with rate of clear cell PHC being the lowest, and that of poorly differentiated or undifferentiated PHC the highest . The positive rate of Pgp was increased as pathological grades increased . Overexpression of Pgp may be responsible for the intrinsic and acquired drug resistance of PHC . Multidrug resistance (MDR) varied with different histological types . Therapy of PHC should be tailored according to individual . Local chemotherapy combined with ethanol-iodized-oil and Sga embolization may become a new way to overcome MDR of PHC.

Cancer Res, 2001 Jan 15, 61(2), 749 - 58
In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576; Mistry P et al.; The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR) . This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs . Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs . XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound . The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines . XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576 . Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date . Accumulation and efflux studies with the P-gp substrates, {3H}daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux . The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium . This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp . Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by {3H}azidopine implying a direct interaction with the protein . In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v . or p.o . In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice . Importantly all of the efficacious combination schedules appeared to be well tolerated . Furthermore, i.v . coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel . These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action . It exhibits potent i.v . and p.o . activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs . Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.

Pflugers Arch, 2001 Jan, 441(4), 529 - 37
Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A; Abrahamse SL et al.; The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux . Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion . Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells . The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux . Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport . Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates . However, reducing intracellular ATP contents completely blocked fluo-3 extrusion . Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not . As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells . This organic anion transport system differs from MDRI and MRP2.

Probl Tuberk, 2000, (6), 57 - 61
{Enhancing the impact of chemotherapy of tuberculosis with parenteral administration of dissolved ozone}; Belianin II et al.; Thirty three patients with acute progressive chronic pulmonary tuberculosis who were admitted to be intolerable since they had gross damage and gained no benefits from chemotherapy due to the multidrug resistance of Mycobacteria tuberculosis were given an additional course of intravenous dissolved ozone administration (PO3) . Before PO3 administration, more than half (57.4%) of the patients received only 1 or 2 antituberculous drugs (ethambutole and ethionamide or ethambutole and oprofloxacin) . PO3 produced a pronounced disintoxifying effect, resulting in cessation of bacterial isolation . The stabilization of a tuberculous process was verified by clinical, X-ray, and laboratory studies in 75.8% of patients . This made it possible to perform bulky operations on the lung . When there was a postoperative progression of the underlying process in the single lung, chemotherapy was supplemented by intravenous PO3, which also promoted the stabilization of the process . The use of PO3 expands the spectrum of used agents and enhances the impact of chemotherapy in the treatment of patients isolating multidrug-resistant M . tuberculosis in both pre- and postoperative periods, this increases the rate of operability in the most serious patients.

Probl Tuberk, 2000, (6), 51 - 4
{Outcomes of surgical interventions in patients with infiltrative pulmonary tuberculosis}; Stepanov SA; Thirty eight patients underwent surgery . The most significant causes of ineffective therapeutical measures in patients were their treatment incompetences (100%), multidrug resistance of Mycobacteria tuberculosis (47.3%), lung tissue destruction (68.4%), concomitant alcoholism (47.3%), relapses of specific lung damage (23.7%) . If medical treatment is ineffective in patients with infiltrative pulmonary tuberculosis for 6 months, it is expedient to make a surgical intervention that promotes recovery (96%) and restore working capacity (84%) in them.

Bioorg Med Chem Lett, 2001 Jan 22, 11(2), 275 - 7
Synthesis and structure--activity analysis of novel dihydropyridine derivatives to overcome multidrug resistance; Tasaka S et al.; The structure activity relationships were studied on newly synthesized 1,4-dihydropyridine derivatives possessing a 1-pentyl group at the 4-position, and 3-pyridylpropylester was found to be one of the effective fragments for overcoming P-glycoprotein mediated multidrug-resistance (MDR) in cultured human cancer cells, in vitro . 3-Pyridylpropylester was also found to be one of the effective fragments for increasing the life span of P-glycoprotein overexpressing MDR P388 leukemia-bearing mice, in vivo . All compounds had weak calcium antagonistic activities, but there appeared no relationship between MDR reversing effect and calcium antagonistic activity.

J Neurooncol, 2000 Sep, 49(2), 105 - 15
Expression of multidrug resistance-associated protein (MRP) in human gliomas; Mohri M et al.; Drug resistance is a major clinical problem in the chemotherapy of human gliomas . The multidrug resistance-associated protein (MRP), a membrane transporter related to non-P-glycoprotein multidrug resistance, is overexpressed in some drug-selected cancer cell lines . To investigate whether MRP is involved in the intrinsic drug resistance of human gliomas, surgical specimens of 20 gliomas (11 glioblastomas, 6 anaplastic astrocytomas, and 3 astrocytomas), 3 normal brain specimens, and 4 glioma cell lines (U87MG, U251MG, U373MG, and T98G) were analyzed . The expression of MRP was studied by RT-PCR and immunohistochemistry in the surgical specimens . The MRP expression levels in the cell lines were assessed by the quantitative RT-PCR and Western blot analyses . Sensitivity to adriamycin (ADM), etoposide (VP-16), cisplatin (CDDP), and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), were determined by MTT assay, and antisense treatment was evaluated in the cell lines . The expression of MRP was detected in 9 of 11 glioblastomas and 3 of 6 anaplastic astrocytomas . The quantitative analyses of the cell lines revealed that the MRP mRNA and protein levels were increased 4.5-fold in the T98G cells as compared to U87MG . T98G cells showed the highest resistance to all drugs . Western blot analysis revealed that treatment with the antisense oligonucleotide reduced the level of MRP expression to 25% of the sense oligonucleotide treatment in T98G cells . The sensitivity to ADM, VP-16 and CDDP was significantly increased in the antisense-treated cells as compared with the sense-treated cells . These results suggest that the MRP expression may be related to the intrinsic multidrug resistance in human gliomas.

Clin Cancer Res, 2001 Jan, 7(1), 145 - 52
Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells; Robey RW et al.; We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family . Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol . Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM . To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol . The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan . Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored . Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found . Northern blot and PCR analysis revealed overexpression of the ABCG2 gene . Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected . These results suggest that ABCG2 plays a role in resistance to flavopiridol.

Anticancer Res, 2000 Nov-Dec, 20(6B), 4627 - 32
PSC 833 induces apoptosis in drug-sensitive human leukemia cell line and modulates resistance to paclitaxel in its multidrug-resistant variant; Duraj J et al.; BACKGROUND: The non-immunosuppressive cyclosporine analog PSC 833 has been shown to reverse multidrug-resistance of neoplastic cells including the MDR-1 gene coded P-glycoprotein (P-gp)-mediated cells resistant to paclitaxel . MATERIALS AND METHODS: Apoptosis was demonstrated in drug-sensitive HL-60 and multidrug-resistant human promyelocytic leukemia HL-60/ADR (MRP) and HL-60/VCR (MDR-1) cells in vitro with the aid of flow cytometry, DNA analysis and western blotting . RESULTS: The techniques used herein determined accumulation of paclitaxel/PSC 833 induced apoptotic cells with sub-G0 (hypodiploid) DNA content and blocked in the G2/M phase of the cell cycle, internucleosomal DNA fragmentation, poly (ADP-ribose) polymerase cleavage, Bcl-2 modulation and Bax up-regulation, without any significant alterations in the levels of Bcl-xL, CD95/Fas or Fas-L proteins . CONCLUSION: Drug resistance modulator PSC 833 abolished the P-gp-mediated multidrug-resistance to paclitaxel and paclitaxel-induced apoptosis in human myeloid leukemia (HL-60/VCR) cells in vitro . Furthermore, PSC 833 alone induced apoptosis in parental drug-sensitive leukemia cells, but not in both multidrug-resistant sublines studied.

Anticancer Res, 2000 Nov-Dec, 20(6B), 4441 - 4
Aspirin enhances multidrug resistance gene 1 expression in human Molt-4 T lymphoma cells; Flescher E et al.; BACKGROUND: We recently found that aspirin induces the expression of P-glycoprotein (P-gp), a protein mediating drug resistance, in human prostate cancer cells . The purpose of this study was to evaluate the effect of aspirin on the expression of P-gp in a different human cancer type, i.e., T lymphoma . Furthermore, we analyzed this effect at the level of the gene encoding P-gp, MDR1, and of the transcription factor (NF-IL6), regulating this gene . MATERIALS AND METHODS: NF-IL6 was assayed by the electrophoretic mobility shift assay, MDR1 mRNA was assayed by the reverse transcriptase polymerase chain reaction (RT-PCR), and P-gp was assayed by Western blotting . RESULTS: aspirin, at plasma attainable levels, induced NF-IL6 DNA-binding activity, and increased MDR1 mRNA expression (by up to 140%), as well as the expression of P-gp, in Molt-4 cells . CONCLUSIONS: This study suggests that treatment with aspirin induces a cellular signal culminating in the enhancement of P-gp expression in T lymphoma Molt-4 cells.

Anticancer Res, 2000 Nov-Dec, 20(6B), 4373 - 7
Modulation of molecular marker expression by induction chemotherapy in locally advanced breast cancer: correlation with the response to therapy and the expression of MDR1 and LRP; Schneider J et al.; PURPOSE: To assess if molecular markers are able to predict the response to induction chemotherapy in locally advanced breast cancer, and if any variation in their expression is associated with the degree of axillary lymph node invasion . METHODS: Between 1995 and 1999, 48 patients with locally advanced breast cancer were submitted to induction chemotherapy at Fundacion Tejerina--Centro de Patologia de la Mama, Madrid, Spain . The patients carried either tumors larger than 5 cm in diameter with clinically positive axillary nodes, T4a or T4b tumors regardless of size, or inflammatory carcinomas . All received between 3 and 6 cycles of CAF standard polychemotherapy (Cyclophosphamide, Doxorubicin and 5-Fluorouracil) with the exception of one patient, who received CMF therapy (Cyclophosphamide, Methotrexate and 5-Fluorouracil), and another one, who received Taxotere-Doxorubicin . After completion of their induction chemotherapy scheme, 1 patient showed a "complete clinical response" (CCR, with disappearance of all clinical and radiological signs of tumor presence), 36 (75.0%) patients showed a "partial response" (PR, > 50%), 10 (20.8%) showed "no response" (NR, < 50%), and finally one progressed under treatment . Core biopsies were performed in all cases prior to treatment for histological diagnosis which allowed for the determination of the following parameters by means of immunohistochemistry: hormone receptors (ER and PR), oncogenes and tumor suppressor genes (c-erb-B2 and p53) and the proliferation marker Ki67 . Initial tumor size, histologic and nuclear grade and histologic variety were also included as variables of the study . After chemotherapy, 37 patients were submitted to a rescue mastectomy at our center . The same aforementioned parameters were determined once again on the operative specimen, together with MDR1 expression (using two different antibodies) and LRP expression . As outcome variables, objective response to treatment and the presence of invaded axillary nodes were considered . RESULTS: Only the expression of the proliferation-associated Ki67 antigen, as well as nuclear grade were affected significantly (p < 0.05) by the previous chemotherapeutic treatment . All other studied parameters showed no significant change in expression . More disappointingly, even, none of the studied variables showed any significant power for predicting either an objective response to treatment, or the presence of invaded axillary nodes at surgery . Both outcome end-points were also unrelated to each other . Overexpression of the multidrug-resistance gene or the LRP gene, finally, showed no correlation whatsoever with the previous response to chemotherapy . CONCLUSION: According to these results, the parameters employed by us are of no practical use for predicting the response to treatment or the presence of invaded nodes at rescue surgery in locally advanced breast cancer . Clinical and surgical assessment remain thus the mainstay of treatment for this group of patients.

Anticancer Res, 2000 Nov-Dec, 20(6B), 4261 - 74
Reversal of multidrug resistance of tumor cells; Szabo D et al.; Drug resistance to chemotherapy is rapidly emerging . Resistance to one drug carries over resistance to unrelated anticancer drugs leading to multidrug resistance (MDR) . A major factor of MDR is P-glycoprotein (P-gp) mediated ABC transport found in many eukaryotic cells . P-gp acts as a drug eMux pump . The mdr1 gene involved in P-gp 170 protein production is localized in the human chromosome 7 band p2 1.0-21.1 . Point mutations after cross-resistance patterns . A variety of stimuli increase the expression of the mdr1 gene: lowered extracellular pH, heat shock, arsenite, cytotoxic agents, anticancer drugs, transfection with oncogenes, HIV-I, and UV-irradiation . An alternative hypothesis to the efflux pump claims that P-gp modifies the intracellular environment to reduce accumulation of anticancer drugs in cancer cells by creating ionic or proton gradients . Chemosensitizers that block P-gp drug extrusion are generally lipid-soluble at physiological pH, possess a basic nitrogen atom and at least two co-planar rings . P-gp blocking does not depend on drug chirality . This opens the way of treating P-gp related MDR with chiral versions of drugs relatively harmless in terms of side-effects . We believe that resistance modifiers combined with cytostatics will chemotherapeutically be more effective for cancer patients.

Anticancer Res, 2000 Nov-Dec, 20(6C), 4809 - 14
Expression of multidrug resistance-related markers in gastric cancer; Fan K et al.; BACKGROUND: To detect the expression of glutathione S-transferase Pi(GST-pi), multidrug resistance-associated protein (MRP), lung-resistance protein(LRP), multidrug resistance gene1 (MDR1) and MGr1 antigen(MGr1-Ag) in the patients with primary gastric cancer and without any prior chemotherapy and to evaluate the correlations between them . PATIENTS AND METHODS: The expression of GST-pi, MRP, LRP and MDR1 in cancer tissue and the adjacent non-cancerous tissue from 50 patients was examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) . The expression of MGr1-Ag in these tissues was also examined by immunohistochemistry . RESULTS: The positive rate of GST-pi mRNA, MRP mRNA, LRP mRNA, MDR1mRNA and MGr1-Ag in gastric cancer tissue was 36.00%, 12.00%, 10.00%, 10.00% and 18.00% respectively . The overall positive rate of their expression was 58.00% . Clinicopathological factors were not significantly related to their expression . No significant correlation was observed between these markers . CONCLUSION: These MDR markers are differently over-expressed and no coexpression exists in gastric cancer . MGr1-Ag was a novel MDR protein.

Vestn Ross Akad Med Nauk, 2000, (12), 21 - 5
{Present-day aspects (diagnosis, clinical course and treatment) of acute progressive pulmonary tuberculosis}; Mishin VIu; A total of 103 patients with acute progressive pulmonary tuberculosis whose age ranged from 18 to 60 years were examined . Caseous pneumonic, infiltrative-caseous, disseminated, and rapidly progressive fibrocavernous tuberculosis was found in 45.6, 17.5, 16.5, and 20.4% of cases, respectively . The clinical picture was characterized by its acute onset with significant intoxication syndrome . Moreover, all the patients had respiratory and immunological failure, varying disseminated intravascular coagulation syndrome, non-specific bronchopulmonary infection; some presented with pulmonary hemorrhage, spontaneous pneumothorax and pleural empyema . The sputum smear test was positive in all the patients . If there was no evidence for drug resistance, patients had a 4-month regimen using isoniazid, rifampicin, pyrazinamide, ethembutol, and kanamycin . In the subsequent 10-12 months, isoniazid, rifampicin, and ethambutol were given . The patients with multidrug resistant tuberculosis were administered protionamide, ofloxacin, amikacin, supplemented by pyrazinamide and ethambutol . The combined chemotherapy could stop bacterial isolation in more than 80% of patients, make the process stable, and prepare them for planned surgical treatment . When complications occurred and the disease was in steady progress, salvage operations were made, which was the only possible way of preventing the progression of disease at times and of saving life in the patient.

Histochem J, 2000 Oct, 32(10), 599 - 606
Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells; Calcabrini A et al.; P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines . A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus . In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated . Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells . Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer . At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells . By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells . Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells . Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.

Glycoconj J, 2000 Mar-Apr, 17(3 -4), 253 - 9
Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences; Lavie Y et al.; The carcinogenic process involves a complex series of genetic and biochemical changes that enables transformed cells to proliferate, migrate to secondary sites and, in some cases, acquire mechanisms that make cancer cells resistant to chemotherapy . This phenomenon in its most common form is known as multidrug resistance (MDR) . It is usually mediated by overexpression of P-glycoprotein (P-gp) or other plasma membrane ATPases that export cytotoxic drugs used in chemotherapy, thereby reducing their efficacy . However, additional adaptive changes are likely to be required in order to confer a full MDR phenotype . Recent studies have shown that acquisition of MDR is accompanied by upregulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably glucosylceramide and caveolin . These changes may be related to the fact that in MDR cells a significant fraction of cellular P-gp is associated with caveolin-rich membrane domains, they may be involved in drug transport and they could have an impact on drug-induced apoptosis and on the phenotypic transformation of MDR cancer cells.

Gan To Kagaku Ryoho, 2001 Jan, 28(1), 55 - 61
{Patient compliance and the quality of life are well maintained in weekly paclitaxel and carboplatin therapy for advanced gynecologic cancers in Japanese women}; Kurihara M et al.; OBJECTIVES: To assess patient compliance and efficacy of a combination chemotherapy consisting of weekly administration of paclitaxel and carboplatin for gynecologic malignancy in Japanese women . METHODS: Fourteen ovarian and three uterine cancer patients received 80 mg/m2 of paclitaxel (paclitaxel) and AUC 1.5 to 2.0 of carboplatin weekly . The toxicity was evaluated and patients' QOL was tested . RESULTS: Neutropenia higher than grade 3 were observed in 29.4% . Four patients received G-CSF support . Grade 1 neurotoxicity was seen in 76.5% of patients . Evaluation of QOL by EORTC-QLQC30 showed significantly better tolerance of a weekly than monthly regimen . Three out of four patients with lung metastasis showed complete disappearance of the lesions . One patient with stage IIIb cervical cancer underwent postchemotherapy-hysterectomy and a complete pathological response was confirmed . The overall response rate was 64.7% including patients previously treated with platinum based multidrug regimens . CONCLUSIONS: A weekly paclitaxel and carboplatin regimen was well tolerated by Japanese women . The regimen was active in over 60% of cases and it also appeared active in multidrug resistant cases.

Anticancer Drug Des, 2000 Aug, 15(4), 303 - 6
Tetramethylpiperidine-substitution increases the antitumor activity of the riminophenazines for an acquired multidrug-resistant cell line; van Rensburg CE et al.; The multidrug resistance (MDR)-neutralizing and cytotoxic properties of five tetramethylpiperidine (TMP)-substituted phenazines were compared with those of their corresponding isopropyl-substituted analogues using a P-glycoprotein (P-gp)-expressing small cell lung cancer cell line (H69/LX4) . All of the TMP-substituted phenazines tested outperformed their isopropyl analogues with respect to both cytotoxic and chemosensitizing properties, indicating the importance of TMP-substitution when designing novel riminophenazines with increased activity against MDR cancer cell lines . Of the TMP-substituted phenazines tested, B4112, chlorinated at position 3 of the phenyl- and anilino-rings, had the most potent anti-cancer activity in vitro, making this agent a potential candidate for evaluation in experimental and clinical oncology.

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