Multidrug resistant

MMWR Recomm Rep, 1993 Nov 12, 42(RR-15), 1 - 28
Tuberculosis control laws--United States, 1993 . Recommendations of the Advisory Council for the Elimination of Tuberculosis (ACET)
Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay.
Department of Internal Medicine, University of Innsbruck, AustriaBACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1) . However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression . PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression) . METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated) . We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump . Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured . Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases . RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors . Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression . Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug . MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26 . MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion . CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs . This enhanced activity does not seem to be associated with more aggressive disease . Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening . IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.

Biochem Pharmacol, 1993 Nov 2, 46(9), 1613 - 9
Rapid stimulation of rhodamine 123 efflux from multidrug-resistant KB cells by progesterone; Jancis EM et al.; Rhodamine 123 is a mitochondrial dye that is retained for prolonged periods by carcinoma cells . While investigating causes of retention of this dye, we found that 10 microM progesterone caused a rapid stimulation of efflux of rhodamine 123 within 15 min from KB V20C cells, which overexpress the multidrug resistance pump . Progesterone did not stimulate efflux from KB cells that do not overexpress the pump, and verapamil blocked rhodamine 123 efflux in the presence or absence of progesterone, indicating that rhodamine 123 is removed from KB V20C cells by the multidrug resistance pump . Progesterone, however, is unlikely to stimulate rhodamine 123 efflux by simply increasing pump activity for two reasons: (1) progesterone inhibited the efflux of daunomycin from KB V20C cells, so it did not stimulate efflux of all drugs, and (2) progesterone inhibited efflux of rhodamine 123 from L1210/VMDRC cells and had little effect on Adr MCF7 cells; both overexpress the multidrug resistance pump . In the experiments with KB V20C cells, progesterone was the most active steroid tested . At 10 microM, progesterone caused a 70-fold stimulation, desoxycorticosterone, testosterone, promegestone and estradiol about 20-fold, and others had little or no effect . Progesterone may act by a non-genomic mechanism to decrease intracellular binding of rhodamine 123, making the dye accessible to the multidrug resistance pump.

Antimicrob Agents Chemother, 1993 Nov, 37(11), 2344 - 7
Therapy of multidrug-resistant tuberculosis: lessons from studies with mice; Klemens SP et al.; The activities of antituberculosis agents were evaluated in a murine tuberculosis model using a drug-resistant isolate . A multidrug-resistant clinical isolate from a recent outbreak of tuberculosis in the New York State correctional system was used for infection . Approximately 10(7) viable Mycobacterium tuberculosis ATCC 49967 (strain CNL) organisms were given intravenously to 4-week-old female outbred mice . Treatment was started 1 day after infection and given for 4 weeks . Spleens and lungs were homogenized, and viable cell counts were determined . Statistical analysis indicated that ethionamide, sparfloxacin, ofloxacin, capreomycin, clarithromycin, and clofazimine are active in the murine test system with this multidrug-resistant tuberculosis isolate . Sparfloxacin is the most active quinolone . Despite in vitro resistance, isoniazid has moderate activity . In vitro susceptibility data coupled with evaluation of agents against drug-resistant isolates in the murine system should provide information necessary to design clinical trials for treatment of infections with these organisms.

Ann Oncol, 1993 Nov, 4(9), 723 - 33
Protracted drug infusions in cancer treatment: an appraisal of 5-fluorouracil, doxorubicin, and platinums; DelaFlor-Weiss E et al.; The feasibility to deliver chemotherapeutic agents by protracted i.v . infusion has greatly increased in the recent past . Indwelling ports, longer lasting central venous catheters requiring less than daily maintenance 'flushing', surgical expertise in placement, use in analgesia and nutrition, and 'smart' pump technology have all contributed to their increasing popularity . Justification for use of infusions in cancer chemotherapy has been slow in appearing with few studies proceeding to the comparative stage . This review will focus on three drugs in common use in cancer treatment, with the purpose of appraising the role of such infusions in cancer therapeutics and of deriving some lessons that might be applicable to other drugs or to drug development in general . For fluorouracil and doxorubicin the rationale and clinical findings favoring further development of infusion regimens is particularly strong . In the case of platinum compounds, some toxicologic advantages have emerged, but other measures designed to protect against the toxicities of cisplatin compete with infusion regimens in this regard . The therapeutic potential for this form of drug delivery, therefore, appears still confined to a subset of patients . Stronger rationales for the use of protracted infusions may be forthcoming from pharmacodynamic findings as in the case of etoposide, combined modality therapy with radiation for FU and cisplatin, biochemical modulation for FU, and reversal of multidrug resistance and its modulation for doxorubicin . While awaiting research into these areas of clinical and pre-clinical investigations, the role of infusion appears most evident in the cardiotoxicity protection of anthracyclines, and in further efficacy exploration (through dose or modulation) of FU . Both mechanistic and pharmacologic considerations could also provide additional stimulus for development of new formulations such as long circulating liposomes, and drugs more suitable for oral administration.

AIDS, 1993 Nov, 7(11), 1453 - 60
A nosocomial outbreak of multidrug-resistant Mycobacterium bovis among HIV-infected patients . A case-control study; Bouvet E et al.; OBJECTIVE: To identify risk factors in a nosocomial outbreak of multidrug-resistant Mycobacterium bovis (MDRMB) tuberculosis (TB) among HIV-infected patients . DESIGN: We evaluated the study period (from the first to the last MDRMB smear-positive patients hospitalized in the unit) using a case-control study with three control groups . Since MDRMB is extremely rare, we assumed that a single strain was responsible for all six cases . SETTING: A 19-bed infectious diseases unit in Paris, France . PATIENTS: The index case was an AIDS patient who was hospitalized in September 1989 because of MDRMB TB . The cases were five HIV-infected patients who developed MDRMB TB between January 1990 and October 1991 . Controls were randomly selected from HIV-infected patients in our unit during the study period (case-control study 1, 15 patients), during the contact period (at least one MDRMB smear-positive patient hospitalized in the unit; case-control study 2,20 patients), and patients matched according to the length of contact (case-control study 3, 24 patients) . INTERVENTIONS: After detecting the nosocomial outbreak, we took respiratory isolation precautions for all patients suspected of having active TB . MAIN OUTCOME MEASURES: Risk factors for MDRMB nosocomial transmission, and the occurrence of new cases of MDRMB infection in HIV-infected patients and health-care workers after the introduction of isolation precautions . RESULTS: The most important predictor of nosocomial transmission of MDRMB to HIV-infected patients was the (mean +/- s.d.) length of contact in days {cases, 22 +/- 15.8; study 1 controls, 11.2 +/- 18.9 (P = 0.07); study 2 controls, 14.6 +/- 8.5 (P = 0.043)} . Only one case of MDRMB TB resulted from exposure to MDRMB-smear-positive patient after the introduction of respiratory isolation measures . The incubation period in the single health-care worker who developed MDRMB TB was longer than in the cases . CONCLUSION: In a nosocomial outbreak of MDRMB TB, the contact time was the main risk factor of transmission to HIV-infected patients . Respiratory isolation measures appear to be effective.

Clin Infect Dis, 1993 Nov, 17 Suppl 2, S442 - 6
Drug-resistant tuberculosis; Riley LW; Multidrug-resistant tuberculosis (MDRTB) is an iatrogenic disease that is emerging as a major infectious disease problem throughout the world . The AIDS pandemic, increased incidence of tuberculosis in populations with easy access to antituberculosis medications, the deterioration of the public health infrastructure, and inadequate training of health care providers in the epidemiology of tuberculosis are some of the factors contributing to the increased incidence of MDRTB . Mortality from MDRTB exceeds 80% in persons infected with human immunodeficiency virus (HIV) but is also high in patients free of HIV . The management of MDRTB is complicated by the lack of methods for rapid detection of resistant strains of Mycobacterium tuberculosis . The risk of infection among close contacts of patients with MDRTB, the probability of the development of active MDRTB after infection, and the likelihood of cure of MDRTB need to be determined quantitatively . Attributable risk estimates for factors associated with MDRTB should be calculated for each community as part of the strategies to prevent MDRTB . Issues regarding chemoprophylaxis in newly infected contacts of MDRTB cases remain unanswered . Basic research on the mechanisms of action of existing antituberculosis drugs may contribute to an understanding of the mechanism of resistance in M . tuberculosis . There is an urgent need to expand the scope of epidemiological, clinical, and laboratory research to address these problems.

Ann Hematol, 1993 Nov, 67(5), 227 - 30
Idarubicin is active on MDR cells: evaluation of DNA synthesis inhibition on P388 cell lines; Petrini M et al.; Multidrug resistance is frequently found in patients affected by hematological malignancies and has been related to a poor prognosis of acute leukemia . In the present paper we report results concerning the activity of idarubicin, an anthracycline derivative, on the leukemic P388 and P388 doxorubicin-resistant cell lines . The results clearly show that idarubicin inhibits DNA synthesis in the resistant cell line more actively than doxorubicin.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10226 - 9
Cationic drug analysis using matrix-assisted laser desorption/ionization mass spectrometry: application to influx kinetics, multidrug resistance, and intracellular chemical change; Rideout D et al.; Highly sensitive and convenient analysis of intracellular cationic drugs has been achieved by applying matrix-assisted laser desorption/ionization mass spectrometry (MALD-MS) . Tetraphenylphosphonium cation was readily identified and quantified (using methyltriphenylphosphonium cation as an internal standard) at subpicomole levels in crude lysate from < 4 x 10(3) FaDu human hypopharyngeal carcinoma cells . A quantitative MALD-MS time course for tetraphenylphosphonium cation accumulation into FaDu cells was comparable to a time course using scintillation counting with tritiated tetraphenylphosphonium . MALD-MS was also capable of demonstrating the reduced accumulation of the cationic drug rhodamine-123 by DoxR MCF7, a multiply drug-resistant human breast adenocarcinoma cell line, relative to the nonresistant parent line MCF7 . In addition, MALD-MS was used to follow a chemical reaction inside intact FaDu cells: the formation of a hydrazone (II-51) from benzaldehyde and an acylhydrazide, 5-{tris(4-dimethylaminophenyl)phosphonio}pentanoyl hydrazide (II-25) . These results suggest that MALD-MS may provide a rapid and practical alternative to existing methods for the analysis of cationic drugs, toxins, and their metabolites in cells and tissues.

Med Clin North Am, 1993 Nov, 77(6), 1391 - 409
The epidemiology of multidrug-resistant tuberculosis in the United States; Kent JH; There has been a significant increase in the number of cases of MDR-TB in the United States . Although cases of MDR-TB have been reported from many areas of the country, the majority of the cases are concentrated in large urban areas . MDR-TB is difficult and expensive to treat . CDC has developed a National Action Plan to Combat Multidrug-Resistant Tuberculosis . The main elements of this plan include (1) greater surveillance and epidemiologic studies of drug-resistant TB; (2) initiatives to make the laboratory diagnosis of MDR-TB more rapid, sensitive, and reliable; (3) education of health care professionals about MDR-TB, its prevention, control, and treatment; and (4) measures to facilitate the development of new antituberculous drugs . CDC has published guidelines for the prevention of nosocomial spread of MDR-TB . to prevent the development and spread of MDR-TB, medical practitioners must suspect TB and make the diagnosis as rapidly as possible . Once a patient is diagnosed with TB, the most important step to prevent the development of drug-resistant disease is to ensure that patients take all of their medication . Directly observed therapy is the best way of ensuring this . In addition, more specific interventions, such as the use of incentives to improve compliance in certain situations, may need to be applied to groups in which high rates of drug resistance have been found, such as HIV-positive persons, IDUs, homeless persons, and persons who have been exposed to persons with MDR-TB . Quick and effective public health interventions targeted at these defined groups should help to control the spread of both drug-susceptible and drug-resistant TB.

Med Clin North Am, 1993 Nov, 77(6), 1335 - 51
Tuberculosis in children; Jacobs RF et al.; The dramatic resurgence and increase in the total number of cases of tuberculous infection and disease in children is alarming in the United States . With poverty, poor access to health care, overcrowding (predominantly in inner-city areas), and an increase in immigration from areas with high endemic rates of TB, the problem in children will continue to increase . If the impact of coinfection with HIV and M . tuberculosis becomes significant in children, as it has in adults in the United States, the increase in the total number of cases of tuberculous disease in children could be staggering . The impact of multidrug-resistant strains of M . tuberculosis and the current crises in availability of effective anti-TB drugs will need a similar resurgence.

Med Clin North Am, 1993 Nov, 77(6), 1277 - 88
The treatment of tuberculosis; Brausch LM et al.; Short-course chemotherapy has made the treatment of TB easier and better than ever, but it works only when patients take the drugs regularly . Compliance is a must for therapy to be successful . Physicians treating patients with tuberculosis should be acutely aware of noncompliance, and every effort to ensure adequate treatment must be put forth . Directly supervised therapy is an excellent option when enough resources are available . Intermittent regimens markedly reduce the manpower required for observed therapy . New agents are being tested for in vitro activity against M . tuberculosis, and clinical studies of those found to be potentially effective are needed to formulate new regimens against the ever-increasing threat of multidrug-resistant TB.

In Vivo, 1993 Nov-Dec, 7(6A), 519 - 23
Modulation of doxorubicin efficacy in P388 leukemia following co-administration of verapamil in mini-osmotic pumps; Slate DL et al.; Co-administration of doxorubicin and verapamil in Alzet mini-osmotic pumps increased the survival of B6D2F1 mice bearing the multidrug-resistant P388/ADR leukemia . A range of doxorubicin and verapamil combinations was studied to define dose-dependent efficacy and toxicity . High doses of doxorubicin (10 mg/kg/day) and verapamil (150 mg/kg/day) could be administered alone without any effect on survival . However, combining high doses of these two agents resulted in host toxicity . Doxorubicin doses of 1 to 10 mg/kg/day in combination with verapamil at 25-100 mg/kg/day were found to improve survival compared with either agent alone . Combination therapy also improved the survival of mice bearing the drug-sensitive P388/0 leukemia when compared to anthracycline treatment alone . The efficacy of the mini-osmotic pump delivery protocol was compared with other regimens delivering the same total cumulative dose of doxorubicin via repeated i.p . injections.

Anal Biochem, 1993 Nov 1, 214(2), 506 - 10
Method for determination of intracellular sodium in perfused cancer cells by 23Na nuclear magnetic resonance spectroscopy; Hansen LL et al.; Changes of intracellular sodium concentrations are often an indication of disease or malfunction . In this work, shift reagent-aided 23Na NMR spectroscopic determination of intracellular sodium was adapted to measurements with perfused cells embedded in agarose gel threads . Ehrlich ascites tumor cells (EHR2) and their multidrug-resistant counterparts (EHR2/DNR+) were immobilized and perfused until the metabolic steady state had been reached as shown by 31P NMR spectroscopy . Subsequent addition of 5 mM dysprosium(III) bis(tripolyphosphate) to the perfusion medium caused a separation of extracellular and intracellular 23Na NMR signals, making quantification of the intracellular sodium possible . The dysprosium shift reagent was apparently nontoxic to the cells, as shown by the unchanged level of ATP and other intracellular phosphates . NMR visibility of the intracellular sodium was determined in suspensions of EHR2 and EHR2/DNR+ cells by treatment with digitonin; the increase of intensity of the extracellular sodium resonance observed after the digitonin treatment corresponded well (97 +/- 3%) to the sum of intracellular and extracellular sodium observed with intact cells prior to the digitonin treatment . The resistant EHR2/DNR+ cells contained a moderately higher intracellular sodium level than the wild-type EHR2 cells, 1.02 +/- 0.10 and 0.77 +/- 0.07 mumol Na/mg protein, respectively . Closely similar levels of intracellular sodium were found by flame photometry . Thus, 23Na NMR offers a reliable method for noninvasive quantification of intracellular sodium in perfused cancer cells.

Cancer Res, 1993 Nov 1, 53(21), 5225 - 32
Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines; Hamaguchi K et al.; We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to > 400-fold) . These cells had strikingly increased (4- to 50-fold) levels of glutathione (GSH) as compared with the drug-sensitive cells of origin (A . K . Godwin et al., Proc . Natl . Acad . Sci . USA, 89: 3070-3074, 1992) . Utilizing this panel of resistant cell lines, we evaluated cross-resistance to classical alkylating agents, natural product drugs, and irradiation . We observed that cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance . Similarly, melphalan cross-resistance continued to increase to > 400-fold and again paralleled the primary cisplatin resistance . Cell lines with low to very high levels of resistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxin derivative etoposide . Cross-resistance is also observed for other natural product drugs, including Adriamycin (approximately 80-fold), mitoxantrone (approximately 440-fold), and taxol (approximately 40-fold) . Cross-resistance to irradiation is, however, modest (< 2-fold) . The cells with the greatest primary resistance to cisplatin most commonly had the highest cross-resistance to the other drugs examined . The cross-resistance to the natural product category drugs was found not to be mediated by the products of either the multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes based on lack of coordinate increased expression or amplification of these genes as assessed by Northern and Southern blot analyses . Furthermore, verapamil failed to markedly increase drug sensitivity . Although there was no indication that these natural product drug efflux pumps were operative, we observed decreased doxorubicin accumulation in these cell lines cross-resistant to natural products . In addition, alternations in DNA topoisomerase II mRNA levels, which have been observed in a variety of human tumor cell lines selected in vitro for resistance to etoposide or teniposide, were not detected . Only intracellular levels of GSH correlated with cross-resistance to these diverse anticancer agents and partial loss of resistance was associated with a marked decrease in glutathione levels . In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least in part, be the direct result of GSH-mediated drug inactivation or may be due to a combination of GSH conjugation to drug and conjugate efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.

Br J Cancer, 1993 Nov, 68(5), 939 - 46
Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells; Versantvoort CH et al.; In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR) . Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein . Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells . In these cells the decreased VP-16 accumulation was also reversed by genistein . Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells . In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines . In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.

Br J Cancer, 1993 Nov, 68(5), 898 - 908
Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells; Schuurhuis GJ et al.; Doxorubicin accumulation defects in multidrug resistant tumour cells are generally small in comparison to the resistance factors . Therefore additional mechanisms must be operative . In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell . This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios) . N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (> 70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance . The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations . N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50 . At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found . This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation . A similar relationship was found for P-glycoprotein-negative MDR cells with low levels of resistance . Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets . Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells . It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.

J Urol, 1993 Nov, 150(5 Pt 1), 1544 - 7
Role of the MDR-1-encoded multiple drug resistance phenotype in prostate cancer cell lines; Theyer G et al.; The treatment of advanced metastatic prostate cancer by hormone manipulation or orchiectomy is frequently followed by the appearance of hormone-insensitive and highly chemoresistant tumor cells . In this study we have investigated the contribution of the P-glycoprotein-mediated drug efflux (multidrug-resistance; MDR) to the cellular resistance of prostate carcinoma-derived cell lines to diverse cytotoxic drugs by detection of P-glycoprotein (P-gp) measurement of P-gp-mediated drug transport and reversal of MDR by chemosensitizers . The in vitro chemosensitivity of three prostate cancer cell lines (PC-3, DU-145 and LNCaP) to doxorubicin was measured in a thymidine incorporation proliferation assay . Growth of the partially hormone-sensitive cell line LNCaP is inhibited by low doses of doxorubicin (IC50:27 ng./ml.), but PC-3 and DU-145 are highly resistant to the drug, with IC50 values of 10 micrograms./ml . and 7.5 micrograms./ml., respectively . The chemosensitivity of the PC-3 and DU-145 cells is increased in response to 1 microM . verapamil, 1 micrograms./ml . cyclosporine A and 2 microM . tamoxifen, which are known to partially reverse the MDR phenotype in other resistant tumors . A verapamil-sensitive drug efflux has been demonstrated for the PC-3 and Du-145, but not for the LNCaP, cell lines, using flow cytometric measurements of the P-gp substrate rhodamine 123 efflux from preloaded cells . In agreement with the functional measurements, the expression of the P-glycoprotein was detected in the PC-3 and Du-145 cell lines in Western blots using the monoclonal C 219 antibody . In conclusion, the chemoresistant and hormone-insensitive PC-3 and Du-145 cell lines express P-gp and exhibit verapamil-sensitive drug efflux, indicative of MDR . However, the low MDR-reversal rates observed in these cell lines in response to chemosensitizers in clinically achievable concentrations (approximately 2- to 3-fold reversal), point to non-MDR-associated cellular mechanisms as dominant factors of chemoresistance in prostate cancer.

Pharmacol Ther, 1993 Nov, 60(2), 215 - 34
Design and tumor targeting of anthracyclines able to overcome multidrug resistance: a double-advantage approach; Priebe W et al.; A novel, 'double-advantage approach' to developing more effective chemotherapies will be reviewed . This approach is based on a presumption that analogs designed on a sound hypothesis, and combined with a rationally selected drug delivery system, will optimize antitumor activity by creating drugs that are more active and that can be more specifically targeted to tumors . In the design of drugs superior to doxorubicin, we have focused on typical multidrug resistance and new anthracycline analogs, whose uptake is not affected by P-glycoprotein . Analysis of structural elements of anthracyclines affecting activity against multidrug resistant tumors and affinity for liposomes will be discussed . Annamycin, a lipophilic anthracycline analog, was selected for further preclinical development as a liposomal formulation and demonstrated, in the initial biological evaluation, high activity against tumors resistant to doxorubicin.

Pharmacol Ther, 1993 Nov, 60(2), 289 - 99
Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals; Borst P et al.; In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam . We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes . The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism . Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile . This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located . These and other results show the importance of the genetic approach for studying drug metabolism . MDR is not only caused by increased activity of Pgps . When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity . Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur . This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms . The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.

Curr Opin Oncol, 1993 Nov, 5(6), 1029 - 34
Doxorubicin and multidrug resistance; Kruh GD et al.; The circumvention of P-glycoprotein function by pharmacologic agents is a major focus of clinical trials aimed at increasing the cytotoxicity of multidrug resistance-associated drugs, including doxorubicin . The success of this approach will likely depend on the clinical significance of P-glycoprotein expression, which has not yet been elucidated for the common solid tumors, and the ability to achieve effective levels of circumventing agents without dose-limiting toxicities . Initial clinical studies suggested that biologically relevant concentrations of multidrug resistance modulators, eg, cyclosporine, can be achieved . Because pharmacologic agents that inhibit drug efflux by P-glycoprotein are themselves pumped out of cells by the transporter, this approach may have an inherent barrier to success . Laboratory studies have suggested alternative strategies for circumventing P-glycoprotein action, eg, the use of monoclonal antibodies directed against P-glycoprotein and liposome-encapsulated drugs.

Anticancer Res, 1993 Nov-Dec, 13(6A), 2059 - 63
The multidrug-resistance modifiers verapamil, cyclosporine A and tamoxifen induce an intracellular acidification in colon carcinoma cell lines in vitro; Hamilton G et al.; In this study we have investigated the effects of the multidrug-resistance (MDR) modifiers verapamil (VPM), cyclosporin A (CsA) and tamoxifen (TMX) on the intracellular pH(pHi) of four colon carcinoma-derived cell lines with low P-glycoprotein expression (CaCo-2, HT-29, SW 620 and SW 480) . Addition of VPM (1 mu M), CsA (1 microgram/ml) or TMX (2 microM) in HEPES- or bicarbonate/CO2-buffered Ringer's solution was followed by dose-dependent and reversible decreases of the pHi (0.1-0.3 units) of all cell lines, as measured ratiometrically by the changes in the pH-dependent fluorescence of bis(carboxyethyl)carboxyfluorescein (BCECF) . Testing the effects of the resistance modifiers on the Na+/H+ antiporter and bicarbonate trans-porters under appropriate buffer conditions and addition of inhibitors (amiloride, DIDS) revealed that the chemomodulator-induced acidification does not interfere with the function of these major pHi-regulating acid-base transporters . The induction of changes in pHi shows no correlation with MDR-reversing activity of the drugs and our data do not support the P-gp-inhibition-mediated accumulation of acidic substrates as underlying mechanism . In addition to the P-gp-directed MDR-reversal, chemomodulator-induced intracellular acidification may enhance the chemosensitivity of the cells especially under alkaline extracellular conditions, and contribute to the decreased efficacy of MDR-modifiers in acidic extracellular environments and to the chemosensitising effect of VPM in P-gp-negative cell lines.

Jpn J Cancer Res, 1993 Nov, 84(11), 1201 - 8
Multidrug resistance in rat colon carcinoma cell lines CC531, CC531mdr+ and CC531rev; Gheuens E et al.; A rat colon carcinoma cell line, CC531, was exposed to stepwise increasing concentrations of colchicine . A cell line, CC531mdr+, which grows in the presence of 0.2 microM of colchicine was obtained . A revertant cell line, CC531rev was isolated upon colchicine withdrawal . The CC531mdr+ displayed a multidrug-resistant phenotype . Marked resistance to the selecting agent colchicine, was found (RF = 37.5) as well as to vinblastine (RF = 11.3) and actinomycin D (RF = 2.6) . Cross resistance to doxorubicin (RF = 8) and daunorubicin (RF = 13.3) was demonstrated . Verapamil was able to reverse drug resistance to colchicine and daunorubicin . The revertant cell line, CC531rev, showed increased sensitivity to colchicine (RF = 0.43), vinblastine (RF = 0.13), doxorubicin (RF = 0.28) and daunorubicin (RF = 0.56) . Marked cross resistance to cis-platinum (RF = 6.9) was also induced in CC531mdr+ and was maintained in CC531rev . We conclude that CC531 displays an intrinsic low-level multidrug-resistant phenotype, which was amplified in the CC531mdr+ variant . This correlates with a higher level of expression of P-glycoprotein . CC531rev lacks the multidrug-resistant phenotype and can be used as the drug-sensitive counterpart of the latter two cell lines . Furthermore, it has been shown that in these cell lines cis-platinum resistance is mediated through a mechanism independent of the multidrug-resistant phenotype, since the revertant cell line CC531rev has lost the multidrug-resistant phenotype while retaining the concomitantly induced cis-platinum resistance of the multidrug-resistant variant CC531mdr+.

Leukemia, 1993 Nov, 7(11), 1888 - 90
Relationship between p53 gene mutations and multidrug resistance (mdr1) gene expression in myelodysplastic syndromes; Preudhomme C et al.; P glycoprotein, the product of multidrug resistance (mdr1) gene, is frequently expressed in advanced myelodysplastic syndromes (MDS) with an excess of bone marrow blasts and could explain their frequent resistance to chemotherapy . P53 gene mutations are also found in 10 to 15% of advanced MDS . Because it has recently been suggested that normal p53 suppressed, but that mutated p53 activated, the mdr1 gene promoter, we tried to correlate p53 mutations and P glycoprotein expression in 34 patients with MDS and an excess of bone marrow blasts (> 5%) . P glycoprotein expression was assessed by immunocytochemistry using JSB1 monoclonal antibody and was found positive in 13 out of the 34 patients . p53 mutations were detected both by immunocytochemistry using three different monoclonal antibodies and by single stranded conformation polymorphism (SSCP) analysis of exons 5 to 8 of the P53 gene . Both methods detected a point mutation in 5 out of the 34 patients . Only one out of the 5 patients with a p53 mutation expressed P glycoprotein, as compared to 12 out of the 29 patients without p53 mutations . This suggested the mutant and normal p53 are not major determinants of the regulation of mdr1 expression in vivo, at least in MDS.

Leuk Res, 1993 Nov, 17(11), 941 - 7
Common expression of the multidrug resistance marker P-glycoprotein in B-cell chronic lymphocytic leukaemia and correlation with in vitro drug resistance; Sparrow RL et al.; The expression of P-glycoprotein (Pgp), which is associated with multidrug resistance (MDR), was investigated in 20 B-cell chronic lymphocytic leukaemia (B-CLL) patients by flow cytometry using two Pgp-specific monoclonal antibodies (mAb), MRK-16 which recognizes an extracellular epitope, and JSB-1 which recognizes an intracellular epitope . Sixteen (80%) patients were positive with MRK-16 whereas all patients were positive with JSB-1 . The proportion of Pgp-positive lymphocytes from each patient sample varied from 2-94% for MRK-16 and 20-93% for JSB-1 . There was no correlation between the level of positivity and disease stage or treatment history . In vitro drug resistance to vincristine (VCR) and doxorubicin (DOX) was determined by the colorimetric MTT assay . All patients were resistant to one or both drugs being consistent with the expression of Pgp . There was no correlation between the level of resistance and disease stage or drug treatment . We investigated the expression of Pgp in the normal counterpart of the B-CLL cells, CD5+CD19+ B-lymphocytes . A minor subpopulation (3%) of CD5+CD19+ lymphocytes isolated from normal controls expressed Pgp suggesting that these cells may be the potential precursors to the B-CLL cell . We conclude that Pgp expression and drug resistance are inherent characteristics of the B-CLL lymphocyte.

J Cell Physiol, 1993 Nov, 157(2), 392 - 402
Differential expression of P-glycoprotein genes in primary rat hepatocyte culture; Lee CH et al.; The multidrug resistance (MDR)-associated protein, P-glycoprotein (Pgp), is expressed on the bile canalicular surface of hepatocytes, where it is thought to function in the detoxification of xenobiotics or in the transport of specific metabolites . Several studies have shown that Pgp expression in rat liver can be perturbed in vivo and in vitro; however, it is not known which of the 3 Pgp genes (class I, II, or III) are involved . In rodents, the class I and II Pgp genes have been shown to mediate MDR while the class III gene apparently does not . In this report, we have used gene-specific probes generated from the 3'-untranslated regions of the three rat Pgp genes (Deuchars et al.: Biochim . Biophys . Acta, 1130:157-165, 1992) to investigate Pgp gene expression in primary rat hepatocytes . We observed that the class II Pgp mRNA, the least abundant in the intact liver, is dramatically increased in culture over a 48 h period, while the class I Pgp showed only a modest increase in mRNA level . In contrast, the class III Pgp mRNA, which is the most abundant in the intact liver, exhibited a gradual decline . In rat liver hepatocytes, different culture conditions, as well as drugs such as cytochalasin D and colchicine, appear to affect the level of the class II Pgp gene expression . Moreover, under all these conditions, there is a strong correlation between the level of the class II Pgp and cytoskeletal (actin and tubulin) mRNAs . Thus, there may be a common mechanism regulating the expression of cytoskeletal protein genes and the class II Pgp gene . These findings have implications for our understanding of the regulation of Pgp gene expression in normal and malignant tissues.

Hepatology, 1993 Nov, 18(5), 1202 - 7
Expression of multidrug resistance genes in rat liver during regeneration and after carbon tetrachloride intoxication; Nakatsukasa H et al.; We analyzed expression of multidrug resistance (mdr) genes in rat liver during regeneration after partial hepatectomy or carbon tetrachloride-induced necrosis . In situ hybridization revealed that in the normal liver the cellular distribution of mdr transcripts and protein is restricted to hepatocytes and that a gradient, highest in zone 1 and lowest in zone 3, exists in the level of the mdr transcripts in the liver acinus . Increased levels of mdr1a and mdr1b transcripts were observed 3 hr after administration of carbon tetrachloride and remained increased for the next 5 days . In contrast, increased expression of mdr1a and mdr1b was first observed 24 hr after partial hepatectomy . Use of gene-specific probes to compare the time courses of mdr1b and mdr2 expression after carbon tetrachloride administration showed distinctly different patterns of expression; mdr1b reached a maximum level of expression at 12 hr, whereas increased mdr2 expression was first observed 48 hr after administration . Nuclear run-on analysis at 12 and 24 hr after carbon tetrachloride administration demonstrated 10-fold and eightfold increases in mdr transcription, respectively . However, 72 hr after carbon tetrachloride treatment the rate of mdr transcription was back to the control level . The cellular patterns of mdr expression after partial hepatectomy and carbon tetrachloride administration were similar; the increase was first observed in zone 1 and gradually extended into zone 3 . These data strongly suggest that the physiological roles of mdr1b and mdr2 are different and that liver regeneration is an appropriate model for elucidating these differences.

Blood, 1993 Nov 1, 82(9), 2829 - 36
p53 and N-ras mutations in two new leukemia cell lines established from a patient with multilineage CD7-positive acute leukemia; Abo J et al.; Two new myeloid cell lines (K051 and K052) were established from a patient with multilineage CD7-positive acute leukemia . The K051 and K052 were established from the patient's bone marrow cells at diagnosis and at relapse, respectively . The K051 cell expressed myeloid-associated antigens (CD13 and CD33), a platelet-associated antigen (CD41), and an erythroid antigen (glycophorin A) . The K052 cell expressed myeloid-associated antigens (CD13, CD14, and CD33), lymphoid markers (CD2, CD5, and CD7), and HLA-DR . Chromosome analysis of both cell lines showed a 17p- chromosome . Both cell lines were investigated for aberrations of the p53 gene and the N-ras gene . A p53 mutation detected in both cell lines consisted of a C-->T substitution in codon 248 . An N-ras mutation detected only in the K052 cell consisted of a G-->C substitution in codon 13 . Expression of the multidrug resistance gene (MDR1) was also investigated by the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) . MDR1-mRNA was more highly expressed by the K052 cell than the K051 cell, being equivalent to that in HEL cells . The functional MDR1-protein against vincristine was also observed, and its function was inhibited by verapamile and Cyclosporin A . The K052 cells were capable of phenotypic or morphologic differentiation after being incubated with granulocyte colony-stimulating factor, interleukin-2, phorbol 12-myristate 13-acetate, or 1,25-dihydroxy-vitamin D3 . In contrast, the K051 cells responded phenotypically to retinoic acid . Thus, the K051 and K052 cell lines will be useful for investigating the cellular and molecular events in leukemogenesis and differentiation, and the mechanism of expression of the MDR1 gene.

Biochim Biophys Acta, 1993 Oct 29, 1154(2), 173 - 81
Inhibition of the multidrug resistance efflux pump; Wigler PW et al.; An ATP-dependent efflux pump is found in the plasma membrane of certain multidrug resistant (MDR) cancer cells . Drug resistance is due to decreased intracellular drug levels that have been reduced to subcytotoxic concentrations . Inhibition of the MDR efflux pump with a reversal agent may 'trap' the cytotoxic drug inside the cell; thus, cellular drug resistance is reversed . Although many different lipophilic substances exhibit reversal activity, inhibition of the pump is stereospecific with respect to the chiral agent cinchonine . In this article, several methods for the estimation of reversal potency are reviewed . Furthermore, information on the transport characteristics of reversal agents is presented . The rate equations for ATP-dependent drug efflux, competitive inhibition of the MDR pump, and noncompetitive inhibition of the pump are derived . A method is presented that discriminates between competitive or noncompetitive inhibition of the pump . These studies show the potential contribution of fundamental inhibition studies to the design of clinical reversal protocols.

Int J Cancer, 1993 Oct 21, 55(4), 667 - 71
Role of cell cholesterol in modulating vincristine uptake and resistance; Pallares-Trujillo J et al.; The relationship between cell-membrane permeability to vincristine and cholesterol/phospholipid levels was studied in L5178Y murine leukemic lymphoblasts and in 2 multidrug-resistant cell sublines, VCR/P60 and VCR/P200, which expressed increasing levels of vincristine resistance . The uptake of 3H-vincristine was measured in all cell lines and in cholesterol-depleted and -reloaded L5178Y and VCR/P200 cells . The initial rate of drug entry in resistant cells was lower than that measured in the parental cell line and it decreased as the relative resistance increased . An increment of cholesterol content, characterized in resistant cells, was directly proportional to the relative resistance to vincristine . Cholesterol depletion in both sensitive and resistant cells resulted in an increase in the rate of vincristine uptake, which reverted to the respective basal levels when each cell line was cholesterol-reloaded . The rate of drug uptake was inversely correlated with the molar ratio of cholesterol to phospholipids . Although both VCR/P cell sublines, but not the sensitive parental cells, expressed the P-glycoprotein in their plasma membrane, there were no differences in drug efflux and retention between resistant and parental cells . These results indicate that cholesterol modulates the permeation of vincristine through the plasma membrane and strongly suggest that increased levels of cholesterol/phospholipid account for the lower drug accumulation and greater resistance in these multidrug-resistant cells.

Int J Cancer, 1993 Oct 21, 55(4), 636 - 9
Flunarizine as a modulator of doxorubicin resistance in human colon-adenocarcinoma cells; Silvestrini R et al.; The potential of the calcium-entry blocker flunarizine in modulating the cytotoxicity of doxorubicin was investigated in human colon-adenocarcinoma cell lines sensitive to (LoVo) or with experimentally induced resistance (LoVo/DX) to doxorubicin . Exposure to 1 to 2 micrograms/ml flunarizine for intervals of up to 24 hr did not affect cell survival in either line . Simultaneous exposure to flunarizine and doxorubicin for 1 hr selectively enhanced doxorubicin activity in the resistant cell line and not in the sensitive cell line . In particular, the doxorubicin concentration able to reduce cell survival by 50% dropped to one third . Moreover, simultaneous exposure to flunarizine significantly increased intracellular doxorubicin accumulation, as evaluated by fluorescence spectrophotometry . Again, flow-cytometric analysis showed hyperpolarization of the membrane in resistant cells, starting from 15 min of exposure to 2 micrograms/ml flunarizine . Finally, in LoVo/DX cells, which normally express gp170, a 24-hr treatment with flunarizine markedly reduced the immunoreactivity of cells with 2 monoclonal antibodies (MAb57 and MRK16) directed against different external epitopes of the glycoprotein . The results from our study indicate the ability of flunarizine to positively modulate doxorubicin-resistance in human colon-adenocarcinoma cells expressing the multidrug-resistance phenotype.

J Natl Cancer Inst, 1993 Oct 20, 85(20), 1685 - 90
Measurement of cremophor EL following taxol: plasma levels sufficient to reverse drug exclusion mediated by the multidrug-resistant phenotype; Webster L et al.; BACKGROUND: Paclitaxel (Taxol) is the first of a new class of cytotoxic agents with activity against tumors resistant to other drugs . For clinical use, paclitaxel is currently formulated in a vehicle of 50% ethanol and 50% polyethoxylated surfactant Cremophor EL (Cremophor) . We have previously shown that Cremophor will block the P-glycoprotein drug efflux pump responsible for the multidrug-resistant phenotype . Overexpression of P-glycoprotein is one mechanism of in vitro resistance to a number of currently used cytotoxic agents including paclitaxel . PURPOSE: Our aim was to develop a bioassay to measure plasma levels of Cremophor and to determine whether or not plasma levels of Cremophor achieved during paclitaxel therapy are sufficient to inhibit the activity of the P-glycoprotein . METHODS: All patients studied had histologically proven, advanced ovarian carcinoma with measurable or evaluable disease and had received at least one prior platinum-containing regimen . The bioassay used flow cytometry to measure the increase in equilibrium intracellular daunorubicin levels in multidrug-resistant human T-cell leukemia cells (CEM/VLB100) in the presence of a series of concentrations of Cremophor . Levels of Cremophor were measured in plasma from 21 patients after a 3-hour infusion of 135 or 175 mg/m2 paclitaxel . Both dose levels were given following premedication with oral dexamethasone, intravenous promethazine hydrochloride, and intravenous cimetidine . The Cremophor bioassay involved incubation of CEM/VLB100 cells (5 x 10(5)) for 1 hour with 2 micrograms/mL daunorubicin in 0.5 mL HL-1 medium plus 0.5 mL plasma prior to flow cytometric analysis . Pretreatment plasma was used to derive a standard curve for the effect of Cremophor on equilibrium daunorubicin levels . All measurements were done in triplicate . RESULTS: In vitro experiments indicated that, for maximal inhibition of P-glycoprotein activity, concentrations of Cremophor of 0.1% (vol/vol) were required . At the end of a 3-hour infusion of paclitaxel, plasma levels of Cremophor in 19 of 21 patients were 0.1% or higher and 0.09% in the remaining two . Concentrations of 5-20 microM paclitaxel dissolved in ethanol without Cremophor did not inhibit P-glycoprotein in this assay . CONCLUSION: The concentrations of Cremophor measured in plasma drawn from patients after a 3-hour infusion of paclitaxel at 135 or 175 mg/m2 were found to be sufficient to inhibit P-glycoprotein activity in vitro . IMPLICATIONS: The efficacy of paclitaxel against some tumors may be aided by its administration in a vehicle solution containing Cremophor in quantities that reach concentrations in the plasma sufficient to reverse multidrug resistance of neoplastic cells.

Biochem Pharmacol, 1993 Oct 19, 46(8), 1421 - 4
Chloroquine resistance in Plasmodium falciparum is not reversed by BIBW-22, a compound reversing the multidrug resistance phenotype in mammalian cancer cells; Dieckmann-Schuppert A et al.; The pteridine derivative BIBW-22 (4-{N-(2-hydroxy-2-methyl-propyl)-ethanolamino}-2,7-bis(cis-2,6-di methyl-morpholino)-6-phenylpteridine), which had been developed for the treatment of multidrug-resistant cancer and binds to P-glycoprotein, was tested against chloroquine resistant Plasmodium falciparum strains in culture . Based on the result that BIBW-22 enhanced rather than lowered chloroquine resistance in vitro, it is concluded that chloroquine resistance in malaria parasites may not be mechanistically linked to the multidrug-resistant phenotype of chloroquine resistant P . falciparum.

Biochemistry, 1993 Oct 19, 32(41), 11042 - 56
Lower electrical membrane potential and altered pHi homeostasis in multidrug-resistant (MDR) cells: further characterization of a series of MDR cell lines expressing different levels of P-glycoprotein; Roepe PD et al.; Recently {Roepe, P.D . (1992) Biochemistry 31, 12555-12564}, increased steady-state levels of chemotherapeutic drug efflux from multidrug-resistant (MDR) myeloma cells were correlated with intracellular alkalinization . To better understand elevated pHi in MDR cells, Na(+)- and Cl-dependent recovery of pHi upon intracellular acid or alkaline shock has been examined for this same series of MDR cell lines . In agreement with another recent report {Boscoboinik, D., Gupta, R.S., & Epand, R.M . (1990) Br . J . Cancer 61, 568-572}, we find that the rate of Na(+)-induced alkalinization after an intracellular acid shock is increased in the MDR cells, relative to the drug-sensitive parent . Interestingly, we also now find that mRNA encoding the human Na+/H+ exchanger (NHE) is overexpressed in these MDR cells, but the level of overexpression does not correlate with the relative drug resistance or steady-state pHi . It is also found that the efficiency of Cl(-)dependent reacidification of pHi, after an intracellular alkaline shock is reduced in the MDR cells . This effect appears to correlate with the relative expression of MDR protein, but not the relative expression of Cl-/HCO3- exchanger (AE), which we now find is also altered in the series of cells . Since elevated pHi will increase delta pH across the plasma membrane, we have also measured the electrical potential for these cells using three different methods . Most interestingly, the magnitude of the plasma membrane electrical potential (delta psi) decreases concomitant with increased expression of the MDR protein . Energy provided by increased delta pH compensates for the lowered delta psi, such that the total electrochemical membrane potential (delta mu H+) remains similar among the cells in this series (delta mu H+ = delta psi - Z delta pH) . These data, along with other recent experiments that associated an increased Cl- conductance with the expression of MDR protein {Valverde, M., Diaz, M., Sepulveda, F.V., Gill, D.R., Hyde, S.C., & Higgins, C.F . (1992) Nature 355, 830-833}, are consistent with a model for MDR protein-mediated multidrug resistance that does not entail direct active transport of lipophilic drugs by the MDR protein.

Biochem Pharmacol, 1993 Oct 19, 46(8), 1317 - 26
The efflux of anthracyclines in multidrug-resistant cell lines; Coley HM et al.; In order to address the association of enhanced drug efflux with the multidrug-resistant (MDR) phenotype, we have studied the cellular pharmacokinetics of anthracyclines in the P-glycoprotein (Pgp)-positive MDR cell lines H69/LX4 (human small cell lung cancer) and EMT6/AR1.0 (mouse mammary tumour) . Both doxorubicin (DOX) and daunorubicin (DNR) were accumulated to a lesser extent and effluxed at a higher rate by MDR cells than by their drug-sensitive counterparts . In contrast, the 9-alkyl substituted compound, aclacinomycin A (ACL), was accumulated and effluxed from parent and MDR cells at an identical rate . In experiments designed to examine energy-dependent efflux, DOX and DNR were shown to be efficiently effluxed against the concentration gradient in the presence of glucose . However, in the same experiments the analogues ACL and Ro 31-3294 (9-alkyl and morpholinyl substituted), which have previously been shown to retain activity against MDR cell lines, were accumulated and effluxed at identical rates in parent and MDR EMT6 cells . Hence, 9-alkyl and morpholinyl substituted compounds appear to behave less favourably as substrates for energy-driven drug efflux by Pgp-positive MDR cells than do DOX or DNR . Resistance modifiers verapamil and cyclosporin A appeared to abolish energy-dependent efflux for DOX and DNR in both the EMT6 and H69 MDR lines whereas they had no effect on the cellular efflux of ACL . The altered cellular pharmacology in MDR cell lines may provide a rational basis for the use of modified anthracycline analogues (e.g . 9-alkyl and morpholinyl (substituted) and resistance of modifying agent in the treatment of tumours expressing a Pgp-mediated phenotype.

Cancer Lett, 1993 Oct 15, 74(1-2), 37 - 41
Inhibition of MDR1 gene expression by H-87, a selective inhibitor of cAMP-dependent protein kinase; Kim SH et al.; N-{2-(p-bromocinnamylmethylamino) ethyl}-5-isoquinolinesulfonamide (H-87), a newly synthesized inhibitor of protein kinase A, significantly decreased the drug resistance of multidrug resistant human U937/M cells, mouse FM3A/M and P388/M cells . Northern blot analysis showed MDR1 gene expression was decreased in H-87-treated P388 M cells . H-87 inhibited the activity of MDR1 (multidrug resistance-1) promoter in a dose-dependent manner in transient expression assay . In contrast, the expression of c-raf-1 gene significantly enhanced the activity of MDR1 promoter . We therefore examined the effect of H-87 on MDR1 gene expression in c-raf-1 transfected CV-1 cells (CV-1/raf) . A significant decrease in the level of MDR1 mRNA was observed after H-87 treatment of the CV-1/raf cells . These results suggest that inhibition of MDR1 gene expression by H-87 is associated with circumvention of drug resistance in MDR cells.

Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9735 - 8
Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance; Altenberg GA et al.; P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) is thought to result from active extrusion of lipid-soluble, titratable chemotherapeutic agents . Given the lack of demonstration of coupling between ATP hydrolysis and drug transport, the resistance to chemically unrelated compounds, and findings of elevated intracellular pH (pHi), it has been proposed that reduced intracellular accumulation of drugs in MDR is due to changes in the pH difference across the plasma membrane . Elevation of pHi or decrease in local extracellular pH (pHo) could reduce the intracellular accumulation of the protonated chemotherapeutic drugs and account for Pgp-mediated MDR . Alternatively, changes in pHi or pHo could increase drug efflux by other mechanisms, such as coupled transport involving H+ or OH-, or allosteric effects on Pgp or other proteins . Both mechanisms could operate independently of the charge of the substrate . The possibility of a role of pHi in drug efflux is important to test because of the clinical significance of the phenomenon of MDR of tumors . We tested this hypothesis and found that MDR can occur in cells with low, normal, or high pHi . Further, resistant cells exhibited reduced steady-state drug accumulation and increased efflux without changes in local pHo . Finally, acute changes in pHi had no appreciable effect on Pgp-mediated drug efflux . We conclude that Pgp-mediated MDR is not a consequence of changes in pHi or pHo.

J Biol Chem, 1993 Oct 15, 268(29), 21493 - 6
Fluorescent cellular indicators are extruded by the multidrug resistance protein; Homolya L et al.; In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators . This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g . by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody . The hydrophilic free acid forms of the indicators are not exported by MDR1 . We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect . Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds . These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.

Schweiz Rundsch Med Prax, 1993 Oct 5, 82(40), 1108 - 11
{Disseminated tuberculosis with a multiresistant strain of Mycobacterium tuberculosis in an HIV-infected Swiss male}; Gunthard H et al.; Multidrug-resistant M . tuberculosis in HIV-infected people has not yet been reported in Switzerland, and there have been no nosocomial epidemics as they have recently occurred in the USA . We present the case of a 38-ear-old HIV-infected man who developed disseminated tuberculosis as AIDS-defining disease . Initially he was treated with isoniazid, pyrazinamide and rifampin . Due to the emergence of resistance to isoniazid and streptomycin, ethambutol was added for one month . Later the therapy was changed back to the initial three drugs . The patient responded well to this therapy, but five months later developed a relapse . In addition to the originally diagnosed double-drug resistance, a reduced susceptibility to rifampin appeared . Ethambutol, ciprofloxacin and amikacin were added to the original three-drug regimen . This resulted in rapid clinical improvement, although sputum cultures remained positive for M . tuberculosis two months later . This isolate was resistant to pyrazinamide . For that reason pyrazinamide was replaced by clofazimine . 14 months after diagnosis the patient died of hepatic failure . Because there was a delay in isolation of one week, 37 potentially exposed health care workers were tested by the Mantoux skin test . No conversions were observed . This case report demonstrates that tuberculosis in HIV-infected patients in Switzerland may be caused by multidrug-resistant M . tuberculosis . We propose that until the results of a susceptibility assay are known, a four-drug combination should be used initially in this patient group.

Schweiz Rundsch Med Prax, 1993 Oct 5, 82(40), 1105 - 7
{Multiresistant tuberculosis in New York}; Salfinger M; New Yorkers have to deal with a tuberculosis situation which is of greater extent and a health-care system which is less prepared than in the sixties . Poverty and the HIV epidemic have had a negative impact on the control of tuberculosis . The emergence of nosocomial multidrug-resistant tuberculosis has created an explosive situation . In 1991, rifampin resistance was 33% in patients who had received antituberculosis therapy and 10% among the patients who had never been treated.

Schweiz Rundsch Med Prax, 1993 Oct 5, 82(40), 1096 - 100
{Multiresistant tuberculosis in the Zurich Höhenklinik Wald from 1984 to 1992}; Linnenberg HS et al.; Out of a total of 386 patients with culture-proven tuberculosis, ten cases of multidrug-resistant tuberculosis were treated in a high altitude chest clinic in Switzerland between 1984 and 1992 . Two patients, Swiss, were alcoholics, the other eight foreigners . None of the patients were HIV-positive . Both Swiss had primary INH resistance, then developed secondary INH/RMP resistance due to poor compliance . All patients were treated with at least two additional in-vitro-sensitive drugs as well as with INH . Five patients underwent resection of lung parenchyma . One of these patients has had three operations and remains the sole therapy failure . One patient died before an operation could be performed . All others are well . Possible reasons for multidrug-resistance and the management of the ten cases are discussed in accordance with the literature.

Lancet, 1993 Oct 2, 342(8875), 841 - 4
Molecular approach to identifying route of transmission of tuberculosis in the community; Genewein A et al.; There is growing concern that tuberculosis is spread in Europe in the way that it is in the USA . We have used DNA "fingerprinting" in a systematic evaluation of tuberculosis cases notified in our community to uncover foci of transmission . An IS6110 probe was used to test all isolates from culture-confirmed tuberculosis cases (163 patients) notified in 1991-92 in the Canton of Berne . In total, 45 patients (27.6%), potentially linked on the basis of restriction fragment length polymorphism, were investigated epidemiologically . The largest group (n = 22) included members of a defined social group (drug addicts, homeless persons, alcoholics), from whom tuberculosis spread to the general population . A key patient developed multidrug-resistant tuberculosis during the surveillance period . This population study showed that (i) extensive transmission of Mycobacterium tuberculosis is now taking place in Europe in the same social setting as in the USA; (ii) there is definite "spillover" to the general population; (iii) the dimensions of the problem cannot be recognised easily by routine public health service activities because of the complexity of the transmission network; and (iv) multidrug-resistant tuberculosis develops in this setting.

J Cell Physiol, 1993 Oct, 157(1), 110 - 8
Increased epidermal growth factor receptor in an estrogen-responsive, adriamycin-resistant MCF-7 cell line; Dickstein B et al.; We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer . The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-glycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype . The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis . Estrogen-binding studies demonstrate 1.4 x 10(6) sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 x 10(5) sites per cell . An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression . Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression . We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure.

Cancer Immunol Immunother, 1993 Oct, 37(5), 337 - 42
Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells; Van de Vrie W et al.; The development of resistance to anticancer drugs urges the search for different treatment modalities . Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility . We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS) . In a 4-h 51Cr-release assay we found no difference in susceptibility to NK cell lysis . No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay . In a prolonged 20-h 51Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS) . None of the cell lines was completely resistant to lysis by aLAK cells . Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.

Cancer Res, 1993 Oct 1, 53(19), 4658 - 64
Effect of verapamil on doxorubicin cardiotoxicity: altered muscle gene expression in cultured neonatal rat cardiomyocytes; Akimoto H et al.; Verapamil reverses multidrug resistance acquired by cancer cells during treatment with chemotherapeutic agents such as doxorubicin by inhibiting the function of P-glycoprotein . Verapamil has also been suggested to potentiate the cardiotoxicity of doxorubicin . We have recently demonstrated that selective inhibition of cardiac muscle gene expression is among the earliest events in doxorubicin cardiotoxicity . To explore the influence of verapamil on doxorubicin cardiotoxicity, we evaluated {14C}-doxorubicin accumulation, cardiac muscle gene expression by Northern blot analysis, and ultrastructural changes in cultured cardiomyocytes in the presence and absence of verapamil . Treatment with a combination of doxorubicin and verapamil for 24 h did not augment doxorubicin accumulation in cardiomyocytes, although substantial augmentation of doxorubicin accumulation by verapamil in cardiac fibroblasts was observed . Further, treatment with verapamil for 24 h did not augment the decrease in expression of muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M isoform creatine kinase) . However, we found that verapamil reduced alpha-actin gene expression in a direct, doxorubicin-independent manner . Furthermore, the effect of doxorubicin plus verapamil on alpha-actin gene expression was additive over a wide range of doxorubicin and verapamil concentrations, resulting in a selective augmentation of doxorubicin-induced inhibition of gene expression for this single muscle protein gene . This was reflected in a substantial increase in cardiac myocyte damage when treatment with verapamil and doxorubicin was compared to treatment with doxorubicin alone by thin section electron microscopy . This suggests a possible mechanism by which verapamil may potentiate doxorubicin cardiotoxicity.

Cancer Res, 1993 Oct 1, 53(19), 4595 - 602
In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative; Hyafil F et al.; N-(4-{2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl}- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR) . The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM . The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype . In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of {3H}daunorubicin and blocks the efflux from preloaded cells . It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein . GF120918 effectively competes with {3H}azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action . After i.v . administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10 . It is eliminated from organs and blood with a half-time of approximately 2.7 h . It is well absorbed after p.o . administration . In mice implanted i.p . with the MDR P388/Dox tumor, a single i.v . or p.o . dose of GF120918 restores sensitivity of the tumor to a single i.p . dose (5 mg/kg) of doxorubicin administered 1 h later . A statistically significant effect is observed at 1 mg/kg GF120918 i.v . and maximal effect is reached at 5 mg/kg . Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c . as assessed by tumor size at day 19 . GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.

Br J Cancer, 1993 Oct, 68(4), 691 - 4
MDR1 gene expression in primary colorectal carcinomas; Pirker R et al.; The expression of the MDR1 gene, a multidrug resistance gene, was prospectively determined in 113 primary colorectal carcinoma specimens and correlated with clinical data including survival durations of the patients . MDR1 RNA was detected in 65% of the carcinomas . No expression of the MDR2 gene was seen, MDR1 gene expression was independent of age and sex of the patients, size and histologic grading of the tumour, lymph node involvement and distant metastasis . Kaplan-Meier analysis revealed that the durations of both relapse-free survival and overall survival were not different between patients with MDR1 RNA positive tumours and those with MDR1 RNA negative tumours.

Ann Trop Med Parasitol, 1993 Oct, 87(5), 443 - 9
Resistance of Plasmodium falciparum to antimalarial drugs in Equatorial Guinea; Roche J et al.; One hundred and sixty-six children from Equatorial Guinea, all under 10 years of age and with acute uncomplicated falciparum malaria, were randomly allocated to four groups and treated with one of the following regimens: chloroquine or amodiaquine (25 mg base/kg body weight over 3 days), quinine (8 mg/kg every 8 h for 3 or 5 days), and sulphadoxine-pyrimethamine (25-1.25 mg/kg, in one dose) . The parasite clearance rates up to day 14 were 28% with chloroquine, 74% with amodiaquine, and 95% with quinine or sulphadoxine-pyrimethamine . The times required to clear asexual blood forms of Plasmodium falciparum in sensitive cases were 64, 70, 73 and 65 h, respectively . Although quinine and sulphadoxine-pyrimethamine are equally effective, quinine is recommended for treatment of multidrug-resistant malaria in paediatric patients, essentially because of the risk of serious reactions to sulpha drugs . Health providers are, however, encouraged to keep supplies of sulphadoxine-pyrimethamine as an option and to refer patients quickly, if required.

Cell Biol Int, 1993 Oct, 17(10), 907 - 17
Multidrug resistance and behavioural phenotype of cancer cells; Bashir I et al.; Resistance to cytotoxic chemotherapy is a major obstacle preventing successful treatment of cancer, allowing dissemination of tumour metastases, and may be viewed as the ultimate cause of death in the majority of patients with a malignant disease . Although cytotoxic chemotherapy is classically employed to produce maximal killing of malignant cells, the therapeutic doses of individual drugs required to achieve this objective are, in general, highly toxic to non-neoplastic host tissues . However, there are several different aspects of cancer cell biology, distinct from their susceptibility to cytotoxicity, that might be exploited in order to alter the behavioral phenotypes of malignant neoplasms . Such features include regulation of cell proliferation, tumorigenicity and metastatic potential . Non-cytotoxic modulation of malignant cells may provide an alternative, and more effective, method of controlling the aggressive behaviour of cancer cells while exhibiting less iatrogenic morbidity and mortality than the therapeutic regimens presently employed.

Arzneimittelforschung, 1993 Oct, 43(10), 1118 - 21
Increase of alkaline phosphatase in multidrug-resistant tumor cells and their cross-resistance to 6-thioguanine; Efferth T et al.; The expression of alkaline phosphatase (AP) was analyzed in multidrug-resistant (MDR) tumor cells (sarcoma 180, lung carcinoma, and renal cell carcinoma cell lines) by means of immunocytochemistry . MDR cell cultures showed an overexpression of AP and a cross-resistance to 6-thioguanine (6-TG, CAS 154-42-7) . Significant correlations between AP expression and doxorubicin or vincristine resistance and P-glycoprotein (P-170) expression were found in these cell cultures . A specific AP inhibitor, levamisole, reversed resistance to 6-TG, but not to doxorubicin . This indicates that 6-TG resistance is certainly associated to P-170 but a causal function of AP for the development of MDR does not exist.

Md Med J, 1993 Oct, 42(10), 981 - 7
Tuberculosis: optimism, pessimism, frustration; Matuszak DL et al.; The re-emergence of tuberculosis as a serious public health threat has captured the nation's attention . Tuberculosis more frequently affects the ethnic minorities and the socially and economically disadvantaged residents of Maryland . Effective regimens are available to treat and prevent tuberculosis . Consistent application of proven infection control measures and of treatment and prevention regimens will prevent the development and spread of multidrug-resistant tuberculosis.

Antimicrob Agents Chemother, 1993 Oct, 37(10), 2054 - 8
Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis; Telenti A et al.; A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr) . The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products . Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus . All 17 different Rifr mutations known to date were consistently detected . PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250) . Implementation of this technique could result in rapid detection of rifampin resistance in M . tuberculosis, a marker of multidrug-resistant tuberculosis.

Photochem Photobiol, 1993 Oct, 58(4), 623 - 6
Sites of photodamage by the iminium salt of a copper octaethylbenzochlorin; Kessel D; Because the benzochlorin derivative copper (II) alpha-meso-N,N'-dimethyloctaethylbenzochlorin iminium chloride (CDS1) is not fluorescent, sites of drug localization in L1210 cells were detected by indirect methods involving using a series of fluorescent probes . The CDS1-mediated cytotoxicity was associated with mitochondrial damage, a decreased membrane potential and an increase in the heterogeneity of membrane sites of binding of a polar analog of diphenylhexatriene . Although CDS1 is a cationic compound, its accumulation was not impaired in a cell line exhibiting the multidrug resistance phenotype.

Br J Surg, 1993 Oct, 80(10), 1259 - 61
Multidrug-resistant colonic cancer cell line LoVoDx is efficiently killed by lymphokine-activated killer cells from patients with carcinoma of the colon; Mooney EF et al.; Multidrug-resistant (MDR+) cancer cells have the ability to grow in the presence of cytotoxic concentrations of antineoplastic drugs as a result of possessing the transmembrane drug efflux pump p-glycoprotein . The MDR+ colonic cancer cell line LoVoDx (derived from the drug-sensitive line LoVo) was tested for sensitivity to lymphokine-activated killer (LAK) cell-mediated toxicity . LAK cells were cultured from patients with colonic cancer and from matched controls with benign disorders . LAK cells from patients with cancer were as effective as those from controls in mediating cytotoxicity . The MDR+ cell line was significantly more sensitive to LAK cell-mediated cell killing than its parental drug-sensitive line LoVo (P < 0.05) . These results indicate a possible role for adoptive immunotherapy in MDR+ tumours expressing p-glycoprotein.

Mol Pharmacol, 1993 Oct, 44(4), 767 - 74
Dual topoisomerase I and II inhibition by intoplicine (RP-60475), a new antitumor agent in early clinical trials; Poddevin B et al.; The mechanisms of action of intoplicine (RP-60475), a 7H-benzo{e}pyrido{4,3-b}indole derivative that is presently in early clinical trials, have been investigated . Intoplicine induced both topoisomerase I- and II-mediated DNA strand breaks, using purified topoisomerases . The topoisomerase cleavage site patterns induced by intoplicine were unique, relative to those of camptothecin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and other known topoisomerase inhibitors . Both topoisomerase I- and II-induced DNA breaks decreased at drug concentrations higher than 1 microM, which is consistent with the DNA-intercalating activity of intoplicine . DNA damage was investigated in KB cells in culture by using alkaline elution . Intoplicine induced single-strand breaks (SSB) in a bell-shaped manner with respect to drug concentration (maximum frequency at 1 microM approximately 220 rad-equivalents) . SSB formation was fast, whereas reversal after drug removal was slow . Similar bell-shaped curves were obtained for DNA double-strand breaks (DSB) and DNA-protein cross-links . SSB and DNA-protein cross-link frequencies were approximately equal, and no protein-free breaks were detectable, indicating the protein concealment of the breaks, as expected for topoisomerase inhibition . Comparison of SSB and DSB frequencies indicated that intoplicine produced a significant amount of SSB not related to DSB, which is consistent with concomitant inhibition of both DNA topoisomerases I and II in cells . Data derived from resistant cell lines indicated that multidrug-resistant cells were cross-resistant to intoplicine but that m-AMSA- and camptothecin-resistant cells were sensitive to intoplicine . Hence, intoplicine might circumvent topoisomerase I-mediated and topoisomerase II-mediated resistance by poisoning both enzymes simultaneously.

Clin Pharmacol Ther, 1993 Oct, 54(4), 421 - 9
Modulation of vinblastine resistance with cyclosporine: a phase I study; Samuels BL et al.; OBJECTIVE: Tumor cell resistance is a major cause of failure to cure advanced malignancies . Multidrug resistance is thought to be an important mechanism of such resistance . Our aims were to identify doses of cyclosporine that would achieve blood levels effective in modulating multidrug resistance to vinblastine and to evaluate the toxicities and maximum tolerated dose of cyclosporine when administered in conjunction with vinblastine . METHODS: We conducted a phase I trial of vinblastine and escalating doses of cyclosporine . Cyclosporine was given by continuous intravenous infusion over 120 hours and vinblastine was administered by continuous infusion from hour 12 to hour 108 . Sixty-two patients entered the trial, of whom 60 were evaluable . RESULTS: Cyclosporine was escalated from 1 to 15.6 mg/kg/day . Vinblastine doses were reduced to 1.6 and then 1.2 mg/m2/day because of increasing vinblastine toxicity at higher cyclosporine doses . The maximum tolerated dose of cyclosporine at 1.2 mg/m2/day vinblastine was 12.5 mg/kg/day; at this dose level, mean blood cyclosporine level was 1.25 +/- 0.41 mumol/L . Significant nephrotoxicity was observed at higher cyclosporine doses in two of four patients . Nephrotoxicity was not significant at doses at or lower than this maximum tolerated dose and was not cyclosporine dose dependent . Myelosuppression, neurotoxicity, and transient hyperbilirubinemia were observed and were cyclosporine dose dependent . CONCLUSIONS . Cyclosporine by continuous infusion may be safely given in high doses concurrently with continuous-infusion vinblastine . Plasma levels of cyclosporine > or = 1 mumol/L can be sustained during vinblastine administration . No sustained effect on T-cell subsets was observed . Vinblastine toxicity is enhanced by cyclosporine in a dose-dependent fashion and correlates with cyclosporine-induced hyperbilirubinemia.

J Environ Pathol Toxicol Oncol, 1993 Oct-Dec, 12(4), 193 - 7
Biological effects of PEMF (pulsing electromagnetic field): an attempt to modify cell resistance to anticancer agents; Pasquinelli P et al.; Pulsing Electromagnetic Field (PEMF) effects lead to a modification of the multidrug resistance (MDR) of cells in vitro and in vivo . The murine leukemic doxorubicin-resistant cell line, P388/Dx, subjected to PEMF irradiation in vitro, showed a significant difference in thymidine incorporation when the concentration of doxorubicin reached a level of 1 microgram/mL, which corresponds to the inhibition dose 50 (ID50) . The human lymphoblastic leukemia vinblastine-resistant cell line, CEM/VLB100, also showed a significant modification under the same experimental conditions at the in vitro ID50 corresponding to a vinblastine concentration of 100 ng/mL . BDF1 mice transplanted with P388/Dx cells also had an increase in their life span when doxorubicin was injected intraperitoneally in fractionated doses, while being subjected to PEMF irradiation.

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 9209 - 13
Functional expression of mouse mdr1 in Escherichia coli; Bibi E et al.; We describe functional expression of the mouse multidrug-resistance protein (P-glycoprotein; P-gp) in an Escherichia coli mutant defective in the outer membrane protease ompT . Heterologously expressed mdr1 appears as an unglycosylated species with an apparent molecular mass of 140 kDa in the membrane of the mutant . Unglycosylated mdr1 retains the ability to bind the photoactivatable drug analog {125I}iodoarylazidoprazosin and confers resistance to tetraphenylphosphonium (TPP+) and tetraphenylarsonium (TPA+), known mdr1 substrates . In vivo resistance is linked to reduced cellular accumulation and energy-dependent efflux of the lipophilic cations . Surprisingly, discrete mutations in the predicted nucleotide binding folds of mdr1 that abolish drug resistance in mammalian cells have no apparent effect on TPA+ efflux via mdr1 in E . coli.

J Infect Dis, 1993 Oct, 168(4), 1052 - 5
Transmission of multidrug-resistant Mycobacterium tuberculosis among persons with human immunodeficiency virus infection in an urban hospital: epidemiologic and restriction fragment length polymorphism analysis; Coronado VG et al.; From January 1990 to December 1991, 16 patients with multidrug-resistant tuberculosis (MDR-TB) caused by Mycobacterium tuberculosis resistant to isoniazid, rifampin, and streptomycin were diagnosed at Elmhurst Hospital . Compared with other TB patients, MDR-TB patients were more likely to have human immunodeficiency virus (HIV) infection (14/16 vs . 21/204, P < .001) and a prior admission (10/16 vs . 3/204, P < .001) . HIV-infected patients hospitalized for > 10 days within three rooms of an infectious MDR-TB patient had higher risk of acquiring MDR-TB than did HIV-infected patients with shorter hospitalizations or locations further from the MDR-TB patient(s) (6/28 vs . 2/90, P < .001) . Isolates of 6 of 8 MDR-TB patients in a chain of transmission were identical by restriction fragment length polymorphism DNA typing . Ambulation on the wards of inadequately masked TB patients and lack of negative pressure in isolation rooms probably facilitated transmission . This report documents nosocomial transmission of MDR-TB and underscores the need for effective isolation practices and facilities in health care institutions.

Lung Cancer, 1993 Oct, 10(1-2), 1 - 12
Immunohistochemical study of P-glycoprotein distribution in lung cancer; Pujol JL et al.; Indirect immunoperoxidase was used to determine the reactivity of C219 (P-glycoCHEK C219, Centocor Diagnostics, Malvern, PA), a monoclonal antibody (Mab) with high affinity for an internal epitope of the P-glycoprotein encoded by the multidrug resistance (MDR1) gene, in 40 surgically resected primary lung tumours . C219 reactivity was qualitatively classified in seven small cell lung cancers (SCLC), 29 non small cell lung cancers (NSCLC), and four carcinoid tumours . Ploidy was analysed by means of static cytometry using a computer-assisted image processor following Feulgen staining of cytologic prints of 32/40 lung tumours . Indirect immunoperoxidase reactivities of Mabs S-L 11.14 and MOC-1 were also studied to characterize the expression of cluster 1 lung cancer antigens and hence to determine among the NSCLC those which expressed the neural cell adhesion molecule (NCAM) . Eighteen (45%) lung tumours strongly expressed P-glycoprotein as an immunostaining of many islets of malignant cells or almost all malignant cells . In addition, 8/40 tumours (20%) showed a weak reactivity (few immunostained cells) and 14/40 (35%) no reactivity . There was no difference of reactivity when NSCLC were compared with SCLC . The expression of P-glycoprotein in NSCLC did not vary significantly when the stage of disease was considered . Among the 29 NSCLC, 10 (36%) expressed S-L 11.14 and MOC-1 . The NCAM positive NSCLC did not show any difference of P-glycoprotein expression in comparison with NCAM negative ones . Finally, C219 immunoperoxidase reactivity did not significantly differ according to the ploidy status . In conclusion, the internal epitope of the P-glycoprotein encoded by the MDR1 gene is frequently expressed by lung tumours of any histological type . This expression is not higher in Stage III and IV lung cancers in comparison with Stage I and II ones, or in NSCLC in comparison with SCLC either . Thus, the C219 related epitope seems to have a weak implication in the lower chemosensitivity of both advanced stages and NSCLC.

Leuk Lymphoma, 1993 Oct, 11(3-4), 239 - 48
Flow cytometric assessment of multidrug resistance (MDR) phenotype in acute myeloid leukaemia; Hart SM et al.; Forty one patients with acute myeloid leukemia (AML), including 27 at presentation and 14 relapsed or resistant cases, were assessed for laboratory evidence of the MDR phenotype . Leukaemic cells from all 41 cases were studied by immunocytochemistry using the JSB-1 monoclonal antibody and simultaneously by reverse transcription polymerase chain reaction (RT-PCR) to evaluate expression of the mdr 1 gene . Cells from 32/41 cases were also assessed for daunorubicin (DNR) accumulation and retention by flow cytometry (FC) . Nineteen of the 41 (46%) patients were positive for MDR by JSB-1 immunocytochemistry (11/27 {41%} at presentation and 8/14 {57%} relapsed or resistant cases) . Nine of the 19 (47%) P-gp positive, de novo patients achieved complete remission . 22 patients were negative by JSB-1 immunocytochemistry (16/27 {59%} at presentation and 6/14 {43%} of the relapsed or resistant cases) and 11/22 (50%) P-gp negative patients achieved a complete remission . Of the 32 patients assessed by FC, 7 (22%) were positive for the MDR phenotype with increased DNR accumulation and retention in the presence of the MDR reversing agent verapamil (VPM) . 6/7 of the FC positive cases were also JSB-1 positive, and 6 had additional poor risk features . Of the twenty five FC negative patients, 6 had received previous chemotherapy and 15 (60%) achieved complete remission . Mdr 1 mRNA levels were increased in all seven FC positive cases whereas only 7/19 JSB-1 positive cases had raised mdr 1 mRNA levels . These results suggest that the assessment of MDR status by immunocytochemistry using JSB-1 is not predictive of response to chemotherapy . Flow cytometric analysis of blast cells appears to correlate well with mdr 1 mRNA levels and may be a better predictor of treatment outcome.

Oncology (Huntingt), 1993 Oct, 7(10), 23 - 8, 32; discussion 32, 35-8
Multidrug resistance: clinical relevance in acute leukemia; List AF; The multidrug resistance (MDR) phenotype represents an important major mechanism of resistance to anthracyclines and related natural products that lends itself to clinical exploitation . Overexpression of the mdr1 gene or its glycoprotein product has been linked to a number of poor prognostic factors in acute myeloid leukemia and adversely affects treatment outcome . Clinical trials are now under way testing pharmacologic modulators of MDR in acute leukemia . Although preliminary results are encouraging, MDR modulators may alter the clearance of some antineoplastics and thereby confound interpretation of randomized trials . This review examines diagnostic methods of MDR detection, its prevalence and impact in acute leukemia, and strategies for MDR reversal.

J Histochem Cytochem, 1993 Oct, 41(10), 1573 - 7
Ultrastructural localization of daunomycin in multidrug-resistant cultured cells with modulation of the multidrug transporter; Rutherford AV et al.; We localized the chemotherapeutic drug daunomycin inside cultured cells by taking advantage of its inherent fluorescence . Multidrug-resistant cultured cells, in which the accumulation of daunomycin can be reversibly controlled with verapamil to block the multidrug transporter, were incubated in daunomycin and verapamil and the accumulated daunomycin was visualized with epifluorescence optics . After fixation under a variety of different conditions to make cells permeable to diaminobenzidine (DAB), the internal daunomycin was illuminated under the fluorescence microscope in the presence of DAB . Photooxidation of DAB in sites of fluorescing daunomycin (photoconversion) resulted in intracellular deposition of oxidized DAB product . These sites were then visualized by transmission electron microscopy . In cells in which the multidrug transporter was inhibited by verapamil, daunomycin was localized in the nucleus of cells by mild fixation conditions such as formaldehyde . Increasing amounts of glutaraldehyde in the fixative caused apparent quenching of the nuclear fluorescence but still allowed fluorescence to occur in other cell organelles, which were then well preserved . Daunomycin was found in the nuclear envelope, the endoplasmic reticulum, and in lysosomes in cells in which the multidrug transporter was inhibited . Lysosomal accumulation has been previously described and was expected because of the known accumulation of positively charged molecules in organelles with low pH . However, the accumulation of daunomycin in the nuclear envelope and endoplasmic reticulum has not been previously observed . These results clearly demonstrate the utility of fluorescence photoconversion methodology for the high-resolution ultrastructural localization of fluorescent materials.

Cytometry, 1993 Oct, 14(7), 736 - 46
Evaluation of flow cytometry for multidrug resistance detection in low resistance K562 cells using daunorubicin and monoclonal antibodies; Van Acker KL et al.; We compared the effectiveness of two flow cytometric methods for the detection of the multidrug resistance (MDR) phenotype . The sensitivity of both methods depended on the ability to discriminate low resistance cells from sensitive ones . Therefore, K562 cells with decreasing vinblastine (VLB) resistance levels were examined, the lowest resistance level being nonmeasurable with a colorimetric MTT assay . The fluorescent drug daunorubicin (DNR) was measured in combination with two modulators of MDR, cyclosporin-A (CsA) and verapamil (Vp) in a functional flow cytometric assay . When compared to sensitive cells, DNR uptake levels at steady state were reduced in all resistant cell lines, except for the lowest resistant cell line . The effect of modulator, CsA, on DNR uptake was seen in all resistant sublines, compared to sensitive cells, except for the lowest resistant cells . In another assay, the P-glycoprotein (Pgp) expression was analysed with monoclonal antibodies, MRK16 and C219 . MRK16 was found to be the most sensitive antibody to screen for MDR+ cells, since we could show Pgp hyperexpression in all resistant cells . C219 reactivity became evident in cells possessing resistance factors higher than 5 . These results indicate that both the functional assay and the Pgp assay are sensitive to be used for screening of MDR+ cells.

Int J Cancer, 1993 Sep 30, 55(3), 478 - 84
Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein; Schinkel AH et al.; Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance . Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4 . The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs . In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected . None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein . The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

Nature, 1993 Sep 30, 365(6445), 451 - 3
Convergent combination therapy can select viable multidrug-resistant HIV-1 in vitro; Larder BA et al.; The reverse transcriptase enzyme of human immunodeficiency virus type 1 (HIV-1) is the target for many inhibitors . Amino-acid substitutions in functional regions of the enzyme that abolish reverse transcriptase activity also prevent HIV-1 replication . But selection pressure by drugs such as AZT (3'-azido-3'deoxythymidine, zidovudine), ddI (2',3'-dideoxyinosine) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) causes outgrowth of resistant variants due to non-lethal mutations in the enzyme . Reports of synergy and lack of cross-resistance between reverse transcriptase inhibitors (refs 7, 9, 10, 12-14, 17, 18, 20, 21), plus the reversal of AZT resistance by mutations induced by ddI and NNRTIs, have indicated that specific drug combinations directed at reverse transcriptase might curtail resistance . Chow et al . extended this concept in a report that specific multiple combinations of resistance mutations in the reverse transcriptase can significantly impair HIV-1 replication . They concluded that evolutionary limitations may exist to prevent the emergence of multidrug resistance to inhibitors of reverse transcriptase . We report here that HIV-1 co-resistant to AZT, ddI and the NNRTI nevirapine can be readily selected in cell culture starting with dual AZT- and ddI-resistant virus . We found no evidence for 'replication incompatible' combinations of resistance mutations, although a mutation (M184-->V) conferring oxathiolane-cytosine nucleoside resistance in reverse transcriptase completely suppressed AZT resistance in a triple-resistant background . These in vitro observations suggest that triple drug combination therapy might ultimately result in co-resistant HIV-1, although they do not preclude assessment of such combinations for treatment of HIV-1 disease.

Lancet, 1993 Sep 25, 342(8874), 756 - 7
Tuberculosis recurrence in Africa: true relapse or re-infection?
Daley CL.
PIP: HIV-seropositive patients respond to rifampicin-containing anti tuberculosis (TB) regimens as well as HIV-seronegative patients . In Nairobi, Kenya, 90% of HIV-positive patients who suffered a recurrence of TB first received a non-rifampicin-containing regimen . The overall unadjusted recurrence rate for HIV-positive patients was 16.7% while it was .5% for HIV-seronegative patients . An earlier, similar study in Zaire also showed a higher recurrence rate in HIV positive patients . A study in the US found a low recurrence rate among HIV-positive patients on rifampicin-containing regimens . A possible explanation for the higher recurrence rates may be that the thiacetazone-containing regimen is not as potent as the rifampicin containing regimen . Another possible explanation may be that no one knows the optimum duration of therapy for HIV-infected patients . 70% of HIV-positive patients in nairobi who suffered a recurrence of TB experienced a cutaneous-hypersensitivity reaction, resulting in a change in therapy and maybe affecting compliance . The researchers of the Nairobi study used DNA fingerprinting to determine whether the patients truly relapsed or were reinfected (cultures were available from only 3 HIV-positive patients) . 1 patient was reinfected by a different strain of Mycobacterium tuberculosis . 4 of 17 AIDS patients in New York City were reinfected with a different multidrug resistant strain of M . tuberculosis . Reinfection is more likely to happen in sub-Saharan Africa where TB an HIV are very prevalent . Physicians cannot accurately determine a treatment regimen in HIV-infected patients in an area of high prevalence of TB . Thus, we need to determine reinfection rates in HIV infected patients to plan a response .

Cancer Res, 1993 Sep 15, 53(18), 4156 - 60
Inwardly rectifying K+ channels and volume-regulated anion channels in multidrug-resistant small cell lung cancer cells; Jirsch J et al.; Studies of multidrug-resistant H69AR cells which overexpress the multidrug resistance-associated protein, compared with drug-sensitive parental H69 cells and revertant H69PR cells, revealed an inwardly rectifying K+ channel current (conductance, 231 pS/pF) and increased volume-regulated anion current (limiting conductance, 2 nS/pF) . The anion current was selective for Cl- ions and sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1-1 mM) but ATP was not required for initial current activation even in excised patch experiments . K+ current reversal potential varied 52 mV/10-fold change in the external K+ concentration and current was blocked by BaCl2 (0.1-1 mM) . The results indicate that overexpression of multidrug resistance-associated protein is accompanied by increases in both K+ channel and volume-regulated Cl- channel current in the multidrug-resistant cell line H69AR.

Cancer Res, 1993 Sep 15, 53(18), 4238 - 42
Bovine serum albumin-doxorubicin conjugate overcomes multidrug resistance in a rat hepatoma; Ohkawa K et al.; A bovine serum albumin-conjugated doxorubicin via the glutaraldehyde bridge (BSA-DXR conjugate) showed potent dose-dependent inhibition of cell growth against daunorubicin-resistant AH66 (AH66DR) cells as well as parental AH66 (AH66P) cells in vitro as compared to treatment with DXR or BSA-glutaraldehyde conjugate without DXR (BSA-GA) . In the culture of AH66DR with BSA-DXR conjugate, drug accumulation in the AH66DR cells increased as a function of time up to 24 h reaching approximately the same drug level as AH66P cells treated with DXR . The intracellular accumulation of the BSA-DXR conjugate was inhibited by the addition of ammonium chloride, while that of DXR alone was not inhibited . Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate or pharmacologically active DXR adduct remained in the cells at a relatively high concentration over a 36-h time period . The life-prolonging effect of the conjugate was assessed using rats inoculated i.p . with AH66P or AH66DR . The rats were treated with the BSA-DXR conjugate, DXR, a mixture of DXR with BSA, or BSA-GA by either the i.p . or i.v . route . Treatment with DXR had no significant surviving effect as compared to that with saline in AH66P-bearing rats . By contrast, BSA-DXR conjugate showed a significant life-prolonging effect as compared with DXR alone in the same degree both in AH66P- and AH66DR-bearing rats . BSA-GA did not show any toxicity in vivo as well as in vitro . These results indicate that the BSA-DXR conjugate allows DXR to escape from the multidrug resistance mechanism.

J Biol Chem, 1993 Sep 15, 268(26), 19505 - 11
SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity; Cornwell MM et al.; To define sequences in the human multidrug resistance (MDR1) promoter that influence transcription, we measured the activity of MDR1 promoter constructs using luciferase as a reporter gene . Deletion of promoter sequences to -121 (relative to the transcription initiation site) had very little effect on promoter activity in transiently transfected cells . Further deletion to -88 increased activity about 4-fold, while deletion to -51 decreased activity about 10-fold . The data indicated that between -121 and -88 and between -73 and -51 were sequences that modulated promoter activity . Because these two regions contain G-rich sequences, which are important for function in other promoters, the effect of mutation of the G-rich regions (-110 to -103 and -61 to -43) on MDR1 promoter activity was measured . Mutation of the -110 G-box (-110 to -103) resulted in a 6-fold increase in promoter activity and inhibited the formation of a specific nuclear protein complex, suggesting that this region functions as a transcriptional "repressor" binding site in cycling cells . In contrast, mutation of the -50 G-box (-61 to -43) reduced promoter activity 5-fold . DNase-I footprinting and electrophoretic mobility shift analysis indicated that specific but distinct protein-DNA complexes formed at each of these two G-rich regions . The -50 G-box contains overlapping sites to which both SP1 and EGR-1 bind specifically . Co-transfection of MDR1 promoter constructs with SP1 into cells that lack SP1 (Drosophila Schneider 2 cells) activated equivalently both the wild type MDR1 promoter and a synthetic promoter containing the MDR1 -50 GC box and MDR1 initiator element.

Biochem Pharmacol, 1993 Sep 14, 46(6), 1096 - 9
Restricted transport of cyclosporin A across the blood-brain barrier by a multidrug transporter, P-glycoprotein; Tsuji A et al.; The blood-brain barrier permeability of cyclosporin A (CsA), an immunosuppressive cyclic peptide, is restricted despite its highly lipophilic nature . In this study, the uptake of CsA by primary cultured bovine brain capillary endothelial cells (BCEC) was investigated in order to clarify whether P-glycoprotein (P-gp), an ATP-dependent drug efflux pump expressed in the luminal surface of BCEC, causes the decreased transport of CsA into the brain . P-gp, having a molecular mass of 130-140 kDa, was detected with anti-P-gp monoclonal antibody, C219, using western blot analysis of the membrane fraction isolated from the bovine brain capillary . The uptake of CsA by primary cultured bovine BCEC was time-dependent, and the steady-state uptake of CsA was increased in the presence of several multidrug resistance reversing agents including verapamil and steroid hormones and the substrate of P-gp in BCEC, vincristine . The steady-stage uptake was increased significantly to approximately 4-fold when cellular ATP was depleted by treating with 2,4-dinitrophenol, suggesting that the efflux process is ATP dependent . Furthermore, in the presence of an anti-P-gp monoclonal antibody, MRK16, at a 10 micrograms/mL concentration, the uptake of CsA was increased approximately 3-fold . These results suggest that the low permeability of CsA into the brain is caused by the active efflux from BCEC by P-gp present in the luminal surface of cells.

N Engl J Med, 1993 Sep 9, 329(11), 784 - 91
Treatment of multidrug-resistant tuberculosis; Iseman MD; The frequency of infections with M . tuberculosis resistant to antituberculous drugs is increasing in the United States and globally . This increase is a major threat to tuberculosis treatment and control programs . To prevent this situation from worsening, initial treatment programs that entail directly observed therapy supported by effective inducements or enforcements must be used . Retreatment of patients who have multidrug-resistant tuberculosis should be carried out in programs with comprehensive microbiologic, pharmacokinetic, psychosocial, and nutritional support systems . Regimens of multiple drugs, which generally are poorly tolerated and more toxic than traditional regimens, must be administered for 18 to 36 months . Resectional surgery may be required for substantial numbers of patients . For patients with AIDS who acquire tuberculosis caused by multiply-resistant strains, the disease may prove lethal before effective therapy can be implemented . Ultraviolet irradiation systems should be used to protect health care personnel and other patients in high-risk environments . Enhanced federal, state, and local programs for prevention and control are urgently needed, and research to identify new medications and systems for their delivery is essential.

Biochemistry, 1993 Sep 7, 32(35), 9156 - 64
Differential modulation of P-glycoprotein transport by protein kinase inhibition; Bates SE et al.; Previous studies of P-glycoprotein have demonstrated that its function can be modulated by phosphorylation . In the present study, inhibition of protein kinase C with calphostin C or stauroporine or prolonged treatment with the phorbol ester TPA decreased phosphorylation of P-glycoprotein, and impaired transport of vinblastine . Calphostin C also inhibited transport of actinomycin D, vincristine, rhodamine, and azidopine in SW620 Ad300 multidrug-resistant human colon carcinoma cells . Photoaffinity labeling of P-glycoprotein with azidopine was decreased by calphostin C, suggesting that dephosphorylation alters the affinity of P-glycoprotein for its substrates . Impaired transport of rhodamine in normal T lymphocytes treated with staurosporine demonstrates that modulation of P-glycoprotein function is not limited to cells selected for drug resistance in vitro . Transport of P-glycoprotein antagonists in SW620 Ad300 cells was also affected by calphostin C . Cyclosporin A transport decreased, while verapamil transport increased . Cyclosporin A in calphostin C-treated cells resulted in additive P-glycoprotein antagonism, while no additive effect could be demonstrated with verapamil, suggesting that the increase in verapamil transport makes it a poorer P-glycoprotein antagonist . These studies suggest that transport by P-glycoprotein is a dynamic process which can be modulated by phosphorylation, and that antagonists may block P-glycoprotein differently in different phosphorylation states.

Nippon Rinsho, 1993 Sep, 51(9), 2343 - 52
{PMP70, the 70-kDa peroxisomal membrane protein: a member of the ATP-binding cassette transporters}; Kamijo K et al.; We have isolated the cDNA of the 70-kDa peroxisomal membrane protein (PMP70) from rat and human liver cDNA libraries . The nucleotide sequence of the cDNA of PMP70 contains an open reading frame of 1977 bp which encodes an amino acid sequence of 659 residues . Possible two domains were identified by hydropathy analysis . One is a hydrophobic region, which presumably contains six transmembrane segments . The other is a hydrophilic domain, which shows striking similarity to the sequences of the ATP-binding cassette (ABC) transporter proteins, including bacterial periplasmic transport proteins, the human multidrug resistance P-glycoprotein (MDR1), cystic fibrosis transmembrane conductance regulator (CFTR), and the putative adrenoleukodystrophy gene product (ALDP) . Based on its transmembrane structure and the homology to ABC proteins, PMP70 may be involved in ATP-dependent transport through peroxisomal membrane.

Ann Hematol, 1993 Sep, 67(3), 107 - 9
Itraconazole and multidrug resistance: possible effects on remission rate and disease-free survival in acute leukemia; Vreugdenhil G et al.; Itraconazole, a triazole antifungal agent, has been reported to reverse drug resistance against daunorubicin in acute leukemia . In a subanalysis from a double-blind, placebo-controlled trial examining the effects of itraconazole on the prevention of fungal infections in neutropenic patients, we studied the effects of itraconazole on remission rate and disease-free survival in patients with acute lymphoblastic (ALL) and acute myelogenous leukemia (AML) receiving remission induction treatment schedules containing daunorubicin (DNR) . Eleven ALL and 17 AML patients received itraconazole and 12 ALL and 25 AML patients were given placebo . Among AML patients the remission rate was slightly higher in the itraconazole group, whereas the disease-free survival was higher among ALL patients given itraconazole . In AML patients DFS was comparable in both groups but the number of high-risk patients in the itraconazole group was higher . These preliminary results may suggest a role for itraconazole in reversing multidrug resistance . Larger trials, however, are required to substantiate these findings and to correlate them with its in vitro effects on multidrug resistance.

Biochem Pharmacol, 1993 Sep 1, 46(5), 851 - 61
Altered topoisomerase I activity and recombination activating gene expression in a human leukemia cell line resistant to doxorubicin; Riou JF et al.; We examined the expression of the genes encoding topoisomerases I and II and those associated with V(D)J {variable(diversity)joining} recombination in two human T-cell acute lymphoblastic leukemia (T.ALL) cell lines, CEM and CEM/DOX . In CEM/DOX cells, which are resistant to doxorubicin, the topoisomerase I gene was found to be 4-fold overexpressed and nuclear topoisomerase I relaxation activity was 2-fold greater in CEM/DOX than in CEM cells . Furthermore, the cleavable complex reaction induced by camptothecin, a specific topoisomerase I inhibitor, was found to be 2.5-increased in the presence of topoisomerase I extracted from CEM/DOX, in comparison to that in CEM cells . Conversely, the topoisomerase II mRNA levels, nuclear decatenation activities and (mAMSA) 4'(9-acridinylamino)methanesulfon-m-anisidide-induced cleavable complex formation in CEM/DOX were similar to those of the doxorubicin-sensitive cells . The results indicate that topoisomerase I activity is elevated in CEM/DOX cells . Nevertheless, CEM/DOX cells were 11-fold more resistant to camptothecin than were CEM cells, and cross-resistance to camptothecin was not reversed by verapamil . Furthermore, using an intact cell assay for DNA-protein complexes, we found that camptothecin-stimulated cleavable complexes formed in CEM/DOX cells were increased in correlation with the elevated topoisomerase I activity . These results suggest that camptothecin resistance in CEM/DOX cells is due to different mechanism(s) than topoisomerase- or P-glycoprotein-associated multidrug resistance . The recombination activating gene, RAG1, which is one of the components of the site-specific V(D)J recombination complex, was 20-fold overexpressed in CEM/DOX cells . In contrast, RAG2 and T160 gene transcripts, other components of the V(D)J complex, were at best poorly detected in both sensitive and resistant cells . No specific V(D)J recombinase activity was found in CEM or CEM/DOX cells when the pJH201 transfection assay was used . The results indicate that CEM/DOX cells failed to generate V(D)J recombination although RAG1 gene is overexpressed . The mechanism of the RAG1 gene activation was not gene amplification, and no rearrangement was detected in the RAG1 gene locus . RAG1 presents homology with the yeast gene HPR1, itself homologous to yeast topoisomerase I and responsible for the control of recombination in somatic cells . Since DNA topoisomerases are themselves involved in the control of DNA topology, recombination and DNA repair, the possible coactivation of RAG1 and topoisomerase I genes in CEM/DOX cells is discussed.

J Infect Dis, 1993 Sep, 168(3), 537 - 51
Tuberculosis symposium: emerging problems and promise; Ellner JJ et al.; Between 1985 and 1991, 39,000 cases of tuberculosis occurred in excess of those expected based on previous trends . Immigration from high-prevalence countries, coinfection with human immunodeficiency virus (HIV), and outbreaks in congregative facilities are most responsible for the increase . Coincident with the increase in tuberculosis, outbreaks of multidrug resistant (MDR) tuberculosis have occurred . Clinical and epidemiologic data support nosocomial transmission . MDR tuberculosis occurred late in the course of HIV infection and was refractory to treatment . Compounding the problems of rising incidence and increasing resistance was the sudden recognition of shortages of antituberculous drugs . The problems currently posed by tuberculosis require new approaches to diagnosis and rapid sensitivity testing as well as assuring an adequate supply of licensed drugs and development of new drugs . A number of steps have been taken by governmental agencies to assure that the challenge is met.

J Ethnopharmacol, 1993 Sep, 40(1), 47 - 51
Antimalarial activity from 'Mhekara' (Uapaca nitida Müll-Arg.), a Tanzanian tree; Kirby GC et al.; An aqueous decoction of the root bark of Uapaca nitida Mull-Arg . is currently used locally at the Benedictine Mission at Peramiho in Tanzania to treat malaria . We have now demonstrated that extracts of root bark and leaves of this tree are active against the multidrug-resistant K1 strain of Plasmodium falciparum in vitro . An ethanolic extract of root bark showed activity against P . berghei in mice but at a dose which also showed toxic effects . The use of this plant in treating malaria appears to be novel and further studies would be of value.

Anticancer Res, 1993 Sep-Oct, 13(5A), 1557 - 63
Detection of human leukemia cells with multidrug-resistance phenotype using multilabeling with fluorescent dyes; Lautier D et al.; Reduced accumulation of multiple drugs is a characteristic of cells overexpressing P-glycoprotein . This phenotype is referred to as multidrug-resistance (MDR) . A protocol based on reduced accumulation of fluorescent dyes is proposed for discriminating MDR cells in cell populations . The combination of three fluorescent dyes, Hoechst 33342, rhodamine 123 and Nile red, with different intracellular targets, has been designed to characterize cells with different levels of resistance, using image cytometry . The fluorescence intensity of each dye was quantified in living cells . The protocol was applied to human leukemia cell lines, (K562, K562/ADR, CCRF-CEM, CEM/VLB100, CEM/VM-1) . The effect of verapamil on dye accumulation is emphasized.

Anal Biochem, 1993 Sep, 213(2), 414 - 21
Quantitative polymerase chain reaction analysis of mdr1 mRNA in multiple myeloma cell lines and clinical specimens; Futscher BW et al.; We have designed a new polymerase chain reaction (PCR) protocol for the quantitation of mdr1 mRNA in cell lines and clinical specimens . This protocol uses an in vitro-generated RNA molecule as an internal standard . This synthetic RNA contains the same mdr1 primer sequences as the cellular mRNA, but yields a different-sized PCR product after amplification . Since a single primer set is used in quantitation, differences in primer efficiency are not a concern . We have used this assay to measure mdr1 expression in a multiple myeloma cell line, 8226/S, its drug resistant variants 8226/dox6 and 8226/dox40, and tumor samples from 10 patients with B-cell malignancies (9 multiple myeloma, 1 chronic lymphocytic leukemia) . 8226/S does not express mdr1 mRNA . 8226/dox6 is 10-fold resistant to doxorubicin, and expresses 32 mdr1 mRNA/10 pg cellular RNA . 8226/dox40 is 140-fold resistant to doxorubicin, and expresses 890 mdr1 mRNA/10 pg cellular RNA . Seven of the 10 patients had levels of mdr1 mRNA expression below that seen in the multidrug-resistant, human multiple myeloma cell line, 8226/dox6 . Three patients had levels of mdr1 expression comparable to those seen in 8226/dox6 . No patient had levels of mdr1 expression close to that seen in 8226/dox40 . Sample RNA integrity is assured by PCR analysis of a different, ubiquitous, cell cycle independent, histone variant, H3.3 . This assay will be useful for studying low level mdr1 expression in cell lines and clinical specimens.

Southeast Asian J Trop Med Public Health, 1993 Sep, 24(3), 505 - 7
Mefloquine level monitoring in patients with multidrug resistant Plasmodium falciparum on the Thai Myanmar border; Karbwang J et al.; A total of 42 patients with uncomplicated falciparum malaria who attended the malaria clinic in Mae Sot, Tak Province were treated with single oral dose of MSP 3 tablets (Fansimef, equivalent to 750 mg of mefloquine) concurrently with primaquine (30 mg) . They all contracted the infection from Cambodia . The aim of the study was to monitor the efficacy of MSP 3 tablets for the treatment of this highly multiple drug resistant strains of Plasmodium falciparum in this area . Of the 39 patients included for efficacy assessment, 13 (33.3%) patients had sensitive responses, whereas 15 (38.5%) and 8 (20.5%) had RI and RII types of response, respectively . Melfoquine concentrations on Day-3 after treatment in patients with sensitive and treatment failure groups were comparable; the respective mean (SD) values were 665 (279) and 772 (264) ng/ml.

Mol Pharmacol, 1993 Sep, 44(3), 552 - 9
Pharmacological characterization of N-substituted phenoxazines directed toward reversing Vinca alkaloid resistance in multidrug-resistant cancer cells; Horton JK et al.; Previously we reported the synthesis and partial characterization of 21 N10-substituted phenoxazines in reversing Vinca alkaloid resistance . Here we report on a subset of these compounds; we have compared their activities in increasing Vinca alkaloid accumulation and reversing drug resistance in KB-ChR8-5 and GC3/c1 (human colon carcinoma) cell lines . Results demonstrated that 1) N-substituted phenoxazines increase accumulation of vinblastine; 2) within this series, there is little correlation or ranking of activity between the two cell lines when Vinca alkaloid accumulation is compared at equal concentrations of modulator; 3) N-substituted phenoxazines demonstrate both quantitative and qualitative differences, compared with verapamil, a standard modulator; and 4) the series includes at least two compounds, 10-{3'-{N-bis(hydroxyethyl)amino}propyl}phenoxazine and 10-(N-piperidinoacetyl)phenoxazine, which increase Vinca alkaloid accumulation but do not significantly inhibit efflux . Additionally, certain of these multidrug resistance modulators significantly enhance accumulation (8-50-fold) of Vinca alkaloids in cell lines with very low or undetectable P-glycoprotein levels, where verapamil has little activity . It is concluded that at least part of the activity of some of these N-substituted phenoxazine modulators may be mediated through a P-glycoprotein-independent mechanism.

J Clin Oncol, 1993 Sep, 11(9), 1652 - 60
Phase I/II trial of cyclosporine as a chemotherapy-resistance modifier in acute leukemia; List AF et al.; PURPOSE: To determine the toxicities and maximum-tolerated dose of cyclosporine (CsA) administered with daunorubicin as a modulator of multidrug resistance (MDR) in acute leukemia, and to evaluate response to treatment and its relationship to mdr1 gene expression . PATIENTS AND METHODS: Patients with poor-risk acute myeloid leukemia (AML) received sequential treatment with cytarabine (3 g/m2/d intravenously {i.v.}) days 1 to 5, and daunorubicin (45 mg/m2/d) plus CsA as a 72-hour continuous infusion (CI) days 6 through 8 in a phase I/II trial . A loading dose of CsA administered over 1 to 2 hours preceded the CI . CsA dose escalations ranged from 1.4 to 6 mg/kg (load) and 1.5 to 20 mg/kg/d (CI) . Whole-blood concentrations of CsA were monitored by immunoassay; plasma concentration of daunorubicin and daunorubicinol were determined by high-pressure liquid chromatography (HPLC) . Specimens were analyzed for P-glycoprotein expression, and results confirmed by a quantitative RNA polymerase chain reaction (PCR) assay for the mdr1 gene transcript . RESULTS: Forty-two patients are assessable for toxicity and response . P-glycoprotein was detected in 70% of cases . Dose-dependent CsA toxicities included nausea and vomiting (22%), hypomagnesemia (61%), burning dysesthesias (21%), and prolongation of myelosuppression . Transient hyperbilirubinemia developed in 62% of treatment courses and was CsA-dose-dependent . Reversible azotemia occurred in three patients receiving concurrent treatment with potentially nephrotoxic antibiotics . Steady-state blood concentrations of CsA > or = 1,500 ng/mL were achieved in all patients receiving CI doses > or = 16 mg/kg/d . Mean plasma daunorubicin, but not daunorubicinol, levels were significantly elevated in patients who developed hyperbilirubinemia (P = .017) . Twenty-six (62%) patients achieved a complete remission (CR) or restored chronic phase and three patients achieved a partial remission (PR) for an overall response rate of 69% (95% confidence interval, 54% to 84%) . The response rate was higher in patients who developed hyperbilirubinemia (P = .001), whereas MDR phenotype did not influence response to treatment . Among five patients with MDR-positive leukemia, cellular mdr1 mRNA decreased (n = 1) or was absent from relapsed specimens (n = 4), while mdr1 RNA remained undetectable at relapse in two patients who were MDR-negative before treatment . CONCLUSION: High doses of CsA, which achieve blood concentrations capable of reversing P-glycoprotein-mediated anthracycline resistance in vitro, can be incorporated into induction regimens with acceptable nonhematologic toxicity . Transient hyperbilirubinemia occurs commonly with CsA administration and may alter daunorubicin pharmacokinetics . Recommended doses of CsA for phase II and III trials are a load of 6 mg/kg and CI of 16 mg/kg/d.

Mol Microbiol, 1993 Sep, 9(6), 1239 - 46
Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot; Finken M et al.; Multidrug-resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks . Recognition of drug resistance is important both for treatment and to prevent further transmission . Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multidrug-resistant M . tuberculosis . We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance . The mutations found either lead to amino acid changes in ribosomal protein S12 or alter the primary structure of the 16S rRNA . The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.

Zhonghua Zhong Liu Za Zhi, 1993 Sep, 15(5), 329 - 31
{Analysis of mdr-1 mRNA and P 170 glycoprotein in cytotoxic resistant sublines}; Wang SW; Multidrug resistant sublines KBV200 and HCTV2000 were established by continuous selection from human KB and HCT cell lines cultured in medium in the presence of vincristine . The mdr-1 mRNA in total RNA from the two sublines was detected by dot blotting with HRP-labelled mdr-1 cDNA probe and enhanced chemiluminescent autography and the P170 glycoprotein in their cells was detected by immunohistochemistry . The mdr-1 gene expression level of KBV200 and HCTV2000 was higher than their wild counterparts . It was shown that the drug resistant properties of the two sublines were related to mdr-1 gene expression . The non-radioactive technique for analysis of specific mRNA and protein could be generalized for clinical research of gene expression.

Rev Invest Clin, 1993 Sep-Oct, 45(5), 481 - 92
{Multiple drug resistance: a problem in cancer chemotherapy}; Lizano Soberon M et al.; One of the main problems in clinical oncology is an acquired cellular drug resistance . Special attention deserves the multidrug resistance phenomenon (MDR) involving tumors which become resistant to a wide spectrum of non-related drugs to which they have never been exposed . Several mechanisms responsible for this phenomenon have been described . Among them is the increased expression of the MDR1 gene which encodes the plasma membrane glycoprotein P-gp . This glycoprotein is an energy-dependant multidrug efflux pump of wide specificity . It seems to have a normal physiological function but in some tumors resistant to chemotherapy its expression is increased . In cell lines the increased expression of P-gp is correlated with a decreased accumulation and retention of drugs inside the cells . In addition to P-gp, at least two other mechanisms of multidrug resistance have been described: a decreased expression and changes in the catalytic activity of topoisomerase II enzyme, and changes in glutathione transferase levels . Through biochemical and molecular methods researchers continue to look for a correlation between non-responding tumors and changes in the known drug-resistance mechanisms . These studies suggest that several factors are involved in the cellular drug resistance observed in human tumors, and probably are interacting between them . In clinical practice, the need of controlling MDR phenomena has led to the creation of alternate therapeutic strategies.

In Vivo, 1993 Sep-Oct, 7(5), 399 - 405
Antitumor activity of the new vinca-alkaloid S 12363 alone or in combination with verapamil on a human multidrug resistant renal carcinoma xenograft; Berlion M et al.; We have established a model of human renal cell carcinoma, Kgg2, transplanted into athymic nude mice which expressed P-glycoprotein (P-gp) (detected by flow cytometry) and a high level of mRNA transcript of mdr1 gene (Northern blot analysis) . We have evaluated the antitumor activity of a new highly potent vinca-alkaloid derivative, S 12363, in comparison with the activity of the reference compound vinblastine (VLB), when used alone or in combination with verapamil (VRP) . The influence of the calcium influx blocker verapamil on the activity of the combination of S 12363 with adriamycin (ADR) was also determined . The results showed that S 12363 at a dose of 0.05 mg/kg/day, administered alone by intraperitoneal route daily on days 1 to 5, induced a tumoral regression of 50% during the first days after treatment . This effect was potentialized by simultaneous treatment with verapamil at 20 mg/kg/day for 5 days, leading to a long-term reduction of 70% of tumor growth . Vinblastine at a dose of 0.4 mg/kg/day administered alone or in combination with verapamil, using the same protocol, was less efficient . The association of S 12363 at 0.075 mg/kg/day (on days: 1-5, 11, 21 and 31), adriamycin at 2 mg/kg/day (on days: 11, 21 and 31) and verapamil at 20 mg/kg/day (on days: 0-5, 11, 21 and 31) induced an important reduction of tumor growth of 80% at the end of the experiment . In conclusion, the new vinca-alkaloid derivative S 12363 could present a therapeutic advantage over the reference compound vinblastine in the treatment of renal cell carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Haematologica, 1993 Sep-Oct, 78(5), 261 - 3
The expression of the multidrug resistance related glycoprotein in adult acute lymphoblastic leukemia; Savignano C et al.; BACKGROUND . More than 50% of adults with acute lymphoblastic leukemia (ALL) actually die with resistant leukemia . The multidrug resistance (MDR) related P170 glycoprotein may be involved in treatment failure . METHODS . P170 content of leukemic cells was assayed by immunocytochemistry with the MRK-16 monoclonal antibody in 41 consecutive cases of adult ALL (27 at onset and 14 at relapse) . RESULTS . Positive cells were found in 11/14 cases at relapse and in 18/27 cases at onset . All cases with a high WBC, FAB L3, B-mature phenotype, and t(9; 22) were positive . In the 27 patients who were studied at onset and were treated with the same protocol, the overall failure rate was 44% in negative cases and 83% in positive cases . CONCLUSION . P170 overexpression may contribute to drug resistance and to chemotherapy failure in adult ALL.

Zhonghua Yi Xue Za Zhi, 1993 Sep, 73(9), 552 - 4, 576
{Oncogene expression in adriamycin and platinum resistant cell lines}; Liu YB; Using dot hybridization and southern blot technique, we studied the oncogene expression in P388/ADR and L1210/cis-platinum resistant cell lines . The amplification of three oncogene probes (C-myc, v-erb-B, N-ras) controlled with beta-actin in P388/ADR resistant cells was decreased by 1.335, 1.695, 1.35-fold (P < 0.05) (drug-resistant 44.5-fold), and 2.195, 2.02, 1.854-fold (P < 0.05) (drug-resistant 23.6-fold) respectively compared with P388/0 sensitive cell lines, and 1.950-, 2.075-, 2.285-fold (P < 0.05) (drug-resistant 3.5-fold) in L1210/cis-platinum compared with L1210/0 sensitive cell lines . The three oncogene amplification in resistant cell lines was not changed when southern blot hybridage was used . These results show that the more resistant the multidrug resistant cell lines is the worse the potential multiplication and prognosis of the tumor with be.

Anticancer Res, 1993 Sep-Oct, 13(5C), 1863 - 6
Multidrug resistance gene transcript level, and P-glycoprotein expression in paediatric malignant mesenchymal tumours; McDowell H et al.; Twenty-four malignant mesenchymal tumour specimens were analysed for human multidrug resistance (MDRI) gene transcript levels using Northern and slot blot techniques . The presence of P-glycoprotein was assessed in 12 of the 24 samples by immunohistochemistry using the monoclonal antibody (MAb) C219 . Increased MDRI transcript levels were found in 2 (8.3%), while, using immunohistochemistry, 2 samples were positive and 3 faintly positive (41.6%) . Overall, elevated P-glycoprotein or MDRI transcript levels were found in tumours of 6 patients, 3 of whom are dead . The relationship to MDRI expression and subsequent resistance to chemotherapy has to be established.

Anticancer Res, 1993 Sep-Oct, 13(5A), 1425 - 30
Characterization of four doxorubicin adapted human breast cancer cell lines with respect to chemotherapeutic drug sensitivity, drug resistance associated membrane proteins and glutathione transferases; de la Torre M et al.; Four human breast cancer cell lines with or without estrogen and progesterone receptors were adapted to growth in the continuous presence of doxorubicin (Dox) at 10 (Zr-75-1), 15 (HTB-122), or 50 (MDA-MB-231 and Hs578T) ng/ml . The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16) . The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16) . The sublines maintained or slightly increased their cis-platinum (CDDP) sensitivity . The sublines of HTB-122 showed resistance only to VP16 combined with a paradoxical increased sensitivity to VCR . The phenotypic alteration in the sublines with respect to Dox sensitivity was maintained for at least two months in the absence of Dox . The glutathione depletor buthionine sulfoximine (BSO) and the calcium channel blocker verapamil (Ver) increased the Dox sensitivity slightly only in the MDA-MB-231 and Hs578T sublines, respectively . Ver also tended to protect some of the sublines from CDDP . The sublines of Zr-75-1 and Hs578T showed increased expression of the 170-kDa permeability glycoprotein (P-gp), whereas expression of a 85-kDa membrane protein determined by the MRK20 antibody was increased in the sublines of Zr-75-1, and HTB-122 . Class pi glutathione transferase (GST) levels varied greatly between the cell lines but increased during Dox selection only in the subline of Zr-75-1 . Class mu GST was detectable in the MDA-MB-231, Hs578T and HTB-122 cell lines, whereas class alpha GST was detectable in these sublines but undetectable in their parental cell lines . The Zr-75-1 subline showed a 5-fold increase in the class alpha concentration . Except for a correlation between increased P-gp expression and resistance to Dox, VCR and VP16, no obvious correlations between receptor status, increased P-gp expression, membrane proteins, GST levels and acquired drug resistance were found . Thus, except for a possible role for P-gp in multidrug-resistance, these findings indicate a pronounced mechanistic heterogeneity responsible for cytotoxic drug sensitivity also in cells with a common histologic origin and exposed to the same drug.

FEBS Lett, 1993 Aug 23, 329(1-2), 63 - 6
Early steps of the P-glycoprotein expression in cell cultures studied with vital fluorochrome; Egudina SV et al.; This study shows that flow cytometry analysis of the rate of fluorochrome Rh123 efflux may be used for detection of cells at initial steps of P-glycoprotein expression and of minor subpopulations of multidrug-resistant (MDR) variants in human cell lines . This method also evaluates the fraction of low-level MDR cells among peripheral blood leukocytes of patients with chronic myeloid leukemia . The alterations in Pgp function were revealed in rat hepatoma cells after short treatment with colchicine.

Int J Cancer, 1993 Aug 19, 55(1), 115 - 21
Differential over-expression of mdr1 genes in multidrug-resistant rat glioblastoma cell lines selected with doxorubicin or vincristine; Schott B et al.; We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line) . Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold) . Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line . This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multidrug-resistance mechanism . Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min . This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 0.5 cells . Northern and Western blot analysis of the mdrI gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdrI gene and the P-glycoprotein to a slightly higher level than the C6 IV line . Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdrIa gene, whereas the C6 IV cells preferentially over-expressed the mdrIb gene . This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines . It can be concluded therefore that the mdrIa gene product is more efficient than the mdrIb gene product in extruding anti-cancer drugs from the cells; and that the mdrIb gene product might preferentially extrude vincristine rather than doxorubicin.

Cancer Res, 1993 Aug 15, 53(16), 3681 - 6
Ultrastructural appraisal of the multidrug resistance in K562 and LR73 cell lines from Fourier transform infrared spectroscopy; Le Gal JM et al.; Two different cell lines sharing the multidrug resistance (MDR) phenotype were investigated for 8 months by means of Fourier transform IR spectroscopy on cell smears . We studied (a) a human leukemic doxorubicin-sensitive K562 cell line, from which a doxorubicin-resistant K562 cell subline was subsequently derived; (b) a Chinese hamster LR73 drug-sensitive line, subsequently transfected with the expression plasmid pDREX4 containing the mdr1 gene, to produce a multidrug-resistant LR73 subline (MDR-LR73) . The sensitivity of Fourier transform IR spectroscopy has allowed differentiation between sensitive and MDR phenotypes among the above lines, even in double blind studies . The MDR phenotype is characterized by three combined features in spectra: (a) a decrease in the intensities of the amide I and II bands; (b) a shoulder on the high wave numbers slope of the amide I bands; (c) a shift toward the high wave numbers of the amide II bands . Furthermore, computational treatment of Fourier transform IR spectra (deconvolution and Gaussian curve-fitting techniques), has evidenced, in MDR-K562 and MDR-LR73 cell sublines, a conformational change involving the same protein in both sublines . It is hypothesized that the protein implicated in the conformational change may be related to the MDR phenotype.

Blood, 1993 Aug 15, 82(4), 1288 - 99
Enhancement of daunorubicin accumulation, retention, and cytotoxicity by verapamil or cyclosporin A in blast cells from patients with previously untreated acute myeloid leukemia; Ross DD et al.; This work was designed to discern the frequency of expression of classical multidrug resistance (MDR) in acute myeloid leukemia (AML) at the time of diagnosis, using Western blotting for P-glycoprotein (Pgp) and functional assays for an MDR phenotype (enhancement of daunorubicin {DNR} accumulation/retention and cytotoxicity by the known MDR modulators verapamil, cyclosporin A, and progesterone) . Blast cells were studied from 49 newly diagnosed AML patients who were subsequently treated with the "3 and 7" combination of cytosine arabinoside (ara-C) and DNR . DNR accumulation (1 microgram/mL, 3 hours) and retention (16 hours) were determined by flow cytometry . Cyclosporin A (CsA, 5 mumol/L) or verapamil (6.6 mumol/L) each caused significant enhancement of DNR accumulation and retention in these blast cell samples (P < .001, Wilcoxon's test) . Verapamil or CsA caused greater than 20% enhancement of DNR accumulation or retention in over 25% or 50% of these patients, respectively; however, there was no correlation with the presence or degree of enhancement and response to treatment . Progesterone (10 mumol/L) caused no significant enhancement of DNR accumulation or retention . The effects of the MDR modulators on the cytotoxicity of DNR was also determined in blast cells from 40 of the patients, using a flow cytometric assay . CsA alone was cytotoxic (caused an approximate 20% decrease in cell survival compared with control, P < .001); CsA or verapamil caused enhancement of 1 mumol/L DNR cytotoxicity (P < .001) . Greater than 40% enhancement of cell kill by CsA or verapamil was observed in over 75% of patients studied . There was no difference in the degree of enhancement of cytotoxicity between patients clinically sensitive or resistant to treatment . Progesterone caused no enhancement in DNR cytotoxicity . In contrast to the functional assays, highly sensitive immunoblots using the C219 antibody to Pgp showed evidence of low level expression of Pgp in blast cells from only 3 of these patients: 1 was chemotherapy resistant, 2 were sensitive . Thus, although the functional assays suggest a high frequency of expression of a classic MDR phenotype in AML patients at the time of diagnosis, with enhancement by CsA obtained at a clinically relevant concentration (5 mumol/L), the frequency of Pgp expression detectable by C219 Western blots was low in these patients . This could be interpreted either that the method used was not sufficiently sensitive to detect Pgp in all of the blast cell specimens that actually overexpressed mdr1, or that the accumulation/efflux-based MDR phenotype observed is not always mediated by Pgp in these previously untreated patients.

Biochim Biophys Acta, 1993 Aug 15, 1150(2), 147 - 54
High levels of P-glycoprotein detected in isolated brain capillaries; Jette L et al.; P-glycoprotein (P-gp) is a highly-conserved membrane protein expressed in various multidrug-resistant cell lines . P-glycoprotein was detected in capillaries isolated from human, beef and rat brains with a Western immunoblotting procedure using the monoclonal antibody C219 (mAb C219) specific for P-gp . The mAb C219 detected a 180 kDa protein in brain capillaries isolated from all three species . The largest amount of antigen was detected in capillaries isolated from human brain . Specific binding was assessed by competitive inhibition of mAb C219 binding by the synthetic epitope VQEALD . The glycoprotein nature of the brain capillary proteins was confirmed by its sensitivity to N-glycanase treatment, which reduced their apparent molecular mass by 5 to 10 kDa . In addition, immunohistochemical studies using the antibodies C219, JSB-1 and C494 were performed . Bovine and rat capillaries showed reactivity only with the mAb C219 . Heavy staining of human brain capillaries was observed with both antibodies C219 and JSB-1, while only weak staining was observed with antibody C494 . These results clearly show that P-glycoprotein is strongly expressed at the blood-brain barrier (BBB) site and suggest that this protein may play a physiological role in regulating the access of certain molecules to the central nervous system, or in the secretory functions of the BBB.

Cancer Res, 1993 Aug 15, 53(16), 3658 - 61
The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein; Krishnamachary N et al.; HL60 cells isolated for resistance to Adriamycin (HL60/ADR) overexpress a 190-kDa ATP binding protein which has a minor sequence homology with P-glycoprotein . It has also been observed that HL60/ADR overexpress the MRP gene which was first identified as a component of a non-P-glycoprotein mediated multidrug resistance of H69/ADR cells {Cole et al., Science (Washington DC), 258: 1650, 1992} . A complementary DNA of MRP has been cloned and based on the deduced sequence encodes a member of the superfamily of proteins which bind ATP and function in various transport processes {Cole et al., Science (Washington DC), 258: 1650, 1992} . In view of this it was of interest to identify the protein encoded by MRP and determine if it may be related to p190 . In the present study we have prepared antisera against three synthetic peptides which correspond to the deduced sequence of the MRP protein . Proteins reactive with the antisera have been examined in HL60/ADR cells using Western blot analysis . All antisera react with a 190 kDa protein contained in membranes of resistant but not sensitive cells . One antiserum used for further studies is not reactive with P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine . Analysis of subcellular fractions demonstrates that p190 is present primarily in the endoplasmic reticulum with lower levels also present in plasma membranes . Treatment of HL60/ADR cells with tunicamycin results in the appearance of a 165-kDa resistance associated protein which reacts with the antipeptide serum . The results of this study therefore demonstrate that the MRP gene encodes a 190-kDa membrane bound glycoprotein.

JAMA, 1993 Aug 11, 270(6), 694 - 8
From the Centers for Disease Control and Prevention . Initial therapy for tuberculosis in the era of multidrug resistance: recommendations of the Advisory Council for the Elimination of Tuberculosis; Malaria prophylaxis in long-term expatriate mineworkers in Ghana; Ashanti Goldfields Corporation Hospital, Obuasi, GhanaThe role of malaria chemoprophylaxis for long-term expatriates has not been re-evaluated since the emergence of widespread multidrug resistance . A survey of 106 expatriates working in a mine in Ghana (holoendemic for malaria) was conducted to determine the compliance with malaria chemoprophylaxis . Overall 64 per cent took regular chemoprophylaxis . Of the long-term expatriates (5 or more years in areas with holoendemic malaria), 48.4 per cent either took malaria prophylaxis very irregularly or not at all . The main reasons for failing to comply were fear of long-term side effects and conflicting advice on prophylaxis . This reluctance to take long-term chemoprophylaxis highlights the need to re-emphasise the importance of anti-mosquito measures, prompt treatment of fevers, and perhaps consider abandoning chemoprophylaxis in those expatriate workers with ready access to hospital care.

Br J Cancer, 1993 Aug, 68(2), 342 - 51
Interaction of multidrug-resistant Chinese hamster ovary cells with amphiphiles; Loe DW et al.; The interaction of membrane-active amphiphiles with a series of MDR Chinese hamster ovary (CHO) cell lines was investigated . Cross-resistance to cationic amphiphiles was observed, which was effectively sensitised by verapamil . MDR cells showed collateral sensitivity to polyoxyethylene amphiphiles (Triton X-100/Nonidet P-40), which reached a maximum at 9-10 ethylene oxide units . Resistant lines were also highly collaterally sensitive (17-fold) to dibutylphthalate . mdrl transfectants showed cross-resistance to cationic amphiphiles, but no collateral sensitivity to nonionic species . Triton X-100/Nonidet P-40 inhibited 3H-azidopine photoaffinity labelling at low concentrations, perhaps reflecting a specific interaction with P-glycoprotein . Further investigation of the molecular basis of collateral sensitivity revealed that association of 3H-Triton X-100 with MDR cells reached steady state levels rapidly, and occurred by a non-mediated mechanism . The equilibrium level of X-100 uptake was inversely related to drug resistance . Collateral sensitivity is thus not a result of decreased Triton X-100 association with the cell . The fluorescent probe merocyanine 540 was used to examine the MDR plasma membrane microenvironment for physicochemical changes . Increasing levels of drug resistance correlated with a progressive shift in the mean cell fluorescence to lower levels, which suggests that the packing density in the outer leaflet of MDR cells is increased relative to that of the drug-sensitive parent.

Mol Biochem Parasitol, 1993 Aug, 60(2), 195 - 208
Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii; Chow LM et al.; The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases . Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells . The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis . This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L . donovani . The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene . Here we report the identification, cloning and sequencing of an mdr-like gene from L . enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L . enriettii . The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa . The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules . Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L . donovani ldmdr1 gene . The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L . enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

Endocrinology, 1993 Aug, 133(2), 521 - 8
Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells; Zhao JY et al.; Considerable evidence has accumulated indicating that overexpression of P-glycoproteins encoded by the multidrug-resistance (mdr) genes is responsible for the development of collateral resistance to a number of structurally and functionally dissimilar cytotoxic compounds in animal cells . There are three mdr genes (mdr1, mdr2, and mdr3) in the mouse genome and two (MDR1 and MDR2) in the human genome; however, only two mouse genes (mdr1 and mdr3) and one human gene (MDR1) can confer multidrug resistance upon transfection into otherwise drug-sensitive cells . Using RNase protection assay we report here that the steady-state levels of mdr1 and mdr3 messenger RNA were elevated in mouse hepatoma cells treated with dexamethasone (Dex); whereas no induction of mdr2 gene was found . Western blot analyses using anti-mdr1 and anti-mdr3 antibodies revealed that the encoded proteins appeared to be increased, but at much reduced levels . The induction was time and Dex concentration dependent . Nuclear run-on experiments demonstrated that the induction was at least in part by transcriptional control . The induction apparently required new protein synthesis since no increases in mdr1 and mdr3 transcripts was found when cultured cells were simultaneously treated with Dex and cycloheximide . Neither mdr1 nor mdr3 gene was induced in the Dex-treated nonhepatoma cell lines, LMtk- and NIH3T3 . Similarly, MDR1 messenger RNA levels were elevated in the Dex-treated human hepatoma line, HepG2, but not in the nonhepatoma, HeLa . This study demonstrated that the hormonal regulation of mdr gene expression is gene and cell type specific.

J Urol, 1993 Aug, 150(2 Pt 1), 505 - 9
Evaluation of multiple drug resistance in human bladder cancer cell lines; Shinohara N et al.; We evaluated multidrug resistance (MDR) in human bladder cancer cell lines UM-UC-2, UM-UC-6, UM-UC-9 and the UM-UC-6dox subline induced to doxorubicin resistance by in vitro doxorubicin exposure . We compared the profile of multidrug resistance in these cell lines with that of the UM-UC-3 human renal cancer cell line . Of these cell lines, UM-UC-2 was most sensitive to both doxorubicin and etoposide, while UM-UC-6, UM-UC-9 and UM-UC-3 showed 1.5-, 2.1-, and 5.4-fold more resistance to doxorubicin than UM-UC-2 cells . These cell lines were also more resistant to etoposide than UM-UC-2 . Addition of verapamil at 10 microM . reduced the doxorubicin resistance in UM-UC-6 and UM-UC-6dox cells, but UM-UC-9 cells showed little change in doxorubicin sensitivity in the presence of verapamil . In a model of intravesical (short-term) treatment verapamil increased the doxorubicin sensitivity of UM-UC-6dox but not that of UM-UC-6 cells . This effect in UM-UC-6dox cells was enhanced by continuously treating with verapamil after doxorubicin had been removed . Western blot analysis with rabbit anti-human P-glycoprotein polyclonal antibody demonstrated a distinct increase in P-glycoprotein in the resistant cell lines as compared with UM-UC-2 . P-glycoprotein expression was roughly proportional to the degree of resistance to both doxorubicin and etoposide, but did not always correlate with the effect of verapamil on decreasing doxorubicin resistance . These results suggest that multidrug resistance is an important phenomenon in bladder cancer and that more than one pathway of multidrug resistance may be present in human bladder cancer cell lines.

Infect Agents Dis, 1993 Aug, 2(4), 193 - 200
Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance; Cao C et al.; Macrophages possess organic anion transporters that carry membrane-impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxy-fluorescein, from the cytoplasm into endosomes and out of the cells . Probenecid, an organic anion transport inhibitor, blocks these processes . Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells . To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid . When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells . This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity . Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells . To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1) . LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug-resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells . The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical.(ABSTRACT TRUNCATED AT 250 WORDS)

Curr Opin Cell Biol, 1993 Aug, 5(4), 684 - 7
The multidrug resistance P-glycoprotein; Higgins CF; P-glycoprotein plays an important role in the resistance of cancers to chemotherapy . Thus, an understanding of the mechanism by which it functions, and its 'normal' physiological role, is of clinical relevance as well as intrinsic interest . Considerable progress towards this goal has been made in the last year or so . In addition, the finding that P-glycoprotein is associated with both a channel and a transporter activity has, potentially, far-reaching implications for an understanding of the nature of channels and transporters.

J Cell Biochem, 1993 Aug, 52(4), 384 - 95
Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells; Sampson KE et al.; Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells . Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes {Ferrell and Martin: J Biol Chem 264:20723-20729, 1989; Mol Cell Biol 10:3020-3026, 1990} . Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells . The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line . The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720 . The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor . Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr(R) and murine melanoma B16/Adr(R) cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.

Cancer Lett, 1993 Jul 30, 71(1-3), 97 - 102
A marine natural product, patellamide D, reverses multidrug resistance in a human leukemic cell line; Williams AB et al.; A cyclic octapeptide (patellamide D) isolated from the marine tunicate, Lissaclinum patella, acts as a resistance-modifying agent in the multidrug resistant CEM/VLB100 human leukemic cell line . A three-day microculture tetrazolium proliferation assay was used to determine the 50% inhibitory concentration (IC50) for vinblastine, colchicine and adriamycin and calculate the degree of resistance modulation . Patellamide D at 3.3 microM was compared with 5.1 microM verapamil in modulating drug resistance in vitro . The IC50 for vinblastine was reduced from 100 ng/ml to 1.5 ng/ml in the presence of patellamide D or to 2.1 ng/ml when exposed to verapamil . Colchicine cytotoxicity was enhanced only 1.4-fold by verapamil, as compared with 2.8-fold using patellamide D (IC50 was reduced from 140 ng/ml to 100 ng/ml or 50 ng/ml) . Adriamycin toxicity was reduced from IC50 > 1000 ng/ml to 110 ng/ml and 160 ng/ml when coexposed to patellamide D and verapamil, respectively . Our results indicate that patellamide D acts as a selective antagonist in multidrug resistance and stresses the importance of investigating marine-derived compounds as a potential new source for modulators of the drug-resistance phenotype.

J Biol Chem, 1993 Jul 25, 268(21), 16059 - 64
Synthetic and natural opiates interact with P-glycoprotein in multidrug-resistant cells; Callaghan R et al.; The ability of P-glycoprotein (P-gp) to transport naturally occurring and synthetic opiate analgesics was investigated . Multidrug-resistant Chinese hamster ovary cells (B30) were found to accumulate significantly lower amounts of morphine than their drug-sensitive counterparts (B1) . This decreased accumulation was reversed upon depletion of cellular stores of ATP . In addition, morphine bound to plasma membranes from B30 cells in a specific, saturable fashion . Verapamil and vinblastine, compounds transported by P-gp, were able to increase accumulation and displace the binding of morphine in B30 cells . In turn, the synthetic opiates meperidine, pentazocine, and methadone were able to increase the accumulation of vinblastine in resistant cells . These compounds were also able to displace the specific binding of vinblastine and the photoaffinity labeling of P-gp in plasma membranes by the radioiodinated anthracycline, iodomycin . The implications of these findings in relation to the distribution, tolerance, and gastrointestinal side effects of opiates are discussed.

Cancer Res, 1993 Jul 15, 53(14), 3221 - 5
Localization of a novel multidrug resistance-associated gene in the HT1080/DR4 and H69AR human tumor cell lines; Slovak ML et al.; Two doxorubicin-selected human tumor cell lines, H69AR and HT1080/DR4, display a multidrug resistance phenotype but do not overexpress P-glycoprotein . Recently, a 6.5-kilobase mRNA encoding a novel member of the ATP-binding cassette superfamily of transport proteins, designated multidrug resistance-associated protein (MRP), has been identified in the H69AR cell line . In the present study, the levels of MRP mRNA were found to be 14-fold higher in HT1080/DR4 cells relative to sensitive HT1080 cells . Southern blotting indicates that gene amplification contributes to the overexpression of MRP in HT1080/DR4 cells . Using a 4-kilobase MRP complementary DNA probe, MRP genes were localized to 2-5 chromosomes bearing homogeneously staining regions and to multiple double minute chromosomes in H69AR cells . Resistant H69AR cells also contained a new der(16) with a structural aberration affecting 16p13.1, the normal cellular locus of the MRP gene . The MRP probe hybridized to two small homogeneously staining regions (hsr) in HT1080/DR4 cells including hsr(7)(p12p15) . MRP localization was restricted to the normal cellular locus, 16p13.1, in the parental H69 and HT1080 cells and the drug-sensitive H69PR revertant cells . Our data provide combined evidence that amplification of the MRP gene is associated with the expression of drug resistance in selected solid tumor cell lines.

Cancer Res, 1993 Jul 15, 53(14), 3262 - 5
Selective enhancement of vincristine cytotoxicity in multidrug-resistant tumor cells by dilantin (phenytoin)
Ganapathi R, Hercbergs A, Grabowski D, Ford J.
Tumor cell resistance to chemotherapeutic agents of diverse structure and mechanism of action is thought to be due to efflux of drug by P-glycoprotein, which is overexpressed in tumor cells with the multidrug-resistant phenotype . Agents generally associated with the multidrug-resistant phenotype include inhibitors of topoisomerase II, e.g., doxorubicin, etoposide, and the microtubule poisons such as vinblastine, vincristine (VCR), and taxol . The antiepileptic drug phenytoin (DPH), an inhibitor of tubulin polymerization, potentiates (P < 0.05) the cytotoxicity of the chemotherapeutically useful microtubule poison VCR in tumor cells with the wild-type or multidrug-resistant phenotype . Among agents associated with the multidrug-resistant phenotype, the modulation of cytotoxicity by DPH was selectively effective with the microtubule poison VCR but not the topoisomerase II inhibitor doxorubicin . The potentiation of vincristine cytotoxicity by DPH was not due to binding to P-glycoprotein or by increasing VCR accumulation . We thus propose a novel mechanism for the modulation of resistance based on evidence that DPH at noncytotoxic concentrations can selectively enhance the cytotoxic potential of vincristine without interfering with P-glycoprotein function . Thus, studies with phenytoin could assist in characterizing other molecular determinants of multidrug resistance and the design of trials to modulate drug efficacy.

FEBS Lett, 1993 Jul 12, 326(1-3), 302 - 5
Doxorubicin-induced oxygen free radical formation in sensitive and doxorubicin-resistant variants of rat glioblastoma cell lines {corrected and republished erratum originally printed in FEBS Lett 1993 May 17;322(3):295-8}; Benchekroun MN et al.; We have studied the formation of hydroxyl radical (OH.) induced by doxorubicin in a series of doxorubicin- or vincristine-selected variants of C6 rat glioblastoma cells in culture by electron-spin resonance spectroscopy using 5,5'-dimethyl-1-pyrroline-1-oxide as a spin trap . Wild-type cells, sensitive to doxorubicin, exhibited in the presence of this drug a concentration-dependent OH . formation which could be inhibited by preincubation with superoxide dismutase, catalase or an antibody against cytochrome P450-reductase . In highly doxorubicin-resistant cells, OH . formation was reduced to about 20% of the level obtained in sensitive cells . In cells presenting a very low level of resistance to doxorubicin or in cells selected with vincristine, both presenting a pure multidrug-resistant phenotype, OH . formation was identical to that obtained in sensitive cells . In cells of intermediate resistance or in revertant cells, intermediate levels of OH . formation were obtained . Protection against OH . formation and action can be identified at the levels of superoxide dismutase and glutathione peroxidase activities, which are both enhanced in the resistant cells.

FEBS Lett, 1993 Jul 12, 326(1-3), 11 - 6
Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter; Uchiumi T et al.; The human MDR1 gene can be induced in response to various environmental stimuli . To examine whether such stress-induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter . H-7, a protein kinase C inhibitor, at more than 40 microM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5-fluorouracil or UV irradiation . DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H-7 . Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences . Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter . The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.

Int J Cancer, 1993 Jul 9, 54(5), 851 - 7
M-CSF gene transduction in multidrug-resistant human cancer cells to enhance anti-P-glycoprotein antibody-dependent macrophage-mediated cytotoxicity; Heike Y et al.; A human macrophage-colony-stimulating-factor (M-CSF) gene inserted into an expression vector (pRc/CMV-MCSF) was transfected into multidrug-resistant (MDR) human ovarian cancer cells (AD10) to induce secretion of human M-CSF into the medium . The M-CSF level in the culture medium of the transfected cells reached 100 ng/ml after 7 days, and the ability of the cells to secrete M-CSF was stable for at least 3 months . Transfection of the M-CSF gene did not result in any change in expression of MDRI (P-glycoprotein), proliferation or chemosensitivity of the cells from those of the parent cells . There was also no difference between the transfected and the parent cells in susceptibility to NK cell- or interleukin-2-activated killer-cell-mediated cytotoxicity . Human blood monocytes that had been incubated for 4 days in medium with the culture supernatant of MH-AD10 cells exhibited higher ADCC activity than untreated monocytes against MDRI-positive cancer cells . This effect of the supernatant of AD10 cells was completely abolished by its treatment with a monoclonal anti-M-CSF antibody (MAb) . When transfected human MDR cells were injected into nude mice, an inverse correlation was seen between the ability of the cells to produce M-CSF and their tumorigenicity . Thus, gene modification of MDR cancer cells seems hopeful as a therapeutic method for enhancing anti-MDRI-MAb-dependent macrophage-mediated cytotoxicity against human MDR cancer cells.

JAMA, 1993 Jul 7, 270(1), 65 - 8
Pitfalls in the care of patients with tuberculosis . Common errors and their association with the acquisition of drug resistance; Mahmoudi A et al.; OBJECTIVE--To determine, among a group of patients with multidrug-resistant pulmonary tuberculosis, whether there had been management practices that deviated from established guidelines, and whether these decisions were associated with the acquisition of multidrug resistance and adverse medical sequelae . DESIGN--Case series . SETTING--Referral center . PATIENTS--All patients with pulmonary tuberculosis admitted to the National Jewish Center for Immunology and Respiratory Medicine in 1989 through 1990 . INTERVENTIONS--The records of all patients referred to this institution for the treatment of tuberculosis in 1989 through 1990 were reviewed to ascertain the nature of management decisions that might have been associated with the acquisition of drug resistance . MAIN OUTCOME MEASURES--Standards of practice as defined by the American Thoracic Society, the Centers for Disease Control and Prevention, and the American College of Chest Physicians were compared with these management decisions to determine whether "errors" had been made, resulting in treatment failure and the development of acquired drug resistance . RESULTS--Among the 35 study patients, errors were detected in the management decisions in 28; there was an average of 3.93 errors per patient . The most common errors were the addition of a single drug to a failing regimen, failure to identify preexisting or acquired drug resistance, initiation of an inadequate primary regimen, failure to identify and address noncompliance, and inappropriate isoniazid preventive therapy . The multidrug resistance acquired through the errors resulted in prolonged hospitalizations, treatment with more toxic drugs, and high-risk resectional surgery . The costs for this "salvage therapy" were extraordinary, averaging $180,000 per patient . CONCLUSIONS--Aggressive professional education, tighter control on the provisions of care for tuberculosis patients, and the committing of additional resources to tuberculosis control programs are vital in improving the care of tuberculosis patients and limiting the development of acquired drug resistance.

Br J Cancer, 1993 Jul, 68(1), 74 - 9
Characterisation of a vindesine-resistant human small-cell lung cancer cell line; Ohta S et al.; We established a vindesine-resistant (x 11.6) human small-cell lung cancer cell line (H69/VDS) by stepwise exposure of parent line H69 to vindesine . H69/VDS showed cross-resistance to taxol (x 10.1), vincristine (x 6.9) and colchicine (x 3.4) but not to doxorubicin, cisplatin or etoposide . There was no significant difference in intracellular {3H}-vincristine and doxorubicin accumulation between H69 and H69/VDS cells . The human mdr1 mRNA was not detected in either of the cell lines . These results indicated that H69/VDS did not express a typical multidrug resistant phenotype . Addition of 20 microM verapamil enhanced the growth inhibitory effect of vindesine on both H69/VDS (x 12.0) and H69 cells (x 3.8) . The amount of total tubulin in H69/VDS cells was lower than that in the H69 parental cells . No significant increase was observed in the amount of total and polymerised tubulins of H69 cells . In H69/VDS cells, however, verapamil increased the amount of total tubulin to the level of parental cells, but decreased the amount of polymerised tubulin . Modulation of tubulin may play a role in the resistance to vindesine.

Jpn J Cancer Res, 1993 Jul, 84(7), 776 - 82
Expression behavior of 85-kDa membrane protein in adriamycin-resistant tumor cells and the inhibition of human tumor growth in athymic mice by MRK-20 monoclonal antibody against the protein; Ishii S et al.; Treatment of tumors with monoclonal antibodies against tumor antigen is one of the selective modalities for cancer therapy . We examined the therapeutic effect of MRK-20 (IgG1), a murine monoclonal antibody against resistance-associated 85-kDa membrane protein . The 85-kDa protein is expressed on the surface of multidrug-resistant cells induced by adriamycin . This protein is also expressed in some multiple-drug-resistant cells, including atypical multidrug-resistant cells . The protein, once lost during long-term culture without adriamycin, was rapidly induced by treatment with adriamycin but not with vinblastine or etoposide, suggesting a close relationship of the protein with adriamycin resistance but not with multidrug resistance . The antibody MRK-20 suppressed the growth of subcutaneously implanted tumors expressing the 85-kDa protein . Adriamycin-resistant human ovarian tumor 2780AD and innately resistant human erythroleukemia HEL cells in athymic mice were completely cured by treatment with MRK-20 antibody when the antibody was administered i.v . 2 days after s.c . tumor implantation . On the other hand, MRK-20 did not show any effect on the growth of the 85-kDa protein-negative A2780 human ovarian tumor . These results indicate that the effect of MRK-20 is highly specific to cells expressing 85-kDa protein.

Anticancer Res, 1993 Jul-Aug, 13(4), 985 - 90
Effect of duration of exposure to S9788, cyclosporin A or verapamil on sensitivity of multidrug resistant cells to vincristine or doxorubicin; Perez V et al.; In order to explain the high potency of S9788, a new multidrug resistance modifier currently in clinical development, we investigated its accumulation and retention in sensitive and resistant cells . Our results show that S9788 is 13-fold more accumulated and 23-fold more retained than VRP in the resistant S1/tMDR cell line . We also studied the effect of duration of incubation on the ability of S9788, verapamil and cyclosporin A to overcome the resistance of S1/tMDR and KB-A1 cells to a short exposure of doxorubicin or vincristine . Compared to a single co-incubation of 4 h, a 24 h post-incubation with 5 microM S9788 markedly increased the reversal of S1/tMDR resistance to VCR (fold reversal 4 h = 123; fold reversal 24 h = 4739), while it moderately increased S1/tMDR sensitivity to DOX (fold reversal 4 h = 1.9; fold reversal 24 h = 2.9) . Similar results were obtained on KB-A1 resistance to VCR (fold reversal 4 h = 41, fold reversal 24 h = 21819) and KB-A1 resistance to DOX (fold reversal 4 h = 89; fold reversal 24 h = 160) . This phenomenon also occurred with verapamil and cyclosporin A . These results clearly show that the effect of duration of exposure on the modulating activity of S9788 and of the 2 other modulators depends on the cytotoxic drug . Although the direct transposition of these results to a clinical situation is difficult, they suggest that a continuous infusion of S9788, starting simultaneously with the administration of the cytotoxic drug and ending 24 h later, might be a more effective schedule for clinical administration than a bolus administration.

Anticancer Res, 1993 Jul-Aug, 13(4), 959 - 61
The reversal of multidrug resistance in cancer (review); Kellen JA; A brief review of the "state of the art" (1992) knowledge concerning various strategies and tactics to overcome multidrug resistance is presented . Multidrug resistance remains the main obstacle to long-term successful cancer chemotherapy; its prevention or elimination is probably one of the most promising avenues in cancer research.

Am J Trop Med Hyg, 1993 Jul, 49(1), 121 - 6
PS-15: a potent, orally active antimalarial from a new class of folic acid antagonists; Canfield CJ et al.; A new, orally-active inhibitor of dihydrofolic acid reductase (DHFR), PS-15 (N-(3-(2,4,5-trichlorophenoxy)propyloxy)-N'-(1-methylethyl)- imidocarbonimidic diamide hydrochloride), has significant activity against drug-resistant Plasmodium falciparum . It is not cross-resistant with other inhibitors of DHFR (e.g., pyrimethamine and cycloguanil) . Although it bears similarities to proguanil, PS-15 represents a new antifolate class of drugs that we have named oxyguanils or hydroxylamine-derived biguanides . This compound displays intrinsic antimalarial activity and also is metabolized in vivo to WR99210, an extremely active triazine inhibitor of DHFR . When tested in vitro against drug-resistant clones of P . falciparum, PS-15 was more active than proguanil, and the putative metabolite, WR99210, was more active than the proguanil metabolite cycloguanil . The drug is also more active as well as less toxic than proguanil when administered orally to mice infected with P . berghei . When administered orally to Aotus monkeys infected with multidrug-resistant P . falciparum, PS-15 was more active than either proguanil or WR99210 . In 1973, WR99210 underwent clinical trials for safety and tolerance in volunteers . The trials showed gastrointestinal intolerance and limited bioavailability; further development of the drug was abandoned . Because PS-15 has intrinsic antimalarial activity, is not cross-resistant with other DHFR inhibitors, and can be metabolized to WR99210 in vivo, oral administration of this new drug should circumvent the shortcomings and retain the advantages found with both proguanil and WR99210.

J Bacteriol, 1993 Jul, 175(14), 4364 - 74
The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat; Shrader TE et al.; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . Distinct versions of the N-end rule operate in bacteria, fungi, and mammals . We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway . L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine . The aat gene maps to the 19-min region of the E . coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases . In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine . However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant . Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro . The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp . Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed . The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins.

Br J Cancer, 1993 Jul, 68(1), 8 - 9
Pharmacokinetic interaction between epirubicin and the multidrug resistance reverting agent D-verapamil; Scheithauer W et al.; The potential for a pharmacokinetic interaction between epirubicin and the second-generation multidrug resistance modulating agent D-verapamil (DVPM) has been investigated in six patients with advanced colorectal cancer . Our results indicate that a significant interaction takes place . Enhanced distribution of epirubicin from the serum and altered disposition might, in fact, explain the increased level of myelotoxicity in this pilot as well as in other clinical phase II studies involving DVPM.

Biochem Mol Biol Int, 1993 Jul, 30(4), 743 - 53
Vinblastine transport by membrane vesicles from human multidrug-resistant CCRF-CEM leukaemia cells: inhibition by taxol and membrane permeabilising agents; Syed SK et al.; Inside-out membrane vesicles prepared from multidrug resistant human leukemic cells (CEM/VBL1000), but not from sensitive cells, transported {3H}-labelled vinblastine (VBL) in an ATP-dependent manner, reaching a plateau level by 15 min . The transport occurred with an apparent Km of 60 +/- 20nM . Verapamil (10 microM), and taxol (IC50 = 1 microM) prevented VBL uptake and evoked VBL diffusion from vesicles when added after VBL uptake had reached steady state . The channel forming agent alamethicin prevented net uptake of VBL and addition of alamethicin to the vesicles after the steady-state had been reached resulted in the rapid efflux of {3H}VBL . Very low concentrations of Triton X-100 (0.01 % v/v) also prevented net uptake of VBL, whilst addition of Triton X-100 and making the medium hypo-osmotic after the steady state had been reached caused the {3H}VBL to rapidly diffuse out of the vesicles . These observations indicate that VBL is actively transported into the lumen of inside-out vesicles from multidrug resistant leukaemia cells.

Cell Mol Biol (Noisy-le-grand), 1993 Jul, 39(5), 543 - 51
Scanning electron microscopy of multidrug resistant cells in haematological and mammary malignancies; Galimberti S et al.; Multidrug resistance (MDR) is frequently found in haematologic malignancies . It has been shown that MDR is often related to the expression of a membrane glycoprotein (P-170) which actively pumps many hydrophobic agents out of the cells . Previous electron microscopic investigations revealed morphological differences between P-388 resistant and P-388 sensible cell membranes, but the modulation of the membrane morphology seems to be related to the tumor-cell environment . In order to establish if morphological differences exist between sensitive and resistant cells, both sensitive and resistant strains from three different cell lines were studied by scanning electron microscopy: human leukaemia CEM and vinblastine resistant cells (CEM/VBL100), human breast cancer MCF-7 and mice leukaemia P-388 with the doxorubicin resistant strains (MCF-7/DX and P-388/DX, respectively) . The surface of the membranes of the sensitive cells was regular, unlike the resistant ones which proved to be irregular, endowed with long villus-like processes or numerous folds and ruffles . The addition of albumin to the culture medium induced a shift from the resistant to the sensitive phenotype, thus suggesting that the P-388/DX morphology may be linked to the concentration of protein in the culture medium . Exposure to DX, verapamil (VRP) or monoclonal antibody against P-170 (mAb-57) did not modify the surface of the resistant strains, demonstrating that surface irregularities are probably not linked to P-glycoprotein function . Blasts from four P-170 positive leukaemic patients were also analyzed: an irregular shape was always found.

Mol Biochem Parasitol, 1993 Jul, 60(1), 53 - 64
Molecular characterization of the ldmdr1 multidrug resistance gene from Leishmania donovani; Hendrickson N et al.; The ldmdr1 gene that confers resistance to multiple structurally dissimilar hydrophobic drugs in Leishmania donovani has been isolated within a 5.4-kb XmaI fragment from a genomic library of L . donovani DNA and its protein coding region sequenced . The longest open reading frame within ldmdr1 encodes a 146.5-kDa protein of 1341 amino acids, designated LDMDR1 . The primary structure and predicted membrane topology of LDMDR1 indicates that it is a member of the P-glycoprotein superfamily with the greatest homology to the mammalian multidrug resistance P-glycoproteins . A 2.3-kb SalI fragment derived from a second ldmdr1 allele was also cloned from the L . donovani library . Nucleotide sequence analysis of a portion of the SalI insert revealed 5 single base differences from its counterpart within the 5.4-kb XmaI fragment, one of which created a PvuI restriction site polymorphism . Southern blots of PvuI-digested DNA divulged that the amplified ldmdr1 gene copies in a multidrug-resistant L . donovani strain were all derived from the single ldmdr1 allele whose protein coding segment was sequenced in its entirety.

Anticancer Res, 1993 Jul-Aug, 13(4), 905 - 8
Reversal of inherent multidrug-resistance in primary human renal cell carcinoma cell cultures by S 9788; Efferth T et al.; In a panel of 14 primary cultures of human renal cell carcinomas the reversal of inherent multidrug-resistance by S 9788, a new triazinoaminopiperidine derivative, was analysed . In combination with doxorubicin S 9788 revealed an evident reversal of multidrug resistance in 12 cell cultures . Two cell cultures remained unaffected . An association between reversal and P-glycoprotein (P-170) expression suggests that S 9788 exerts its activity through P-glycoprotein.

Leukemia, 1993 Jul, 7(7), 963 - 9
High expression of the multidrug resistance P-glycoprotein in high-risk myelodysplasia is associated with immature phenotype; Sonneveld P et al.; The expression of the multidrug resistance (MDR-1) gene product, P-170 glycoprotein (P-170) was investigated in 26 patients with low-risk (n = 9) or high-risk (n = 17) myelodysplastic syndrome (MDS), using a panel of monoclonal antibodies to P-170 (C219, JSB1, C494, MRK16) and quantitative analysis of MDR-1 mRNA . P-170 membrane staining was demonstrated in bone marrow blast cells of 14/17 HR-MDS and in 2/9 LR-MDS patients (p < 0.01) . P-170 expression was associated with the presence of blast cells characterized by an immature or early myeloid phenotype as defined by CD34 expression (p = 0.034), CD13 or CD33 expression (p = 0.0006), or CD13/33 plus terminal deoxynucleotidyl transferase (TdT) double expression (p = 0.04) . With double fluorescence analysis, P-170 expression was observed in a subset of CD34+ cells, but not in CD34- cells . P-170 expression was present in 13/15 (86%) patient samples with an abnormal karyotype as compared with 3/10 samples (30%) with a normal karyotype (p < 0.05) . Nine of these 15 patients had a loss or a deletion of chromosome 7 . Thirteen out of 16 (81%) MDR-1 positive patients developed acute leukemia versus two of ten (20%) MDR-1 negative patients (p = 0.025) . It is concluded that MDR-1 expression in MDS is present in cells with an immature phenotype and is frequently observed in patients who have an abnormal karyotype and a high risk of leukemic transformation.

Cancer Res, 1993 Jul 1, 53(13), 3040 - 5
Modulatory effects of tamoxifen and recombinant human alpha-interferon on doxorubicin resistance; Kang Y et al.; Doxorubicin (DOX) resistance is frequently due to the multidrug resistance gene product P-glycoprotein . This study examined the effects of two biochemical modulators, recombinant human alpha-interferon (IFN-alpha) and tamoxifen (TAM), on the DOX sensitivity, DOX retention, and P-glycoprotein expression of the multidrug-resistant Chinese hamster ovary cell line ChR C5 and the parent AuX B1 cell line . In the absence of either modulator, the 50% inhibitory concentration for DOX after 1-h incubation as determined using a microculture tetrazolium assay was 8.3 microM in ChR C5 cells and 0.4 microM in AuX B1 cells . In ChR C5 cells, IFN-alpha (500 units/ml) for 24 h had no affect on DOX cytotoxicity, but tamoxifen (1.0 microM) for 24 h enhanced DOX cytotoxicity with the 50% inhibitory concentration decreased by 2-fold to 4.2 microM . A combination of IFN-alpha (500 units/ml) for the initial 24 h followed by TAM (1.0 microM) for another 24 h was even more effective in ChR C5 cells with the DOX 50% inhibitory concentration decreased by 4-fold to 2.1 microM . The combination IFN-alpha and TAM dramatically increased DOX accumulation in the resistant ChR C5 cells without significantly affecting P-glycoprotein expression as measured using flow cytometric analysis . IFN-alpha and/or TAM had no effect on DOX cytotoxicity or accumulation in parent DOX-sensitive AuX B1 cells . Both cell lines were estrogen and progesterone receptor negative . These data indicate that synergism between IFN-alpha and TAM may partially reverse DOX resistance and may potentially be useful in enhancing the clinical effectiveness of DOX.

Arch Otolaryngol Head Neck Surg, 1993 Jul, 119(7), 753 - 7
Detection of P-glycoprotein with four monoclonal antibodies in normal and tumor tissues; Pavelic ZP et al.; P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1), is an active efflux pump for many structurally diverse lipophilic compounds . Using peroxidase-antiperoxidase immunohistochemistry technique and four anti-Pgp monoclonal antibodies directed against different epitopes of the molecule, we examined the distribution of Pgp in normal human tissues and squamous cell carcinomas of the head and neck . All four antibodies detected Pgp in bronchial cells, mammary ductal epithelium, gallbladder epithelium, epithelia of small and large intestine, bile canaliculi, dermal sweat glands, proximal tubules of kidney, endometrium, trophoblasts, adrenal gland, and capillaries of central nervous system, testis, and papillary dermis . Of the 23 head and neck squamous cell carcinomas, about 60% had detectable Pgp . It is possible that differences noticed between antibodies are due to cross-reactivity to proteins unrelated to MDR1 . Care must be taken in interpreting staining results when only one or two monoclonal antibodies are used.

Oncology, 1993 Jul-Aug, 50(4), 303 - 8
Modulation of P-glycoprotein-mediated multidrug resistance by monoclonal antibodies, immunotoxins or antisense oligodeoxynucleotides in kidney carcinoma and normal kidney cells; Efferth T et al.; In the present investigation we show that both monoclonal antibodies against P-170 (265/F4, MRK 16, and HYB 612) and 265/F4-ricin alpha-chain immunotoxin are useful tools for the eradication of kidney carcinoma and normal kidney primary cell cultures with high P-170 expression, whereas kidney tumor and normal kidney cell lines with low amounts of P-170 are less affected . Furthermore, we found that the expression of P-170 in both kidney tumor and normal kidney cell cultures with high amounts of P-170 was inhibited by antisense oligodeoxynucleotides complementary to base pairs -9 to +6 of the MDR1 gene . These data indicate that these approaches for the eradication of P-170 expressing multidrug-resistant cells are not limited to tumors but also affect P-170 expressing normal cells.

Jpn J Cancer Res, 1993 Jul, 84(7), 703 - 7
Relationships among tenascin expression, DNA ploidy patterns, and multidrug resistance gene product (P-glycoprotein) in human colon carcinoma; Sugawara I et al.; Relationships among tenascin expression, DNA ploidy, and P-glycoprotein were examined in 81 primary human colon cancers and 61 metastatic lymph nodes . First, the DNA ploidy patterns of colon cancerous tissue surrounded (TN+) and not surrounded (TN-) by tenascin immunoreactivity were investigated . Then the expression of P-glycoprotein, one of two multidrug resistance gene products, was examined in TN+ and TN- colon cancer tissues by immunohistochemistry . Aneuploid DNA patterns were observed at high frequency in TN- colon cancer tissues (37/61) and metastatic lymph nodes (44/52) . In contrast, diploid DNA patterns were observed predominantly in TN+ colon cancer tissues (50/56) . Although P-glycoprotein expression was observed in primary TN+ and TN- colon cancer (9/81), the level of P-glycoprotein expression was not correlated with DNA aneuploidy in TN- colon cancer tissues . Overall, reduced tenascin expression was correlated well with DNA aneuploidy, but no significant correlation was found between DNA aneuploidy and P-glycoprotein appearing when cancer cells become resistant to several anti-cancer drugs . Thus, tenascin may play an important role in preventing colon cancer cells from invading surrounding tissues.

Am J Physiol, 1993 Jul, 265(1 Pt 1), L27 - 32
5'-Adenylylimidodiphosphate does not activate CFTR chloride channels in cell-free patches of membrane; Carson MR et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel requires both phosphorylation of its R domain and the presence of nucleoside triphosphates for activation . Our previous work suggested that hydrolysis of nucleoside triphosphates may be required to support channel activity . However, recent studies have suggested that the nonhydrolyzable adenosine triphosphate analogue, 5'-adenylylimidodiphosphate (AMP-PNP), may support some Cl- channel activity in sweat gland duct epithelia in the presence of low ATP concentration and in Cl- channels associated with expression of the P-glycoprotein multidrug resistance transporter . To examine the effect of AMP-PNP, we applied it to the cytosolic surface of phosphorylated CFTR Cl- channels contained in excised, cell-free patches of membrane . We found that preparations of 10 mM AMP-PNP opened phosphorylated CFTR Cl- channels . However, this effect was due to contaminating ATP: high-pressure liquid chromatography analysis of AMP-PNP demonstrated that 10 mM AMP-PNP could contain up to 50 microM ATP, which could account for the observed stimulation of CFTR Cl- channel activity . When contaminating ATP was hydrolyzed with hexokinase, AMP-PNP was unable to support CFTR channel activity . AMP-PNP (10 mM) also failed to attenuate or potentiate the current induced by 0.3 mM ATP . These results suggest that AMP-PNP has no direct effect on CFTR Cl- channels.

Biochemistry, 1993 Jun 29, 32(25), 6470 - 6
The nitrogen of the acetamido group of colchicine modulates P-glycoprotein-mediated multidrug resistance; Tang-Wai DF et al.; The substituents of drug molecules and the specific amino acid residues of P-glycoprotein (P-gp) implicated in drug/protein interactions are largely unknown . We have used a series of colchicine analogs modified on the A, B, and C rings to identify the discrete chemical groups on the colchicine molecule that are required for recognition by P-gp . For this, the toxicity of these analogs was tested on independent cell clones expressing either of the two mouse mdr genes, mdr1 and mdr3, known to confer multidrug resistance . Modifications of the methoxy groups on the A and C rings modulated cellular toxicity but had no effect on P-gp recognition; however, modifications at the C7 position of the B ring, in particular the removal of the nitrogen atom of the acetamido group, had a dramatic effect . Analogs bearing a hydrogen at that position were not substrates for P-gp . The importance of the nitrogen at C7 was independently verified in thiocolchicine and allocolchicine analogs similarly modified, although overall levels of resistance to these compounds were somewhat reduced compared to their colchicine counterparts . The study of allocolchicine congeners bearing a six-carbon C ring and of two other analogs completely lacking a B ring suggested that intact B and C rings were important for interaction with P-gp . These results suggest that the structural determinants for cytotoxicity (tubulin binding) and P-gp recognition map to nonoverlapping sites in the colchicine analogs analyzed . Examination of calculated molar refractivities (CMR) revealed that only compounds showing CMR values greater than 9.7 were P-gp substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Cancer, 1993 Jun 19, 54(4), 663 - 8
Characterization and chemosensitivity of a human epithelioid sarcoma cell line (SARCCR 2); Roche H et al.; A new cell line was derived from the epithelioid sarcoma of a Caucasian woman who had previously received chemotherapy . The cells grew as an adherent monolayer, with a doubling time of 28 hr and had mainly epithelial morphology, but with areas of mesenchymal-like cytoplasmic extensions . The cells were tumorigenic in nude mice, with a short growth time, and a doubling time of 8 days . The cell line showed over-expression of P-glycoprotein by Western blot analysis, and its sensitivity to doxorubicin and vincristine was low . This sensitivity could be enhanced by reversants of multidrug resistance (MDR), such as cyclosporin or verapamil . This cell line constitutes an excellent model for studying compounds able to reverse MDR.

J Biol Chem, 1993 Jun 15, 268(17), 12267 - 73
Defective translocation of protein kinase C in multidrug-resistant HL-60 cells confers a reversible loss of phorbol ester-induced monocytic differentiation; Slapak CA et al.; Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters . This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype . The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation . The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation . By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene . Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine . The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation . Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line . Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction . Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells . Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid . Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.

Eur J Pharmacol, 1993 Jun 15, 246(1), 53 - 8
Reversal of multidrug resistance in Chinese hamster ovary cells by the immunosuppressive agent rapamycin; Hoof T et al.; The ability of cyclosporin A, FK506, and rapamycin to overcome multidrug resistance was investigated in Chinese hamster ovary cells in vitro by growth inhibition experiments . 1-30 microM of immunosuppressant sensitized drug-resistant cells and their drug-sensitive parents in a dose-dependent manner to adriamycin (2-2000-fold), colchicine (2-260-fold), and vinblastine (2-120-fold) . The multidrug resistance-reversing activity increased in the order rapamycin < FK506 < cyclosporin A, irrespective of whether the resistant cells overexpressed hamster or human P-glycoprotein . The interaction of the three macrolides with P-glycoprotein was characterized by their ability to competitively inhibit the photoaffinity labelling of plasma membranes of resistant CHRB30 cells by iodomycin . The three immunosuppressants bound with high affinity to P-glycoprotein.

MMWR Morb Mortal Wkly Rep, 1993 Jun 11, 42(22), 427, 433 - 4
Outbreak of multidrug-resistant tuberculosis at a hospital--New York City, 1991; P-glycoprotein-mediated transcellular transport of MDR-reversing agents; Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University Hospital, Faculty of Medicine, Kyoto University, JapanUnderstanding of the interactions between P-glycoprotein and multidrug resistance (MDR) reversing agents is important in designing more effective MDR modulators . We examined transcellular transport of several MDR modulators by using a drug-sensitive epithelial cell line, LLC-PK1, and its transformant cell line, LLC-GA5-COL300, which expresses human P-glycoprotein on the apical surface . Basal-to-apical transports of azidopine and diltiazem across the LLC-GA5-COL300 monolayer were increased and apical-to-basal transports were decreased compared to those across the LLC-PK1 monolayer, indicating that P-glycoprotein transports azidopine and diltiazem . Movements of nitrendipine and staurosporine across the epithelial monolayer were not affected by P-glycoprotein . These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transport process.

Biochim Biophys Acta, 1993 Jun 6, 1177(2), 117 - 26
Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation; McClean S et al.; Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody . Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi . The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

J Natl Cancer Inst, 1993 Jun 2, 85(11), 897 - 901
Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations; Tsai CM et al.; BACKGROUND: At diagnosis, most small-cell lung cancers (SCLCs) are chemosensitive, whereas non-small-cell lung cancers (NSCLCs) are usually chemoresistant . Activation of ras genes and HER-2/neu genes (also known as ERBB2) is encountered in subpopulations of NSCLC but not in SCLC and has been linked to shortened survival . Therefore, activation of these genes may be associated with intrinsic chemoresistance in NSCLC . Studies have also suggested that the multidrug-resistant phenotype expressed by the MDR1 gene (also known as PGY1) does not correlate with the in vitro chemosensitivity of NSCLC cells or with clinical response to therapy and does not explain the spectrum of cross-resistance to drugs . PURPOSE: The purpose of this study was to investigate the relationships between chemoresistance and the presence of ras gene point mutations and overexpression of the HER-2/neu gene in NSCLC cell lines, which indicates gene activation . METHODS: Using a panel of 20 NSCLC cell lines established from untreated patients, we assessed the differences in HER-2/neu messenger RNA (mRNA) expression in the cell lines with or without ras mutations . We performed in vitro drug sensitivity testing by the tetrazolium-based MTT {i.e., 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide} assay with doxorubicin, carmustine, cisplatin, melphalan, mitomycin, and etoposide, and we determined the differences in IC50 values (i.e., the drug concentrations required to inhibit cell growth by 50%) for the cell lines . RESULTS: We found a statistically significant correlation between the IC50 values for all six drugs and the degree of HER-2/neu gene expression in all 20 cell lines (r = .67-.86; P < .005) as well as in the subpopulation of eight cell lines with ras mutations (r = .83-.98; P < .05) . The IC50 values for doxorubicin, carmustine, cisplatin, and melphalan were not significantly different in the cell lines with or without ras mutations, but the values for mitomycin and etoposide in lines with ras mutations were slightly lower than in those without ras mutations (borderline significance, P = .031) . Levels of HER-2/neu expression in cell lines with ras mutations were lower than those without ras mutations, but the difference was not statistically significant . CONCLUSION: Our findings indicate that overexpression of HER-2/neu is a marker for intrinsic multidrug resistance in NSCLC cell lines . IMPLICATIONS: If the clinical relevance of our findings is confirmed, HER-2/neu gene expression can be used as a predictor of therapeutic failure in NSCLCs . The relationships between HER-2/neu gene expression, cell proliferation, and chemoresistance in NSCLC require further investigation.

J Bacteriol, 1993 Jun, 175(12), 3723 - 9
Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB; Furukawa H et al.; Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step . Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics . Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin . TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition . The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene . Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains . Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.

J Hepatol, 1993 Jun, 18(2), 168 - 72
MDR1 (multidrug resistance) gene expression in human primary liver cancer and cirrhosis; Chenivesse X et al.; MDR1 RNA levels were analysed in patients with hepatocellular carcinoma (HCC), benign liver tumors or cirrhosis . None of the patients with HCC had received chemotherapy . MDR1 RNA levels were increased relative to a normal liver specimen in 15/26 liver cancers, 2/6 benign liver tumors and 0/8 tumor-free cirrhotic livers . MDR1 was also overexpressed in cirrhotic non-tumorous liver tissues of some patients with HCC . The results indicate that the MDR1 gene is overexpressed in liver tumors and some preneoplastic lesions and that might account for the very poor response of primary liver cancers to chemotherapy.

Mol Microbiol, 1993 Jun, 8(6), 1163 - 75
ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB; Koronakis V et al.; The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria . HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains . Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity . GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro . The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein . Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies . Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.

Southeast Asian J Trop Med Public Health, 1993 Jun, 24(2), 216 - 20
Economic analysis of malaria control for migrant workers in eastern Thailand; Kamolratanakul P et al.; A randomized, double-blind field trial was carried out to compare the economic impact of permethrin-treated nets with that of untreated nets as a method of malaria control . The study was conducted in 261 long-term migrant workers and 138 seasonal agricultural migrant workers in the eastern rural areas known to be highly endemic for multidrug-resistant Plasmodium falciparum infection . One hundred and twenty-six longterm migrants and 59 seasonal migrants used treated nets, while 135 and 79, respectively, used untreated nets . The impregnated-nets program was cost-saving as well as offering improved effectiveness . The net benefit of using a treated net was US$1.17 per worker from the Malaria Division's perspective and US$1.61 per worker from the worker viewpoint . The use of impregnated nets with large-scale primary health care programs likely will be the most cost-effective and cost-beneficial method for controlling malaria in eastern Thailand.

Trends Microbiol, 1993 Jun, 1(3), 109 - 13
Molecular mechanisms of isoniazid: a drug at the front line of tuberculosis control; Zhang Y et al.; The resurgence of tuberculosis and emergence of multidrug-resistant isolates has focused attention on the need for an improved understanding of molecular aspects of the disease, and for elucidation of the factors responsible for drug action and resistance . Recent research has probed the mechanism of action of isoniazid (INH), a key drug in the chemotherapy of tuberculosis.

J Surg Res, 1993 Jun, 54(6), 621 - 4
MDR-1 expression in metastatic malignant melanoma; Levine EA et al.; Metastatic malignant melanoma (MMM) carries an ominous prognosis . This poor prognosis is due in part to the remarkable resistance of MMM to a wide variety of antineoplastic agents . One common mechanism of resistance to chemotherapy is that of multidrug resistance (MDR) which is mediated by the MDR-1 gene . Previous efforts to determine MDR-1 expression in MMM have all failed to show expression using relatively insensitive immunohistochemical techniques . The purpose of this study is to evaluate the incidence of MDR-1 expression in MMM using a highly sensitive polymerase chain reaction (PCR)-based assay . Twenty-two clinical snap-frozen MMM specimens were analyzed . Total cellular RNA was extracted and quantitated from which cDNA was synthesized . Sequences of MDR-1 and beta 2-microglobulin (internal control) cDNA were coamplified using PCR . The relative yield of the MDR-1-specific PCR product was quantitated relative to that of a standard series of cell lines with known MDR-1 expression . Analysis revealed that 45% of MMM expressed MDR-1 at low levels . None of the tumors expressed high levels of MDR-1 . Overall survival, disease-free survival, thickness of primary lesion, histology, and sex were not found to correlate with MDR-1 expression . We conclude that MDR-1 is frequently expressed at low levels in MMM . However, this incidence of MDR-1 expression suggests that non-MDR mechanisms of drug resistance are dominant in MMM . Further studies will be required to determine if MDR-1 expression is of clinical significance in MMM.

Neth J Med, 1993 Jun, 42(5-6), 218 - 31
Multidrug resistance mediated by P-glycoprotein in haematological malignancies; Pasman PC et al.; The phenomenon that a tumour, resistant to a cytotoxic drug, is also resistant to a large number of other drugs used in cancer chemotherapy, is called multidrug resistance . A transmembrane protein, known as P-glycoprotein (P-gp), is involved in this resistance pattern . P-gp is able to pump large, lipophilic molecules out of the cell . 'Naturally occurring' drugs such as the anthracyclines and the Vinca alkaloids meet these criteria . To study the future clinical implications of multidrug resistance, we have gathered data in the literature on the presence of P-gp in haematological malignancies . At diagnosis 14-62% of all patients showed P-gp expression . Of previously treated patients 29-62% was positive for P-gp . A slight tendency to find a higher frequency of P-gp positivity in these previously treated patients was observed (so-called 'acquired resistance') . Early mutation and selection by the cytotoxic drug could account for the higher levels in treated patients . Chemotherapy itself could induce the expression of the P-gp pump . With the use of in vitro work various pharmacological agents have been found that can antagonize P-gp's function . Using these agents in clinical trials, some refractory patients showed a response to chemotherapy . We conclude that P-gp is probably just one of many causes of drug resistance in patients with haematological malignancies . Clinical results in some studies look promising, but many problems have still to be solved before common use.

Anticancer Drugs, 1993 Jun, 4(3), 395 - 406
Differential reversal of lipophilic antifolate resistance in mammalian cells with modulators of the multidrug resistance phenotype; Assaraf YG et al.; Chinese hamster ovary (CHO) T19 cells express a stable P-glycoprotein (P-170)-dependent multidrug resistance (MDR) phenotype and display a 24- to 29-fold cross-resistance to the lipophilic antifolates piritrexim (PTX) and trimetrexate (TMTX) . We have examined the ability of various modulators of the MDR phenotype to sensitize T19 cells to TMTX and PTX in a clonogenic assay . An almost complete reversal of TMTX resistance in T19 cells was achieved with several modulators of the MDR phenotype whereas only a partial sensitization of T19 cells to PTX was obtained with the most potent modulator . In an attempt to explore the apparent P-170-independent locus of protection against PTX, resistant T19 sublines were isolated after stepwise selection with PTX and TMTX . Thus, T19 cells made resistant to PTX displayed a dramatic decrease in P-170 mRNA levels despite the maintenance of the parental T19 MDR gene amplification, whereas T19 cells selected for TMTX resistance exhibited a further increase in P-170 mRNA levels . Hence, the modulation experiments together with the established lipophilic antifolate-resistant T19 variants suggest that although T19 cells possess a P-170-dependent MDR phenotype and display a similar cross-resistance to TMTX and PTX, the protective pathway need not be necessarily via P-170 . Rather, a pathway appears to exist that protects T19 MDR cells from the cytotoxicity of PTX without requiring a P-170 function.

Eur J Pharmacol, 1993 Jun 1, 248(1), 49 - 58
Vincristine cellular pharmacokinetic changes associated with multidrug resistance; Domenech C et al.; The correlation between vincristine cellular pharmacokinetics and its biological action was analyzed in L5178Y cells and in a multidrug resistant subline (VCR/G40) at the nM drug concentration range attained in serum during the clinical use of the drug . The reduced rate of drug influx and the higher drug efflux measured in VCR/G40 cells justify the lower drug accumulation characterized in these cells compared to the parental cells . Nevertheless this does not seem to be the sole reason accounting for the resistance expression since similar cytotoxic effects of vincristine on both cell lines were only attained when the intracellular drug concentration was ten times higher in resistant than in parental cells . Bearing in mind that the drug-binding kinetics have not been modified during the resistance development, our results indicate that some vincristine accumulated by resistant cells may be compartmentalized into these cells before its interaction with microtubules.

Mol Pharmacol, 1993 Jun, 43(6), 858 - 62
Expression of the antisense cDNA for protein kinase C alpha attenuates resistance in doxorubicin-resistant MCF-7 breast carcinoma cells; Ahmad S et al.; Multidrug resistance is functionally associated with the expression of a plasma membrane energy-dependent drug efflux pump termed P-glycoprotein, the product of the mdr1 gene . Transfection of P-glycoprotein-expressing doxorubicin-resistant MCF-7 cells with an expression vector containing the cDNA for protein kinase C alpha in the antisense orientation reduces protein kinase C alpha levels and decreases total protein kinase C activity by 75% . This is accompanied by reduced phosphorylation of P-glycoprotein, a 2-fold increase in drug retention, and a 3-fold increase in doxorubicin cytotoxicity . These results provide further evidence that protein kinase C alpha can positively regulate multidrug resistance in MCF-7 cells through posttranslational phosphorylation of P-glycoprotein.

Br J Cancer, 1993 Jun, 67(6), 1316 - 23
Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines; Coley HM et al.; This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines . The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines . For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining . In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein . The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence . For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence . For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus . The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter . The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus . Relatively little effect was seen in the parental lines . Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines . Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.

Br J Cancer, 1993 Jun, 67(6), 1203 - 8
Calmodulin inhibitor trifluoperazine in combination with doxorubicin induces the selection of tumour cells with the multidrug resistant phenotype; Kamath N et al.; Trifluoperazine (TFP) is effective in modulating DNA damage/repair in doxorubicin (DOX) treated cells . In the present study we have characterised the resistance phenotype of parental sensitive L1210 mouse leukaemia cells (L1210/S) adapted to grow in the presence of 0.017 microns DOX+5 microM TFP (L1210/DT) . Although with prolonged exposure, 0.017 microM DOX alone produced < 35% cell kill in L1210/S cells, similar cytotoxicity was achieved at 0.43 microM DOX in L1210/S cells selected in the presence of 0.017 microM DOX+5 microM TFP . L1210/DT cells were > 30-fold resistant to DOX following a 3 h drug exposure in a soft agar colony assay . In contrast, DOX sensitivity in cells adapted to grow in 5 microM TFP alone was comparable to L1210/S cells . Resistance to other inhibitors of topoisomerase II in L1210/DT cells was > 30-fold to etoposide and > 6-fold to amsacrine . The levels of the 170 kDa and 180 kDa isoforms of topoisomerase II in an immunoblot were comparable between the L1210/S and L1210/DT cells . Cross resistance to vincristine in the L1210/DT cells was accompanied by the overexpression of plasma membrane P-glycoprotein . Although a 1.5-2-fold decrease in accumulation of etoposide and DOX was observed in the L1210/DT cells, drug levels for equivalent DNA damage in the alkaline elution assay were > 5-fold higher in the L1210/DT versus L1210/S cells . No abrogation in the modulating effects of TFP on DOX, VP-16 or amsacrine induced cytotoxicity was apparent in the L1210/DT cells . Results suggest that: (a) TFP in combination with low concentrations DOX can induce the selection of cells with the multidrug resistant phenotype; and (b) characteristics of cells selected for resistance to DOX or DOX plus TFP are comparable.

Leuk Res, 1993 Jun, 17(6), 491 - 9
Autologous bone marrow transplantation for leukemia: monoclonal antibody-mediated purging of multidrug-resistant leukemia; Treichel RS; Nine drug-resistant variants of human leukemic/lymphoma cell lines were evaluated for the expression of antigens which are commonly targeted in antibody-dependent purging protocols used for autologous bone marrow transplantation . Flow cytometry revealed that most drug-resistant variants differ in antigen expression from parental lines . Both epipodophyllotoxin-selected T-ALL variants tested expressed lower levels of CD5 than parental counterparts . In contrast, other antigens (CD7, CD3, CD4, CD8, CD38, CD9, CD10, CD24, CD19, CD20) were not consistently altered in drug-resistant variants . Drug-resistant cells were lysed as effectively as drug-sensitive cells by murine antibody and baby rabbit complement, but only when particular monoclonal antibodies or cocktails of monoclonal antibodies were utilized . Two of the nine drug-resistant variants proved to be more resistant than parental cells to antibody-dependent cell-mediated cytolysis (ADCC).

Leuk Res, 1993 Jun, 17(6), 483 - 90
Expression of dihydrofolate reductase and multidrug resistance genes in trimetrexate-resistant human leukemia cell lines; Li XK et al.; Exposure of MOLT-3 human leukemic cells in culture to a lipophilic antifolate, trimetrexate (TMQ), resulted in the development of sublines resistant to antifolates as well as to drugs related to multidrug resistance . The TMQ-resistant sublines had an increase in dihydrofolate reductase (DHFR) activity and overexpression of P-glycoprotein . In these sublines, neither the DHFR gene nor the MDR1 gene were amplified . In these cells, DHFR transcripts were also not overexpressed but DHFR protein was increased, indicative of translational or post-translational control of DHFR activity . In contrast, MDR1 transcripts were found to be overexpressed, in parallel with P-glycoprotein production . Therefore, increases in P-glycoprotein appear controlled at the transcriptional level . These data support evidence that TMQ produced two phenotypic changes independently: the former probably from folate deficiency and the latter from the lipophilic nature of the compound.

FEBS Lett, 1993 Jun 1, 323(3), 191 - 7
The intriguing link between modulation of both multidrug resistance and ligand-toxin conjugate cytotoxicity; Jaffrezou JP et al.; Pharmacological agents which possess a chemosensitizing activity (i.e . the ability to modulate the multidrug resistance phenotype) can equally enhance ligand-toxin conjugate cytotoxicity . By confronting results obtained in both fields of research it appears that quite a number of agents, which are structurally unrelated, possess this bilateral effect . We have therefore attempted to provide a brief review of the literature and to discuss a hypothesis by which a common mechanism such as modifications in intracellular vesicle sorting and/or lipid metabolism may be implicated . We believe that these observations may provide clues for future research.

Exp Hematol, 1993 Jun, 21(6), 779 - 84
Effect of differentiating agents on modulation of MDR1 gene expression in multidrug-resistant hematopoietic HL60/DNR cell line; Zhou DC et al.; The phenomenon of multidrug resistance (MDR) is frequently encountered in clinical situations, and could contribute to the failure of chemotherapy in acute leukemia . Preliminary studies have suggested that MDR1 gene expression in normal hematopoietic stem cells might be downregulated during differentiation . In the present study, we induced a multidrug-resistant promyelocytic leukemia cell line, HL60/DNR, to myeloid differentiation by exposure to all-trans retinoid acid and dimethyl sulfoxide (DMSO) . We found that HL60/DNR cells retained the ability to respond to the differentiation stimulus . However, although MTT assays revealed a slight decrease of IC50 in differentiated cells, neither efflux of daunorubicin (DNR), nor expression of P-glycoprotein (P-gp), nor quantity of MDR1 mRNA has been downregulated in differentiated cells . We can conclude, therefore, that MDR1 gene expression in this multidrug-resistant myeloid cell line is not modified by induction of its differentiation.

Cancer Res, 1993 Jun 1, 53(11), 2544 - 7
Thaliblastine, a plant alkaloid, circumvents multidrug resistance by direct binding to P-glycoprotein; Chen G et al.; The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells . In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7 . Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells . Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block . Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner . TBL partially blocked the photolabeling of P-glycoprotein with {3H}azidopine, and this blocking effect was further enhanced in combination with ADR . Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.

Cancer, 1993 Jun 1, 71(11), 3611 - 9
Expression of the multidrug transporter, P-glycoprotein, in renal and transitional cell carcinomas; Nishiyama K et al.; BACKGROUND . Renal cell carcinomas (RCC) respond poorly to anthracyclines, Vinca alkaloids, and other agents . P-glycoprotein is overproduced in multidrug-resistant cells and thought to function as an energy-dependent drug efflux pump . The authors thus examined the expression level of P-glycoprotein in RCC and transitional cell carcinomas (TCC) . METHODS . P-glycoprotein was detected using immunoblotting with a monoclonal antibody against it, C219 . RESULTS . Thirty-three of 38 patients with RCC and 3 of 17 patients with TCC had P-glycoprotein positive tumors . The expression level of P-glycoprotein in most of RCC was lower than that in the normal kidney tissues and that of P-glycoprotein in the TCC was very low . The size of P-glycoprotein in 14 RCC and 3 TCC was 5-10 kilodaltons smaller than in the normal renal tissues . The variation of P-glycoprotein size in the RCC was attributed to differential N-linked glycosylation . P-glycoprotein in a RCC was photolabeled by tritiated azidopine, and the labeling was inhibited by some organic agents . P-glycoprotein distributed on the apical or marginal cell surface of the RCC . CONCLUSIONS . These data show that P-glycoprotein was expressed in many RCC, and its expression level, glycosylation, and distribution were altered . These data also suggest that the P-glycoprotein in RCC had similar drug binding site(s) to that in multidrug-resistant cells.

Zhonghua Wai Ke Za Zhi, 1993 Jun, 31(6), 330 - 2
{Intravesical instillation of combined adriamycin and verapamil in preventing recurrence of superficial bladder tumor}; Liu NB; Multidrug-resistance of the tumor cells is the main cause for failure of chemotherapy . Such resistance is correlated to P-glycoprotein 170, which can pump the chemotherapeutic agent out of the tumor cells . This action could be reversed by verapamil through competition for closely related binding sites on P-glycoprotein . Twenty-four postoperative cases of superficial bladder tumors were treated by intravesical instillation of combined adriamycin and verapamil, and they were followed up for 17-35 months (average 24.5 months) . Two cases (8.7%) had recurrence . NO marked toxicity and side-effects were observed . Verapamil working as a drug-resistance reversing agent might be used in some other tumors not sensitive to chemotherapy.

J Formos Med Assoc, 1993 Jun, 92 Suppl 2, S69 - 75
{The efflux of intracellular vincristine in drug-resistant human lung cancer cells is not mediated by P-glycoprotein}; Song EJ et al.; A subline (PC-9/VCR) of the human lung adenocarcinoma cell line (PC-9), derived by in vitro exposure to vincristine (VCR), exhibited a 10-12-fold resistance to VCR by MTT and HTCA assay . Compared to the parental cell line (PC-9), PC-9/VCR-resistant cells displayed a reduced accumulation of VCR . The rate of VCR efflux was shown to be enhanced by PC-9/VCR . Unlike multidrug resistance, this efflux was independent of P-glycoprotein overexpression as determined by the Northern blotting method . In addition, PC-9/VCR showed no collateral sensitivity to verapamil . This resistant subline only showed 6.9-fold and 2.5-fold cross resistance to colchicine and vinblastine, respectively . This preliminary result indicates that defective drug accumulation in PC-9/VCR is due to other mechanisms possibly involving the microtubule assembly.

J Protein Chem, 1993 Jun, 12(3), 279 - 90
Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins; Manavalan P et al.; A sequence comparison of the two membrane-associated (MA) domains of the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance transporter (MDR), and alpha-factor pheromone export system (STE6) proteins, each of which are believed to contain a total of 12 transmembrane (TM) segments, reveals significant amino acid homology and length conservation in the loop regions that connect individual TM sequences . Similar structural homology is observed between these proteins, hemolysin B (HLYB) and the major histocompatibility-linked peptide transporter, HAM1, the latter two which contain a single MA domain composed of six TM segments . In addition, there are specific sequences that are conserved within the TM segments of the five different membrane proteins . This observation suggests that the folding topologies of the MA domains of MDR, STE6, and CFTR in the plasma membrane are likely to be very similar . The sequence analysis also reveals that there are three characteristic motifs (a pair of aromatic residues, LTLXXXXXXP and GXXL) that are conserved in MDR, STE6, HLYB, HAM1, but not in CFTR . We propose that although CFTR may be evolutionarily related to these other membrane proteins, it belongs to a separate subclass.

Br J Cancer, 1993 Jun, 67(6), 1189 - 95
Differential modulation of doxorubicin toxicity to multidrug and intrinsically drug resistant cell lines by anti-oestrogens and their major metabolites; Kirk J et al.; The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines . The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells . Modification was observed at clinically achievable anti-oestrogen concentrations . Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.

Cancer Res, 1993 Jun 1, 53(11), 2591 - 6
Overcoming tumor necrosis factor and drug resistance of human tumor cell lines by combination treatment with anti-Fas antibody and drugs or toxins; Morimoto H et al.; Monoclonal mouse anti-Fas antibody is directed against Fas antigen, a M(r) 36,000 encoded polypeptide that belongs to the family of cell surface proteins which includes nerve growth factor receptor, tumor necrosis factor (TNF) receptors, B-cell antigen CD40, and T-cell antigens OX40 . Anti-Fas antibody mimics TNF-alpha in its cytolytic activity but not in other TNF-alpha-mediated activities . Thus, we examined if anti-Fas antibody synergizes in cytotoxicity with toxins and drugs . The present studies demonstrate that anti-Fas antibody in combination with diphtheria toxin (DTX), Adriamycin, or cis-platinum results in enhanced cytotoxicity and synergy and also overrides resistance to TNF, drugs, or toxins when tested against a battery of human tumor cell lines . Synergy with anti-Fas and DTX requires that DTX is enzymatically active, since inhibitors of DTX-mediated protein synthesis inhibition resulted in loss of synergy . When the plant toxin ricin was used, there was no synergy with anti-Fas antibody but rather an additive effect . The synergy was not obtained in a TNF receptor-negative line but was achieved with other anti-Fas-resistant lines . Cell lines resistant to either Adriamycin or cis-platinum were rendered sensitive by the combination of drug and anti-Fas antibody . Further, combination treatment of anti-Fas and Adriamycin overcame resistance of the gp 170-expressing, multidrug-resistant MDR ovarian line . In all cases, cytotoxicity was augmented by pretreatment of target cells with gamma-interferon which upregulates Fas antigen expression . These results show that anti-Fas antibody can synergize in cytotoxicity with toxins and chemotherapeutic drugs, and combination treatment can reverse resistance to TNF, toxins, and/or drugs.

Biochem Pharmacol, 1993 May 25, 45(10), 2025 - 35
Different modes of anthracycline interaction with topoisomerase II . Separate structures critical for DNA-cleavage, and for overcoming topoisomerase II-related drug resistance; Jensen PB et al.; In contrast to the classic anthracyclines (doxorubicin and daunorubicin), aclarubicin (ACLA) does not stimulate topoisomerase II (topo II) mediated DNA-cleavage . This distinction may be important with respect to topo II-related drug resistance, and the aim of this study was to clarify drug-structures responsible for this difference . Various ACLA analogs were tested for: (a) interaction with purified topo II, (b) induction of DNA cleavage in cells, (c) cellular uptake and (d) cytotoxicity . A remarkable distinction was seen between analogs containing the chromophore aklavinone (AKV) (e.g . ACLA) which have a carboxymethyl group (COOCH3) at C-10 and drugs with a beta-rhodomycinone (RMN) chromophore with hydroxyl groups at C-10 and at C-11 . Thus, RMN-containing analogs, including the aglycone RMN itself, effectively stimulated topo II-mediated DNA cleavage . In contrast, AKV-containing drugs inhibited DNA cleavage and antagonized cytotoxicity mediated by RMN-containing drugs . In OC-NYH/VM cells, exhibiting multidrug resistance due to an altered topo II phenotype (at-MDR), cross-resistance was only seen to the RMN-containing drugs whereas no cross-resistance was seen to the non-DNA cleaving AKV-containing compounds . Thus, our data show that one domain in the anthracycline is of particular importance for the interaction with topo II, namely the positions C-10 and C-11 in the chromophore, and further that at-MDR was circumvented by a COOCH3 substitution at position C-10 . These findings may provide guidance for the synthesis and development of new analogs with activity in at-MDR cells.

J Biol Chem, 1993 May 25, 268(15), 11417 - 25
Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P-glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12; Greenberger LM; P-glycoprotein can mediate multidrug resistance in tumor cells and is hypothesized to be an energy-dependent drug efflux pump . The protein is composed of two cassettes; each cassette contains six transmembrane domains followed by a nucleotide binding fold . The {125I}iodoaryl azidoprazosin photoaffinity drug binding sites in P-glycoprotein have been mapped by immunological analysis . After complete digestion of P-glycoprotein with trypsin, two major photolabeled fragments have been mapped . The 5- and 4-kDa fragments are located within, or immediately C-terminal to, the last transmembrane domain of each cassette: transmembranes 6 and 12 of P-glycoprotein, respectively . The 4-kDa fragment maximally extends up to, but not including, the Walker A motif of the second nucleotide binding fold, whereas the 5-kDa fragment maximally extends a few residues beyond the Walker A motif of the first nucleotide binding fold . A minor photolabeling domain is also found between transmembrane domain 4 and up to, but not including, transmembrane domain 6 . These data suggest that a portion of the drug binding site in P-glycoprotein is in close proximity to ATP binding regions . Furthermore, symmetrical regions in each cassette of P-glycoprotein are likely to participate in drug efflux.

MMWR Recomm Rep, 1993 May 21, 42(RR-7), 1 - 8
Initial therapy for tuberculosis in the era of multidrug resistance . Recommendations of the Advisory Council for the Elimination of Tuberculosis; Energy-dependent export of leukotriene B4 in Xenopus oocytes; Department of Biochemistry, Faculty of Medicine, University of Tokyo, JapanThe export of leukotriene (LT) B4 was studied using the Xenopus oocyte system . The oocytes were microinjected intracellularly with {3H}LTB4, and the export/injection ratios were determined . The ratios decreased with increasing doses of LTB4, converging upon 4% . The export was temperature-dependent and decreased with ATP depletion, thereby suggesting that the export is energy-dependent and carrier-mediated . The LTB4 export was inhibited by 6-trans-LTB4 and its 12-epi isomer, but much less by LTD4 . Neither verapamil nor quinidine significantly inhibited LTB4 export . These results suggest that oocytes have an export system specific for LTB4 and its isomers, and that the multidrug-resistant transporter is not involved in this system . By contrast, much of platelet-activating factor, another lipid mediator, was retained in oocytes at different temperatures after injection . It was thus shown that the two potent lipid mediators are exported from cells in a different manner and that oocytes are a good model for analyzing export systems for bioactive mediators.

Int J Cancer, 1993 May 8, 54(2), 309 - 14
A study of cross-resistance pattern and expression of molecular markers of multidrug resistance in a human small-cell lung-cancer cell line selected with doxorubicin; Supino R et al.; A doxorubicin-resistant variant of the human small-cell lung-cancer cell line N592 was selected by in vitro continuous exposure to increasing drug concentrations . The aim of this study was to examine the cross-resistance pattern, cellular pharmacokinetics of doxorubicin and expression of molecular factors of resistance . The sub-line N592/DX exhibited a multidrug-resistance phenotype, which was somewhat atypical, since it included cisplatin . Development of doxorubicin resistance could not be attributed to differential doxorubicin uptake or retention . Verapamil partially reverted doxorubicin resistance without affecting cellular pharmacokinetics . These findings are consistent with undetectable levels of mdr-1-gene expression in these cells . A molecular analysis of other putative mechanisms of multidrug resistance indicated no alterations in GSH levels or GSH-related enzymes, but a marginal reduction of topoisomerase II alpha expression in the resistant sub-line . This reduction, which was associated with an increase in topoisomerase I, does not explain the high degree of resistance . This study supports the view that alternative, unidentified mechanisms, which may be of clinical relevance, must be involved in the development of multidrug resistance of small-cell lung cancer.

Science, 1993 May 7, 260(5109), 819 - 22
Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages; Jacobs WR Jr et al.; Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis . Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene . Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate . Signals could be detected within minutes after infection of virulent M . tuberculosis with reporter phages . Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production . In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced . Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M . tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.

Am J Public Health, 1993 May, 83(5), 766 - 8
Commentary: tuberculosis in New York City--the consequences and lessons of failure; Landesman SH; The resurgent tuberculosis epidemic represents--especially in New York City--a failure to maintain a public health infrastructure that was focused on preventing active disease in high-risk populations (i.e., individuals with the human immunodeficiency virus {HIV}) and on treating active tuberculosis patients until cured . Although the tuberculosis problem in New York City and other localities is worsened by homelessness, poverty, and substance abuse, it is possible to bring tuberculosis under control by directing public health resources into targeted programs that enhance compliance with tuberculosis treatment regimen and expand chemoprophylaxis efforts among HIV-infected individuals . These two avenues will decrease, respectively, the number of cases of multidrug-resistant tuberculosis and the total number of new cases.

Am J Public Health, 1993 May, 83(5), 758 - 66
New York City's tuberculosis control efforts: the historical limitations of the "war on consumption"; Lerner BH; New York City began America's first campaign to control tuberculosis in 1893, and the disease declined until the 1970s . Throughout the 20th century, New York relied on three control strategies: screening, supervised therapy, and detention of noncompliant persons . Officials consistently identified the persistent foci of tuberculosis to be minorities and the poor, and they concentrated efforts among these populations . Recently, however, in the setting of rising human immunodeficiency virus infection and homelessness, tuberculosis--including multidrug-resistant strains--has returned to New York with a vengeance . Tuberculosis control in the city has been limited by two problems that hamper many public health programs: (1) antituberculosis measures, while appropriately targeting the poor, have been inconsistently funded and poorly coordinated; and (2) efforts have emphasized detection and treatment of individual cases rather than improvement of underlying social conditions . Renewed efforts by New York and other cities must address these limitations.

Gan To Kagaku Ryoho, 1993 May, 20(7), 889 - 95
{The present status of chemotherapy for hepatocellular carcinoma}; Itsubo M; In general, chemotherapy does not play an essential role in hepatocellular carcinoma (HCC) because of the low sensitivity to antitumor agents in cancer cells . Additionally, the vast majority of patients with HCC have chronic liver disease, notably cirrhosis, so it is virtually impossible to administer a large amount of antitumor agents . In fact, chemotherapy plays an important part in multimodal treatment for HCC . Whenever chemotherapy is used for patients in the advanced stage, it should be aimed to improve prognosis without impairment of their quality of life . Regarding prognostic factors of chemotherapy for unresectable patients, both reserved hepatic function and tumor advancement are important . Intra-arterial infusion chemotherapy using MMC, ADM, CDDP and/or 5-FU via hepatic artery is appropriately used to improve the therapeutic efficacy . The response rate by one shot injection seems to be approximately 10-20% in general . Although it is not clear which medicine and means of administration are most effective, oral administration is used as a subsidiary treatment for HCC in general . In order to enhance the efficacy of antitumor agents, various drug delivery systems including selective enhancement of tumor blood flow with angiotensin II and drug carriers such as Lipiodol have been applied . Recently, totally implantable arterial access devices have been used for intermittent intra-arterial infusion chemotherapy . It seems to make life easier for the treated patients . Further trials are planned to develop new modes of chemotherapy such as overcoming multidrug resistance by calcium antagonists.

Mol Microbiol, 1993 May, 8(4), 637 - 42
Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids; Silver S et al.; The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F0F1-type H(+)-translocating ATP synthases, the related vacuolar H(+)-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator) . The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins . New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases . Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.

Cell Biol Int, 1993 May, 17(5), 477 - 85
Relationship between multidrug resistance, hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells; Vickers SE et al.; We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines . We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers . Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.

Br J Haematol, 1993 May, 84(1), 83 - 9
Differentiation and multidrug resistance in response to drug treatment in the K562 human leukaemia cell line; Marks DC et al.; The relationship between differentiation and P-glycoprotein expression in response to chemotherapeutic drugs was studied in the K562 human leukaemia cell line by treatment with low, but clinically achievable levels of vinblastine and epirubicin . Resistant sublines were easily generated with the multidrug resistant phenotype being expressed in response to drug treatment as low as 1 ng/ml vinblastine and 10 ng/ml epirubicin . These sublines showed stable but heterogeneous expression of P-glycoprotein as revealed by immunocytochemistry, and confirmed by cloning . This heterogeneity was maintained over 18 months with intermittent drug treatment . While selection for resistance induced erythroid and myeloid differentiation, expression of P-glycoprotein was not correlated with the stem cell antigen CD34 or with specific markers of erythroid or myeloid differentiation.

J Orthop Res, 1993 May, 11(3), 396 - 403
Expression of the multidrug resistance gene in osteosarcoma: a pilot study; Wunder JS et al.; Resistance to combination chemotherapy remains a challenge in the treatment of osteosarcoma yet has not been studied extensively in this tumour . One mechanism of multiple drug resistance is increased expression of the multidrug resistance gene (mdr1) . The level of mdr1 messenger RNA (mRNA) expression has been found to correlate with the degree of drug resistance in a number of tumour cell lines in vitro, which suggests that it also may be useful as a predictor of similar resistance in vivo . Using a highly sensitive assay based on the polymerase chain reaction to measure the amount of mdr1 mRNA, we detected various levels of mdr1 expression in 18 osteosarcoma specimens from 15 patients with resectable nonmetastatic osteosarcoma . At follow-up at a minimum of 30 months later, a trend toward a worse outcome was observed in patients with tumours exhibiting high levels of mdr1 expression . The results of this pilot study suggest that a larger scale prospective investigation of the effect of mdr1 gene expression on outcome in osteosarcoma is warranted.

Jpn J Cancer Res, 1993 May, 84(5), 489 - 92
Enhancement of reversing effect of cyclosporin A on vincristine resistance by anti-P-glycoprotein monoclonal antibody MRK-16; Naito M et al.; The synergistic effect of MRK-16, a monoclonal antibody against P-glycoprotein, and cyclosporin A (CsA) on the modulation of vincristine resistance was studied by isobologram analysis in three different, highly multidrug-resistant tumor cells . In all cell lines, the synergistic effect was demonstrated at various concentrations of MRK-16 and CsA . While MRK-16 alone did not enhance the sensitivity of the moderately resistant KB-8-5 cells to vincristine, it increased two-fold the reversing effect of cyclosporin A at 1 microM, an achievable blood concentration . Since MRK-16 alone showed therapeutic effects against multidrug-resistant tumors, the combined use of MRK-16, CsA and antitumor agents should provide therapeutic benefits for the treatment of resistant tumors.

Eur J Haematol, 1993 May, 50(5), 279 - 85
Clinical significance of P-glycoprotein expression in acute leukaemia as analysed by immunocytochemistry; Tiirikainen MI et al.; Multidrug resistance, mediated by the overexpression of an energy-dependent transport protein, P-glycoprotein, has been one of the major targets of interest in solving the mechanisms of clinical drug resistance of malignant cells . To evaluate the correlation between P-glycoprotein overexpression and the response to chemotherapy, we analysed cytospin preparations of gradient-separated blood or bone marrow mononuclear cells from 79 patients with acute leukaemia by means of the P-glycoprotein-directed monoclonal antibody JSB-1 and immunocytochemistry using the alkaline phosphatase-antialkaline phosphatase technique . P-glycoprotein expression was detected in all disease phases of acute leukaemia . Thirteen out of 51 patients at diagnosis, 10/29 patients in relapse or during residual disease and 8/27 patients in remission overexpressed P-glycoprotein . Seven out of the 8 positive remission samples were collected between the cycles of consolidation treatment . Our results suggest that increased P-glycoprotein expression in samples collected between the cycles of consolidation treatment during remission may be induced in normal leukocytes by cytotoxic drug treatment, infections, or by some physiological mechanisms related to the disease . Patients older than 45 years of age were significantly more often P-glycoprotein-positive (11/25) at diagnosis than younger patients (2/26) . P-glycoprotein expression at diagnosis was significantly correlated with a low remission rate after the first cycle of induction therapy . Of 34 P-glycoprotein-negative patients, 25 achieved remission after the first cycle as compared to 4/12 of the P-glycoprotein-positive patients . Our results indicate that the method used is specific and sensitive enough for the analysis of P-glycoprotein expression and that the expression at initial presentation is inversely correlated with the outcome of induction therapy.

J Clin Microbiol, 1993 May, 31(5), 1293 - 8
Mixed-linker polymerase chain reaction: a new method for rapid fingerprinting of isolates of the Mycobacterium tuberculosis complex; Haas WH et al.; Rapid recognition of multidrug-resistant strains of Mycobacterium tuberculosis is a desirable goal for treatment of patients and protection of health care workers . DNA fingerprints produced with the insertion sequence IS6110 generate restriction fragment length polymorphism (RFLP) patterns that reliably identify M . tuberculosis complex strains . This report describes a rapid technique for RFLP typing using the polymerase chain reaction . The method uses one primer specific for IS6110 and a second primer complementary to a linker ligated to the restricted genomic DNA . In one strand the linker contains uracil in place of thymidine, and specific amplification is obtained by elimination of this strand with uracil N-glycosylase . Mixed-linker fingerprinting clearly differentiated multidrug-resistant isolates from 12 outbreaks and unambiguously assigned them to 26 RFLP groups.

Br J Cancer, 1993 May, 67(5), 959 - 68
Constitutive expression of multidrug resistance in human colorectal tumours and cell lines; Kramer R et al.; In this study we report detection of mdr1 gene expression in the liver metastases of 7/11 patients with colon carcinoma and characterise the MDR phenotype associated with a panel of 19 human colon carcinoma cell lines . Within this panel, mdr1 mRNA biosynthesis and surface localisation of Pgp were assessed with respect to MDR functionality where the cell lines are representative of different clinical stages of tumour progression, metastatic potential and differentiation . The data indicates that constitutive levels of mdr1 mRNA/Pgp expression may not necessarily result in the functional expression of the MDR phenotype . While low levels of mdr1 mRNA/Pgp were detected in 5/8 well differentiated colon cell lines, only 2/8 were functionally MDR . In contrast, 10/11 moderate and poorly differentiated lines expressed mdr1 mRNA/Pgp and of these, 9/11 were functionally MDR . The phosphorylation status of the mature 170 kD P-glycoprotein and the surface localisation of this glycoprotein showed the strongest correlation with functionality . Analysis of cell lines for cross-resistance and chemosensitivity profiles against a battery of chemotherapeutic drugs suggests multiple mechanisms, in addition to Pgp, contribute to the overall resistance of colorectal cancer.

Hepatology, 1993 May, 17(5), 854 - 60
Induction of multidrug resistance gene expression during cholestasis in rats and nonhuman primates; Schrenk D et al.; P-glycoprotein, an energy-dependent plasma membrane drug-efflux pump capable of reducing the intracellular concentration of a variety of hydrophobic xenobiotics, is encoded by mdr1, a member of the multidrug-resistant (mdr) gene family . The physiological function of this protein is unknown . Because of its location on the bile canalicular domain of the hepatocyte, we and others have hypothesized that P-glycoprotein may have a physiological role as a biliary transporter of xenobiotics and endobiotics and that its expression may therefore be altered in cholestasis . Both obstructive and alpha-naphthylisothiocyanate-induced cholestasis increased mdr1a and 1b gene expression in rat liver . Hepatic P-glycoprotein levels were also increased, and the protein remained localized at the biliary hepatocyte domain . Induction of mdr1a and mdr1b gene expression in rat liver was accomplished by means of increased transcription . alpha-Naphthylisothiocyanate-induced cholestasis in cynomolgus monkeys increased hepatic expression of both the mdr1 and 2 genes . To investigate the possible role of P-glycoprotein as a biliary efflux transporter, biliary excretion of vinblastine, a representative substrate of P-glycoprotein, was studied in rats . Increased hepatic mdr messenger RNA and P-glycoprotein levels, mediated by the xenobiotic inducer 2-acetylaminofluorene, resulted in a significant increase in biliary excretion of vinblastine, which was antagonized by the P-glycoprotein inhibitor verapamil . These findings suggest that P-glycoprotein functions as a biliary efflux pump for xenobiotics and, possibly, for unidentified physiological inducers that may mediate increased transcription of the mdr gene observed during cholestasis.

J Clin Invest, 1993 May, 91(5), 2207 - 15
Sensitivity of K562 human chronic myelogenous leukemia blast cells transfected with a human multidrug resistance cDNA to cytotoxic drugs and differentiating agents; Hait WN et al.; The blast crisis of chronic myelogenous leukemia (CML) is refractory to most forms of cancer chemotherapy, but may be amenable to drugs that differentiate rather than kill leukemic cells . One mechanism implicated in resistance to cytodestructive drugs is overexpression of P-glycoprotein, the MDR1 gene product . While several classes of drugs sensitize multidrug-resistant (MDR) cells by interfering with the function of P-glycoprotein in vitro, few sensitizers have been effective in vivo . We have developed a preclinical model of MDR/CML uncomplicated by other mechanisms of drug resistance to evaluate the effects of MDR1 overexpression on cytodestructive and differentiation therapy and the ability of sensitizers to restore chemosensitivity in this disease . The CML-derived cell line K562 was transfected with a human MDR1 cDNA from the pHaMDR1/A expression vector and selected with vinblastine . Resistant K562 clones were 20-30-fold resistant to vinblastine, were cross-resistant to doxorubicin and etoposide, and remained sensitive to cytosine arabinoside, 6-thioguanine, hydroxyurea, and mechlorethamine . Resistance was associated with decreased cellular accumulation of cytotoxic drug and was reversed by cyclosporin A and trans-flupenthixol . The MDR phenotype did not adversely affect the ability of K562 cells to produce fetal hemoglobin in response to hemin, and was associated with increased responsiveness of cells to differentiate with cytosine arabinoside . Upon differentiation, the resistant clones increased MDR1 mRNA and P-glycoprotein . These studies suggest that the overexpression of the MDR1 gene in CML may not adversely affect the ability to undergo erythroid differentiation and that these resistant K562 cell lines are good models for studying drug resistance mediated by P-glycoprotein in CML.

J Cell Physiol, 1993 May, 155(2), 414 - 25
Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II; Campain JA et al.; The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens . To characterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16 . To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11-2933, was added to the selections . The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold) . These cells are also two- to four-fold resistant to m-AMSA, daunorubicin, and mitoxantrone . FVP3 is not resistant to the P-glycoprotein substrates vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H-VP-16 accumulation . Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X . Using live cells treated with VP-16, band depletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X . This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components . In addition, the topoisomerase II present in nuclear extracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1) inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16 . These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3.

Cancer Res, 1993 May 1, 53(9), 1974 - 7
BIBW 22, a dipyridamole analogue, acts as a bifunctional modulator on tumor cells by influencing both P-glycoprotein and nucleoside transport; Chen HX et al.; BIBW 22, a phenylpteridine analogue of dipyridamole (DPM), enhanced vincristine cytotoxicity approximately 10 times more than DPM in a multidrug-resistant (MDR) KB V20C cell line . Using rhodamine 123 accumulation in KB V20C cells as an indicator of MDR phenotype, BIBW 22 was shown to be about 100 times more potent than DPM in inhibiting the MDR-associated efflux of rhodamine 123 . Photolabeling of P-glycoprotein in KB V20C plasma membranes with 0.2 microM {3H}azidopine was strongly inhibited by 1 microM BIBW 22, indicating that this compound reverses the MDR phenotype by interfering with MDR-associated P-glycoprotein . In addition, BIBW 22 at 1 microM could also enhance the cytotoxicity of 5-fluorouracil in KB cells about 20-fold . Its potency in inhibiting nucleoside transport is 7-fold more potent than that of DPM . These results suggest that BIBW 22 is a potent bifunctional modulator which influences both P-glycoprotein and nucleoside transport in tumor cells . Potential use of this compound as a modulator of combination chemotherapy involving antimetabolites and drugs affected by MDR should be explored.

Blood, 1993 May 1, 81(9), 2394 - 8
Expression of the multidrug resistance-associated P-glycoprotein (P-170) in 59 cases of de novo acute lymphoblastic leukemia: prognostic implications; Goasguen JE et al.; Immunocytochemical detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at time of diagnosis in a series of 36 children and 23 adults with acute lymphoblastic leukemia (ALL) using two monoclonal antibodies JSB1 and C219 . Immunophenotypes were obtained in all cases and karyotypes were analyzed in 37 cases . Detection with JSB1 or with C219 led to similar results in terms of positive cells and cases, but the intensity of staining was higher with JSB1 . In the populations studied, the rate of first complete remission differed between MDR-positive and MDR-negative in adult patients only (56% v 93%, respectively, P = .05) . Of the 16 MDR-positive patients who had presented a first complete remission, 13 (81%) relapsed, compared with 13 of 35 (37%) MDR-negative (P = .008) patients . A higher rate of relapse among MDR-positive compared with MDR-negative patients was observed in adults and in children taken separately (adults 100% v 46%; children 73% v 32%, respectively) . The survival rates (Kaplan-Meier method) were significantly higher in MDR-negative compared with MDR-positive populations as a whole (P = .002) and among children (P = .05) and adults (P = .03) taken separately . Event-free survival curves followed this trend . The percentage of second complete remission was very low in the MDR-positive group (15%) compared with 38% for the MDR-negative group . These results were shown by multivariate analysis to be independent of age, immunophenotypes, and karyotypes and clearly show the importance of MDR phenotype detection in ALL.

Cancer Lett, 1993 Apr 30, 69(2), 139 - 48
Mdr1/P-glycoprotein expression in natural killer (NK) cells enriched from peripheral or umbilical cord blood; Wilisch A et al.; The sensitivity to antineoplastic agents of subpopulations of haematopoietic cells during cancer chemotherapy is an open question . The performance of natural killer (NK) cells, possibly assisting the elimination of tumour cells under drug treatment might be of particular interest . We examined the expression of the transmembrane multidrug transporter mdr1/P-glycoprotein in NK-cells (CD56+) enriched from the peripheral blood or the umbilical cord blood from healthy donors by indirect immunocytofluorescence using the monoclonal P-glycoprotein antibody C219 and a polymerase chain reaction (PCR) approach with amplimers specific for the human mdr1 cDNA . As the antibody C219 apparently cross-reacts with the human mdr3 gene product whose functions are as yet unclear we also checked expression of this gene by PCR using mdr3 specific amplimers . Distinct, but rather inhomogeneous mdr1/P-glycoprotein expression was found in NK-cells enriched from the peripheral blood . NK-cells enriched from the umbilical cord blood showed quite strong mdr1 expression levels throughout, exceeding the values found in the moderately multidrug-resistant cell line CCRF VCR 100 which is permanently cultivated in the presence of 100 ng/ml vincristine . Mdr1/P-glycoprotein expression was mirrored by lowered sensitivities of the cultivated NK-cells towards actinomycin D or adriamycin . The drug sensitivity could be modulated by treatment of the cells with the immunosuppressive drug cyclosporin A . Expression of the mdr3 gene was low or absent in all NK-cell samples examined so far.

Lancet, 1993 Apr 24, 341(8852), 1044 - 9
Halofantrine versus mefloquine in treatment of multidrug-resistant falciparum malaria; ter Kuile FO et al.; The continuing spread of multidrug resistance in Plasmodium falciparum malaria makes the search for alternative treatments ever more urgent . We have investigated the relative efficacy of halofantrine and mefloquine in two paired randomised trials on the Thai-Burmese border, a multidrug-resistant area . In the first trial, 198 patients with acute uncomplicated falciparum malaria were randomly assigned either the standard halofantrine regimen (24 mg/kg) or mefloquine (25 mg/kg) . The cumulative failure rates by day 28 were 35% with halofantrine and 10% with mefloquine (p = 0.0002) . In the second study of 437 patients, a higher dose of halofantrine (8 mg/kg every 8 h for 3 days = 72 mg/kg) was both more effective and better tolerated than mefloquine 25 mg/kg; the failure rates were 3% and 8% (p = 0.03), respectively, or 1% vs 6% after adjustment for possible reinfections (p = 0.009) . The rate of failure was higher after retreatment than after primary treatment in all study groups . Halofantrine 72 mg/kg was especially effective in the retreatment of these recrudescent infections; the failure rate was 44% with mefloquine and 15% with high-dose halofantrine (relative risk 3.0 {95% CI 1.2-7.3}, p = 0.008) . Thus, high-dose halofantrine is better tolerated and more effective than mefloquine for the treatment of uncomplicated falciparum malaria in this area . However, evidence of possible cardiotoxicity will need to be investigated fully before a role can be established for halofantrine in the treatment of multidrug-resistant malaria.

J Natl Cancer Inst, 1993 Apr 21, 85(8), 632 - 9
Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs; Chaudhary PM et al.; BACKGROUND: The MDR1 (multidrug resistance) gene (also known as PGY1), which encodes the transmembrane efflux pump P-glycoprotein (Pgp), confers resistance to Pgp-transported cytotoxic drugs in tissue culture . The increase in MDR1 expression in tumors after chemotherapy is usually attributed to selection of pre-existing multidrug-resistant cells by Pgp-transported drugs . MDR1 expression in tissue culture can be increased by several types of stress-inducing treatment, including agents that activate protein kinase C (PKC) . Previous studies, however, failed to demonstrate that short-term exposure to any chemotherapeutic drug can induce the expression of the resident MDR1 gene in drug-sensitive human cells . PURPOSE: This study was designed to utilize highly sensitive assays to determine if transient exposure to chemotherapeutic drugs would have an effect on MDR1 expression in human cells and to assess if PKC inhibitors would influence such an effect . METHODS: We analyzed the MDR1 gene expression in several human cell lines derived from leukemias or solid tumors, after treatment with different cytotoxic drugs, in the presence or absence of PKC inhibitors . Pgp function and expression were studied by flow cytometric assays, and MDR1 messenger RNA (mRNA) was assayed by polymerase chain reaction . RESULTS: Transient exposure to chemotherapeutic drugs, including agents that are not transported by Pgp, induced Pgp and MDR1 mRNA expression in subpopulations of treated cells in most of the tested cell lines . This induction was observed along with microscopically detectable cell damage . The drug-induced MDR1 expression and the associated twofold to threefold increase in resistance to vinblastine were sustained in K562 leukemia cells for at least several weeks after the removal of the drug . Drug-mediated MDR1 induction was blocked by nonspecific protein kinase inhibitors that are active against PKC, but not by a protein kinase inhibitor ineffective against PKC . CONCLUSIONS: Expression of the human MDR1 mRNA and Pgp can be induced in response to cellular damage by cytotoxic drugs regardless of whether the drugs are transported by Pgp . This induction can be prevented by protein kinase inhibitors . IMPLICATIONS: Induction of MDR1 expression in response to cellular damage may account for increased MDR1 expression in treated human tumors . Protein kinase inhibitors may be useful in preventing the emergence of multidrug resistance during cancer chemotherapy.

Cancer Res, 1993 Apr 15, 53(8), 1845 - 52
Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells; Ling YH et al.; The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells . P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent . In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect . By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder . Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures . Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin . Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event . DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid . Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases . In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis . The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.

Cancer Res, 1993 Apr 15, 53(8), 1747 - 50
Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines; Zaman GJ et al.; Human cells can become multidrug resistant (MDR) by an increase in the activity of the MDR1 P-glycoprotein or by other, as yet unknown mechanisms, referred to as non-P-glycoprotein mediated MDR (non-Pgp MDR) . S . P . C . Cole et al . {Science (Washington DC), 258: 1650-1654, 1992} recently reported that in two cell lines non-Pgp MDR was associated with the overexpression of a new putative membrane transporter gene, MRP . Using an RNase protection assay we have analyzed the expression of MRP in non-Pgp MDR sublines of the human lung cancer cell lines SW-1573 (non-small cell lung cancer) and GLC4 (small cell lung cancer) . In all of ten SW-1573 derived lines examined the MRP mRNA level was equal to that in the parental line, whereas MRP was 25-fold overexpressed in a resistant subline of GLC4 . We conclude that overexpression of MRP cannot account for all forms of non-Pgp MDR.

J Biol Chem, 1993 Apr 15, 268(11), 8290 - 7
Identification of 5' and 3' sequences involved in the regulation of transcription of the human mdr1 gene in vivo; Madden MJ et al.; The human mdr1 gene encodes a putative drug efflux pump (P-glycoprotein) whose overexpression is associated with the development of multidrug resistance (MDR) . The promoter and 5'-flanking DNA of this gene were isolated from a human genomic DNA library and used to prepare a series of chloramphenicol acetyltransferase (CAT) fusion vectors under the transcriptional control of the mdr1 promoter (mdrCAT vectors) . Transient transfection of these mdrCAT vectors produced CAT activities similar to those produced by transfection of CAT vectors containing viral promoters . The regulation of mdr1 expression was examined in two MDR tumor cell lines selected for resistance to doxorubicin and their corresponding parental cell lines . Although nuclear run-on analysis indicates that the expression of the mdr1 gene in these two MDR cell lines is regulated by transcriptional mechanisms, mdrCAT expression was not significantly increased in either of these lines relative to parental cells . Thus, the sequences involved in the transcriptional regulation in these cells are apparently not included in the constructs studied (-4741 to +286) . Analyses of a series of deletion constructs show that the basal mdr1 promoter activity is encoded by sequences that span a region adjacent to the transcription start site (-134 to +286) and that sequences 3' to the start of mdr1 transcription are necessary for proper initiation of transcription in vivo . Structural and functional studies indicate that an initiator (Inr) sequence surrounding the major transcription start site governs accurate initiation of mdr1 transcription.

J Biol Chem, 1993 Apr 15, 268(11), 7613 - 6
Progesterone regulates the murine multidrug resistance mdr1b gene; Piekarz RL et al.; P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues . In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus . An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene . R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor . A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form . A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene . Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector . This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.

FEBS Lett, 1993 Apr 5, 320(2), 87 - 91
Reduced influx is a factor in accounting for reduced vincristine accumulation in certain verapamil-hypersensitive multidrug-resistant CHO cell lines; Stow MW et al.; The rates of accumulation, influx and efflux of vincristine have been examined in a series of multidrug-resistant Chinese hamster ovary cell lines which show exceptionally high levels of hypersensitivity (collateral sensitivity) to several resistance modifiers . The more highly resistant members of the series show significantly reduced levels of vincristine influx compared to the control cell line from which they were derived . It is possible that resistance modifier hypersensitivity and reduced vincristine influx may be due to a common change in membrane composition which has arisen during prolonged selection for vincristine resistance in these cell lines.

J Biol Chem, 1993 Apr 5, 268(10), 7520 - 6
Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene; Yu L et al.; Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes . Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein . To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays . These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells . Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein . The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters . Such conservation suggests that this sequence may have an important role in mdr gene expression . The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity . DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein . These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.

J Biol Chem, 1993 Apr 5, 268(10), 7474 - 81
N-glycosylation and deletion mutants of the human MDR1 P-glycoprotein; Schinkel AH et al.; P-glycoproteins are heavily glycosylated plasma membrane proteins, which confer multidrug resistance by pumping a range of different drugs from the cell . To investigate the significance of the conserved N-glycosylation sites present in the putative first extracellular loop of P-glycoproteins, we mutated one, two, or all three of these sites present in the human MDR1 P-glycoprotein . We also deleted a stretch of 20 amino acids, containing two of the three N-glycosylation sites . The effects of these mutations were studied by transfection into drug-sensitive cells . In vincristine-resistant transfected clones selected for similar steady state levels of membrane-bound P-glycoprotein, the absence of N-glycosylation did not alter the level or pattern of (cross-)resistance . However, the absence of N-glycosylation sites drastically reduced the efficiency with which drug-resistant clones could be generated . These findings suggest that N-glycosylation contributes to proper routing or stability of P-glycoprotein but not to drug transport per se . The deletion mutants demonstrated a clearly decreased and altered drug resistance pattern, even with a high level of P-glycoprotein in the plasma membrane . This, and possibly the observed lack of glycosylation of the remaining intact glycosylation sequence, suggests a constrained P-glycoprotein structure . Our findings support the current model for P-glycoprotein structure.

Int J Hematol, 1993 Apr, 57(2), 121 - 30
Action mechanism of idarubicin (4-demethoxydaunorubicin) as compared with daunorubicin in leukemic cells; Fukushima T et al.; To characterize a new anthracycline agent, idarubicin (4-demethoxydaunorubicin) and to establish its reasonable and appropriate use in the chemotherapy of acute leukemia, we examined its mode of action and compared it with the results obtained for daunorubicin . Idarubicin was shown to have the characteristic features of the action mechanism of anthracyclines, such as having a strong binding capacity to DNA, in terms of frequency and efficiency and reducing the template activity of DNA by binding to DNA or inducing DNA strand breaks in leukemic cells and inhibiting the DNA polymerase reaction . Idarubicin was superior to daunorubicin in terms of intracellular accumulation and binding capacity to DNA, which may result in idarubicin having much stronger activity in inducing DNA strand breaks than daunorubicin . These excellent properties of idarubicin were considered to explain idarubicin having stronger antileukemic effects than daunorubicin . The efficacy of idarubicin in multidrug-resistant cells is also discussed.

Antimicrob Agents Chemother, 1993 Apr, 37(4), 761 - 8
The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants; Paulsen IT et al.; Nucleotide sequence analysis of ORF1 from the integron on the broad-host-range plasmid R751 revealed that the first 94 of 110 codons of ORF1 from R751 are identical to ORF4, an open reading frame from the 3' conserved segment of other integrons found in gram-negative bacteria, after which point they diverged completely . The predicted products of both ORF1 and ORF4 share homology with the multidrug exporter QacC . Phenotypic analysis revealed that ORF1 specifies a resistance profile to antiseptics and disinfectants almost identical to that of qacC, whereas ORF4 specifies much lower levels of resistance to these compounds . ORF4, whose product lacks the C-terminal 16 amino acids of the ORF1 protein, may have evolved by the interruption of ORF1 from the insertion of a DNA segment carrying a sulI sulfonamide resistance determinant . Hence, ORF1 was designated qacE, and its partially functional deletion derivative, ORF4, was designated qacE delta 1 . Fluorimetric experiments indicated that the mechanism of resistance mediated by QacE, the protein specified by qacE, is active export energized by proton motive force . Amino acid sequence comparisons revealed that QacE is related to a family of small multidrug export proteins with four transmembrane segments.

Anticancer Drugs, 1993 Apr, 4(2), 265 - 72
Antiproliferative and chemomodulatory effects of interferon-gamma on doxorubicin-sensitive and -resistant tumor cell lines; Borsellino N et al.; Biological agents might offer various therapeutic opportunities in the treatment of cancer, including a direct and/or host-mediated antiproliferative effect and also the possibility to favorably modulate tumor resistance to antineoplastic drugs . We studied the in vitro antiproliferative effects of interferon (IFN)-gamma on the mouse B16 melanoma and Friend erythroleukemia, and the human K562 erythroleukemia, as doxorubicin (DXR)-sensitive and -resistant (multidrug resistant) variants . These effects were marked in B16 melanoma and rather slight in K562 erythroleukemia, without any difference between the DXR-sensitive and -resistant lines . The chemosensitive variant of Friend erythroleukemia showed an intermediate response, which was greater than that seen in its resistant counterpart . There was no apparent relationship between the antiproliferative activity of IFN-gamma and the glutathione content of the cell lines . On the other hand, this activity was enhanced by co-treatment with glutathione-depleting concentrations of buthionine sulfoximine, but only in the cell lines which had responded better to IFN-gamma alone . This result probably confirms that a free radical mechanism plays a part in the antitumor effect of the cytokine . Finally, a range of concentrations of IFN-gamma, including slightly cytotoxic ones, did not substantially improve the antiproliferative effects of doxorubicin on the various cell lines, except in the DXR-sensitive variant of Friend erythroleukemia where a synergistic effect of the combination was observed . Thus, our results are not very promising with regard to a possible favorable modulatory activity by IFN-gamma of DXR (multidrug)-resistance.

Cancer Res, 1993 Apr 1, 53(7), 1657 - 64
Transcriptional activation of the mouse mdr3 gene coincides with the appearance of novel transcription initiation sites in multidrug-resistant P388 tumor cells; Lepage P et al.; In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene . In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification . The mechanism underlying mdr3 overexpression in these cells was investigated . Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene . Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1) . Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon . In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2 . Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Gan To Kagaku Ryoho, 1993 Apr, 20(6), 831 - 3
{Analysis of MDR1 (multidrug resistance) gene expression by RT-PCR}; Nakashima A et al.; Multidrug resistance to anticancer drugs proved to be related to the MDR1 gene which encodes the P-glycoprotein, an energy-dependent drug-efflux pump for lipophilic drugs . We investigated the expression of the MDR1 gene in clinical samples by RT-PCR . The subjects were all resected cases of 14 colorectal cancers, five gastric cancers, two esophageal cancers, two gallbladder cancers and 20 lung cancers . Adrenal gland was used as a positive control . Total RNA was extracted from a fresh tissue sample . The cDNA was synthesized from 1 microgram of total RNA using reverse transcriptase . With the above cDNA as the template, amplification of the 157-bp fragment of the MDR1 gene was performed using PCR . The PCR product was polyacrylamide gel-electrophoresed and ethidium bromide-stained . In addition, a dot blot analysis was performed to quantify the amount of PCR product . Since PCR was performed simultaneously under the same conditions, the PCR product was quantified at four stages, from (3+) to (-), to indicate the degree of expression of MDR1 mRNA . Adrenal gland showed (3+)-(2+) and colorectal cancer exhibited mostly (2+)-(1+) . Both cancerous and non-cancerous areas evidenced a similar degree of expression in the cases of colorectal cancer . The MDR1 gene was expressed at low levels in other digestive tract cancers and in lung cancer . The levels of MDR1 expression revealed no correlation to either histological type or clinical stage . The present method may contribute to designing anti-cancer protocols.

Bull Cancer, 1993 Apr, 80(4), 310 - 25
{In vitro characterization of S9788, a new modulator of multidrug resistance}; Perez V et al.; S9788, a new triazinoaminopiperidine derivative, was 250-fold more active than verapamil in sensitizing DC-3F/AD cells to actinomycin D . This multidrug-resistance modulating activity of S9788 was confirmed on DC-3F/AD, P388/ADR-10, P388/VCR-20, KB/A1, K562/R, S1/tMDR, and COLO320DM cells, with respect to actinomycin D, doxorubicin, vincristine, vinblastine and etoposide . S9788 is 2-255-fold more active than verapamil, depending on the cell line and the cytotoxic agent used, potentiating preferentially the cytotoxicity of the vinca-alkaloid derivatives . S9788 only had a slight effect on vincristine accumulation and retention by parental sensitive cells, as well as on their sensitivity to cytotoxic drugs . S9788 fully restored vincristine accumulation and retention by S1/tMDR cells (S1 cells transfected by the mdr 1 gene) to a level similar to that measured in S1 cells, leading to a blockade in the G2 + M phase of the cell cycle . Therefore, S9788 enhances vincristine activity by restoring its cellular accumulation in resistant cells . After a short incubation period of cells with vincristine (4 h), a condition close to the clinical administration of this cytotoxic agent, a post-incubation for 20 hours more with 5 microM S9788, fully restored the sensitivity of S1/tMDR cells to vincristine . After an exposure of 4 hours at equal concentrations, the accumulation of S9788 was 13-fold that of verapamil in S1/tMDR cells, and 20 hours after the removal of the modulators, S9788 was retained at a concentration 20-fold that of verapamil . S9788 is therefore a powerful new modifier of the P-gp-mediated multidrug-resistance, which by its pharmacological properties (cellular kinetics and activity), might be used in combination with existing chemotherapy against tumors displaying the MDR phenotype.

J Pharm Pharmacol, 1993 Apr, 45(4), 268 - 73
Effects of isoquinolinesulphonamide compounds on multidrug-resistant P388 cells; Nakamura S et al.; The effects of eight isoquinolinesulphonamide compounds on resistance to vinblastine in adriamycin-resistant mouse leukaemia cells (P388/ADR) which overexpress the relative molecular weight (M(r)) 140 kDa P-glycoprotein in the plasma membrane were investigated . N-{2-(Methylamino)ethyl}-5-isoquinolinesulphonamide (H-8) and N-(2-aminoethyl)-5-isoquinolinesulphonamide (H-9) did not reverse vinblastine resistance . N-{2-{N-{3-(4-Chlorophenyl)-2-propenyl}amino} ethyl}-5-isoquinolinesulphonamide (H-86) and N-{2-{N-{3-(4-chlorophenyl)-1-methyl-2-propenyl} amino}ethyl}-5-isoquinolinesulphonamide (H-87) caused accumulation of intracellular vinblastine and inhibition of vinblastine efflux from the cells and reversed the resistance . Addition of an aminoethyl group to the nitrogen atom of the sulphonamide group (W-66) or a formyl group at the terminal amino group (H-85) of H-86 reduced those activities . Conversion of the chlorophenyl group of H-87 to pyridinyl (H-31) or furanyl (H-34) markedly decreased activities against the drug resistance . The activity against vinblastine accumulation closely correlated with the apparent partition coefficient of compounds . These compounds dose-dependently inhibited photoaffinity labelling of a photosensitive analogue of vinblastine, N-(p-azido-(3-{125I)salicyl)-N'-beta-aminoethyl-vindesine ({125I}NASV), and there was a good correlation between inhibition of {125I}NASV-photolabelling and hydrophobicity . Although these isoquinolinesulphonamides inhibited protein kinase A with different magnitudes, this activity did not correlate with the effect on the drug resistance . These results indicate that isoquinolinesulphonamide compounds with a hydrophobic group interact with antitumour drugs on P-glycoprotein and reverse multidrug resistance without involvement of their activity on protein kinase A.

Anticancer Drugs, 1993 Apr, 4(2), 173 - 80
D-verapamil downmodulates P170-associated resistance to doxorubicin, daunorubicin and idarubicin; Damiani D et al.; Verapamil (VRP) is an effective modulator of P170-associated multidrug resistance (MDR), but its clinical application is limited by cardiovascular side-effects . The D-isomer of VRP (D-VRP) is 10 times less active than the racemic mixture on the cardiovascular system, but retains a MDR modulating activity . Daunorubicin (DNR) and doxorubicin (DX) are two anthracyclines whose cytotoxicity is strongly related with the expression of P170, while their respective lipophylic derivatives idarubicin (IDA) and iododoxorubicin (IDX) are less P170-dependent . We studied the effect of D-VRP on intracellular retention and on the cytotoxicity of these four anthracyclines in two MDR cell systems (LOVO and CEM) by flow cytometry and by a microcultured tetrazolium colorimetric assay (MTT) . We found that in MDR cells D-VRP increased intracellular anthracycline concentration and increased the cytotoxicity of DNR, IDA and DX but not of IDX . The effect of D-VRP was dose-related, but it was already consistent at D-VRP concentrations that can be readily maintained in vivo (2-3 microM) . These data suggest that at a clinically tolerable concentration D-VRP can downmodulate the resistance to DNR and DX and can restore full sensitivity to IDA.

FASEB J, 1993 Apr 1, 7(6), 572 - 9
Modulation of doxorubicin resistance in multidrug-resistant cells by liposomes; Thierry AR et al.; In this study, we have confirmed the ability of liposome-encapsulated doxorubicin to modulate drug resistance, as previously observed in CH LZ cells (Thierry et al., Cancer Commun . 1, 311-316, 1989), in two human multidrug-resistant (MDR) cell lines, the breast cancer MCF-7/ADR cell line, and the ovarian carcinoma SKVLB cell line . This effect was specific to MDR cells, as liposomally encapsulated doxorubicin did not enhance cell sensitivity to the drug in the parental cell lines . Cytotoxicity assays demonstrated that empty liposomes in the presence of free doxorubicin (Dox) reversed resistance to the drug at a level that may be higher than that observed when liposome-encapsulated Dox is used . This effect seems to be due to the high affinity of Dox for cardiolipin, one of the liposome components, which leads to the association of the drug and the cardiolipin-containing liposomes in the culture medium before entry into the cells . Neither pretreatment of empty liposomes before drug treatment nor combined incubation of vincristine and empty liposomes alter MDR in CH LZ cells, suggesting that the drug must be encapsulated or complexed to the liposomes to overcome MDR . Because MDR in CH LZ cells does not seem to be related to GSH level, MDR modulation by liposome-encapsulated Dox apparently may not be effected by altering the GSH function . These results suggest that the enhancement of sensitivity of MDR cells using Dox encapsulated in liposomes or complexed with liposomes may be explained by an increase in cell drug incorporation and by an intracellular drug redistribution . Fluorescence confocal microscopy study indicated that Dox is transported and distributed mainly in intracytoplasmic vesicles in SKVLB and MCF-7/ADR cells, whereas in parental cells the drug is located mainly in the nucleus . In addition, presentation of Dox in liposomes modifies the drug distribution pattern in MDR cells by partially shifting the drug to nuclear compartments . Thus, liposome-associated Dox may bypass the vesicular drug transport in MDR cells, resulting in the enhancement of the drug biological activity.

Carcinogenesis, 1993 Apr, 14(4), 781 - 3
P-glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture; Fardel O et al.; P-Glycoprotein (P-gp), the multidrug resistance (MDR) gene product, has previously been shown to be functional and overexpressed in adult rat hepatocytes during primary culture . In the present study, we examined P-gp expression in human, mouse and hamster hepatocytes cultured in conditions similar to those used for rat liver cells . Northern blotting and doxorubicin P-gp-mediated efflux analyses revealed that liver parenchymal cells from these three species exhibited only limited increased MDR mRNA levels with no intracellular decreased drug retention, thus suggesting that the marked functional P-gp induction observed in rat hepatocytes could reflect a species-specific response of these cells to an unfamiliar environment . An 8 h exposure to cycloheximide, which strongly induced MDR mRNAs in rat liver cells, also increased, although to a far lesser extent, MDR mRNA levels in human, mouse and hamster hepatocytes, indicating that P-gp expression in normal liver parenchymal cells could be at least partly negatively regulated by a labile protein factor.

EMBO J, 1993 Apr, 12(4), 1615 - 20
The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells; Lincke CR et al.; P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs . The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown . In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract . We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constructs in which the promoter area of the pgp genes was fused to the coding region of lacZ . Expression of pgp-1 and pgp-3, as inferred from pgp-lacZ transgenic nematodes, was confined to the intestinal cells . The expression patterns of both genes were virtually indistinguishable . Quantitative analysis of pgp mRNA levels during development showed that pgp-1, -2, and -3 were expressed throughout the life cycle of C.elegans, albeit with some variation indicating developmental regulation . The expression of P-glycoprotein genes in intestinal cells is an evolutionarily conserved feature of these genes, consistent with the hypothesis that P-glycoproteins provide a mechanism of protection against environmental toxins.

Arch Otolaryngol Head Neck Surg, 1993 Apr, 119(4), 411 - 4
Detection of P-glycoprotein in squamous cell carcinomas of the head and neck; Kelley DJ et al.; One established mechanism of multidrug resistance is elevated expression of P-glycoprotein . The expression of P-glycoprotein by immunohistochemistry was examined in squamous cell carcinomas of the head and neck using a newly developed monoclonal antibody, UIC2, which is specific to the human MDR1 gene product and recognizes an external epitope of the protein . P-glycoprotein was detected in 60% of the samples . MDR1 expression was compared with clinical response to chemotherapy in eight patients who received MDR1-dependent drugs and response was accurately predicted in seven (89%) of eight patients . Positive P-glycoprotein staining correlated with the degree of tumor differentiation, but not with other clinical factors . Therefore, analyzing the expression of P-glycoprotein may play a role when planning chemotherapeutic regimens for patients with head and neck cancer and may be an additional prognostic and diagnostic tool in these patients.

Cancer Res, 1993 Apr 1, 53(7), 1555 - 9
Inhibition of multidrug resistance by a new staurosporine derivative, NA-382, in vitro and in vivo; Miyamoto K et al.; The effects of a newly synthesized compound, N-ethoxycarbonyl-7-oxo-staurosporine (NA-382), on multidrug resistance in tumor cells were investigated . Protein kinase-inhibitory activity of NA-382 was lower but more selective to Ca2+/phospholipid-dependent protein kinase than that of staurosporine . NA-382 at noncytotoxic concentrations effectively reversed in vitro multidrug resistance of Adriamycin-resistant P388 (P388/ADR) cells, without influencing the drug sensitivity of sensitive P388 cells . NA-382 inhibited extrusion of vinblastine (VBL) and increased intracellular accumulation of VBL, more in P388/ADR cells than in sensitive P388 cells, with higher potency than staurosporine . This compound also reduced VBL resistance of other multidrug-resistant cell lines, AH66 and K562/ADR, by inhibiting VBL efflux and promoting VBL accumulation . NA-382 also dose dependently potentiated the effects of VBL and Adriamycin in P388/ADR-bearing mice . The toxicity of staurosporine was too high to use the combination with VBL in vitro and in vivo . NA-382 accumulated VBL in P388/ADR cells even after desensitization of Ca2+/phospholipid-dependent protein kinase by treatment with 12-O-tetradecanoylphorbol-13-acetate and 18 h, while being suppressed by 12-O-tetradecanoylphorbol-13-acetate added simultaneously or shortly before NA-382 . Both staurosporine and NA-382 inhibited the photolabeling of {3H}azidopine on M(r) 140,000 P-glycoprotein in the plasma membrane from P388/ADR cells . These results indicate that this new staurosporine analogue, NA-382, reverses multidrug resistance by directly inhibiting the drug binding to P-glycoprotein, but not by Ca2+/phospholipid-dependent protein kinase inhibitory action.

Nat Genet, 1993 Apr, 3(4), 317 - 22
Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells; Whelan J et al.; Dominantly acting mutations that eliminate the allosteric regulation of CTP synthetase confer a form of multidrug resistance and a mutator phenotype to cultured Chinese hamster ovary cells . Mutations responsible for this phenotype have been identified in 23 independent strains selected for resistance to arabinosyl cytosine and 5-fluorouracil . All these mutations were due to base substitutions at seven sites within a highly conserved region of the ctps gene . This clustering should make it feasible to assess the role of such mutations in the development of drug resistance encountered in the treatment of malignant disease.

Kitasato Arch Exp Med, 1993 Apr, 65 Suppl, 25 - 36
Methods for detection of MDR1 mRNA expression on acute myelogenous leukemia cells; Sato H et al.; Overexpression of the human multidrug resistance gene (MDR1) on acute myelogenous leukemia (AML) correlates with poor prognosis . We evaluated several methods for mRNA estimation to standardize simple and reliable techniques for identifying MDR1 positive leukemia among untreated AMLs in large scale studies . Northern blot detection of MDR1 mRNA suffered from low signal-to-noise ratio under the conventional conditions, that was improved mainly by removing unincorporated radioactivity . The amount of MDR1 transcripts on positive cells was estimated less than 10% of that of constitutive mRNA species . A modified method seemed useful in estimating the total amount of the MDR1 mRNA in a whole leukemic cell population, and suitable to study stock samples or for large prospective clinical trials . RT-PCR was more sensitive in detecting MDR1 mRNA than Northern blot analysis, and the very feature made it virtually impossible to exclude contamination with normal hematopoietic cells . This procedure showed that FAB M3 leukemias were essentially MDR1 negative, and there existed frequently myelodysplastic syndrome subpopulation which had excessive MDR1 transcripts . In situ hybridization of the mRNA with a FITC-labeled phosphorothioate oligonucleotide probe was visualized using flowcytometry or con-focus lightmicroscopy, enabled us to recognize the difference between multidrug resistant K562/ADM and its wild type.

Anticancer Drugs, 1993 Apr, 4(2), 223 - 9
Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives; Jachez B et al.; FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux . The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype . Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones . FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity . One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.

Cancer Res, 1993 Apr 1, 53(7), 1475 - 9
Overexpression of a M(r) 110,000 vesicular protein in non-P-glycoprotein-mediated multidrug resistance; Scheper RJ et al.; A M(r) 110,000 protein (p110) is overexpressed in P-glycoprotein-negative multidrug-resistant tumor cell lines of different histogenetic origins . These cell lines show an ATP-dependent drug accumulation defect, suggesting the presence of drug transporter molecules different from P-glycoprotein . Immunohistochemical staining with a p110-specific monoclonal antibody (LRP-56) showed that, like P-glycoprotein, the molecule has a high expression in normal epithelial cells and tissues chronically exposed to xenobiotics and potentially toxic agents, such as bronchial cells, cells lining the intestines, and kidney tubules . Staining of LRP-56 is primarily cytoplasmic, in a coarsely granular fashion, indicating that it reacts with a molecule closely associated with vesicular/lysosomal structures . Involvement of p110 in the energy-dependent drug transport process present in the cell lines is unknown.

Biochem Biophys Res Commun, 1993 Mar 31, 191(3), 1252 - 60
Overexpression of a UV-damage recognition protein in a UV-sensitive human colon cancer cell line that features multidrug-resistant phenotype; Chao CC et al.; We have previously hypothesized that the level of UV-damage recognition protein (UVDRP) is a potential indicator of DNA-damage-mediated sensitivity or resistance of human cells to UV radiation {Chao, C.C.-K., Biochem . J . 282, 203-207, 1992} . In this study, we detected a 2-fold increase in the UVDRP binding activity in a UV-sensitive multidrug-resistant (MDR) human colon cancer cells compared to the parental SW620 cells . However, the MDR cells did not display enhanced DNA repair . The data is consistent with the conclusion that DNA excision repair is not the sole determinant of cellular sensitivity to genotoxic agents . The results also suggest that recognition of damaged DNA may be associated with generating a signal which mediates cellular sensitivity to UV radiation.

J Biol Chem, 1993 Mar 25, 268(9), 6077 - 80
Human P-glycoprotein transports cyclosporin A and FK506; Saeki T et al.; Cyclosporin A, a cyclic undecapeptide, and FK506 are efficient immunosuppressive agents . They also attract attention as effective P-glycoprotein modulators that inhibit P-glycoprotein from binding to anticancer drugs and overcome multidrug resistance . Cyclosporin A itself interacts with a common binding site of P-glycoprotein to which Vinca alkaloids and verapamil bind . We were interested to determine whether cyclosporin A and FK506 are substrates for P-glycoprotein to transport, and we studied their transcellular transport . In LLC-PK1 cells, derived from porcine kidney proximal tubule and forming a highly polarized epithelium, cyclosporin A was transported in a saturable manner . LLC-GA5-COL300, a transformant cell line derived by transfecting LLC-PK1 with human MDR1 cDNA isolated from normal adrenal gland, expresses P-glycoprotein specifically on the apical surface and shows a typical multidrug-resistant phenotype . LLC-GA5-COL300 cells showed increased transport of cyclosporin A from the basal to the apical side . Kinetic analysis showed that this transport was a typical saturable transport with the calculated apparent Michaelis constant (Kappm) and the maximum flux (Vmax) as 8.4 microM and 2.4 nmol/mg protein/h, respectively . LLC-GA5-COL300 also showed increased transport of FK506 from the basal to the apical side . These results indicate that P-glycoprotein transports the immunosuppressive agents cyclosporin A and FK506.

J Natl Cancer Inst, 1993 Mar 17, 85(6), 478 - 83
Restoration of taxol sensitivity of multidrug-resistant cells by the cyclosporine SDZ PSC 833 and the cyclopeptolide SDZ 280-446; Jachez B et al.; BACKGROUND: Taxol, a promising agent for the treatment of cancer, has entered phase II clinical trials . Nevertheless, it belongs to the class of compounds that show impaired retention in multidrug-resistant cells expressing P-glycoprotein (Pgp), a drug efflux pump . Chemosensitizers like verapamil modulate multidrug resistance by interfering with the efflux action of Pgp and thus can decrease drug resistance or can restore drug sensitivity by restoring normal drug accumulation and distribution within the multidrug-resistant tumor cell . The two strongest, nearly equipotent chemosensitizers identified to date are the cyclosporine derivative SDZ PSC 833 and the semisynthetic cyclopeptolide SDZ 280-446 . PURPOSE: This study was designed to investigate the capacities of verapamil, SDZ PSC 833, and SDZ 280-446 to decrease resistance of two multidrug-resistant cell lines to taxol . METHODS: We studied in vitro the growth of two multidrug-resistant tumor cell lines displaying high resistance to taxol: multidrug-resistant Chinese hamster ovary cells and murine monocytic leukemia P388 cells . We determined the taxol concentration that produced 50% inhibition of cell growth (IC50) in the two multidrug-resistant cell lines and in the parent cell lines, in the presence of a range of chemosensitizer concentrations (0-30 microM) . IC50 values were determined in the presence and in the absence of verapamil, SDZ PSC 833, or SDZ 280-446 . RESULTS: At nontoxic concentrations (0.3-1 microM), SDZ PSC 833 and SDZ 280-446 produced an almost complete reversal of the high taxol resistance of the multidrug-resistant tumor cells, whereas only partial restoration of sensitivity to taxol was achieved with verapamil . CONCLUSION: SDZ PSC 833 and SDZ 280-446 can restore the normal taxol sensitivity of highly resistant multidrug-resistant tumor cells . IMPLICATIONS: The combination of taxol with SDZ PSC 833 or SDZ 280-446 may be recommended for treatment of multidrug-resistant cancers.

Am J Epidemiol, 1993 Mar 15, 137(6), 676 - 84
Sample size and power determination for a binary outcome and an ordinal exposure when logistic regression analysis is planned; Bull SB; General methods of sample size determination for logistic regression analyses are now available, but these will often require substantial information for their application . The author presents methods useful in the special case of a binary outcome and a three-level quantitative exposure, which includes application to a three-level ordinal exposure for a specified scaling . The computationally simple methods were developed in planning an investigation of the prognostic value of multidrug resistance gene (mdr1) expression in sarcoma . Because logistic regression was planned for the analysis, calculations were based on the ability to detect a linear trend in the log odds of tumor response to chemotherapy associated with increases in the level of mdr1 expression from negative to low positive to high positive . Closed form expressions were used to assess sensitivity to the ordinal scaling and the distribution of the mdr1 levels, and to the assumption of a linear trend in the log odds versus a linear trend in the proportions.

FEBS Lett, 1993 Mar 15, 319(1-2), 133 - 7
Evidence for binding of extrachromosomal DNA sequences to nuclear matrix proteins in multidrug-resistant KB-V1 cells; Thiebaut F et al.; Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes) . Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression . Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins . This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis . Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.

J Biol Chem, 1993 Mar 15, 268(8), 5894 - 8
Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase; Inoue S et al.; Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases . Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s) . However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R . C., Inoue, S., Roitelman, J., Schimke, R . T., and Simoni, R . D . (1992) J . Biol . Chem . 267, 5731-5734) . To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein . These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity . Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily . The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones . Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.

Cancer Res, 1993 Mar 15, 53(6), 1354 - 9
Multidrug resistance is a component of V79 cell resistance to the alkylating agent adozelesin; Bhuyan BK et al.; Adozelesin is a highly potent alkylating agent which has entered clinical trials based on its unique mechanisms of action and broad-spectrum antitumor activity in vivo . V79 cells resistant to adozelesin (V79/AdoR) were not resistant to the alkylating agent cisplatin but showed the phenotypic and genotypic characteristics of multidrug resistance . Thus V79/AdoR was cross-resistant to several structurally and functionally unrelated drugs, resistance was reversed by verapamil, and the resistant cell line expressed mdr mRNA and p170 glycoprotein . Also, adozelesin uptake and the amount of drug alkylated to DNA was much lower in the resistant cell line as compared to the sensitive parent . However, even with the same amount of drug bound to DNA (10 fmol/micrograms DNA) the survival of V79/S approximately 15% survival) was much lower than that of V79/AdoR (approximately 80%) . Therefore the resistance of V79/AdoR cannot be explained solely by the multidrug resistance mechanism (i.e., lower drug uptake and less drug alkylation to DNA), which suggests that multiple mechanisms may account for resistance to adozelesin . V79/AdoR showed different levels of cross-resistance to several adozelesin analogues . The analogues could be divided into 2 groups; those with very low partition coefficients (log P < 2 as compared to 2.74 for adozelesin) had low levels of cross-resistance, whereas analogues with higher partition coefficients (log P > 2.4) were cross-resistant to adozelesin.

FEBS Lett, 1993 Mar 15, 319(1-2), 71 - 4
Cleavage of human MDR1 mRNA by a hammerhead ribozyme; Kobayashi H et al.; We designed a hammerhead ribozyme which site-specifically cleaved the GUC sequence in codon 179 of MDR1 mRNA . The cleavage site was 6 amino acids upstream from the drug binding site and was considered sufficiently close to the essential locus for P-glycoprotein function . The ribozyme cleaved the MDR1 mRNA under physiological conditions in vitro . The cleavage was dependent on ribozyme concentration and on incubation time . Mg2+ ion was essential for the cleavage . These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

J Biol Chem, 1993 Mar 15, 268(8), 5856 - 60
A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene; Goldsmith ME et al.; Basal transcription of the human multidrug resistance (mdr1) promoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines . Deletion of mdr1 DNA sequences to -89 relative to the start of transcription (at +1) had little effect on expression . Deletion of nucleotide sequences from -89 to -70, however, resulted in a 5-10-fold reduction in mdrCAT expression . DNase I footprint analysis demonstrated that the region from -85 to -70 was protected from nuclease digestion using nuclear extracts from these cell lines . The sequence between -82 and -73 is perfectly homologous with the 10-base pair Y-box consensus sequence found in the promoters of all major histocompatibility complex class-II (MHC II) genes . The Y-box sequence in MHC II genes is required for accurate and efficient transcription and contains the sequence CCAAT in the reverse orientation (Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C., and Mathis, D . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 6249-6253) . Mutations in the reverse CCAAT sequence of the Y-box consensus substantially reduced expression of an mdrCAT vector and eliminated nucleoprotein binding in an electrophoretic mobility shift assay . These results suggest that proteins which bind to the putative Y-box consensus sequence are critical for basal transcriptional regulation of the human mdr1 gene.

Cancer Res, 1993 Mar 15, 53(6), 1343 - 7
Scytophycins, novel microfilament-depolymerizing agents which circumvent P-glycoprotein-mediated multidrug resistance; Smith CD et al.; Cells demonstrating the multidrug resistance phenotype because of overexpression of P-glycoprotein, a drug efflux pump, are resistant to the cytotoxic effects of most natural product drugs . To determine if P-glycoprotein confers resistance to the syctophycins, a family of natural cytotoxic macrolides recently isolated from cyanobacteria of the family Syctone-mataceae, we have characterized the effects of these compounds on drug-sensitive (SKOV3) and drug-resistant (SKVLB1) human ovarian carcinoma cells . While SKVLB1 cells demonstrated > 150- and 10,000-fold decreases in sensitivity to Adriamycin and vinblastine, respectively, they were equally sensitive as SKOV3 cells to the antiproliferative effects of tolytoxin and certain related scytophycins . The SKVKB1 cells were 4- to 11-fold resistant to other scytophycins and were 14-fold resistant to cytochalasin B . Microfilaments in SKOV3 and SKVLB1 cells were depolymerized by similar concentrations of tolytoxin, while cytochalasin B was less potent toward SKVLB1 cells than SKOV3 cells . Both tolytoxin and cytochalasin B enhanced the cytotoxicity of vinblastine toward SKVLB1 cells; however, neither compound affected the sensitivity to Adriamycin or cisplatin . Verapamil markedly increased the accumulation of {3H}vinblastine by SKLVB1 cells, while cytochalasin B caused only modest increase, and tolytoxin had no effect on {3H}vinblastine accumulation . These results suggest that some of the scytophycins, including tolytoxin, are not subject to P-glycoprotein-mediated efflux from cells exhibiting multidrug resistance due to overexpression of this transport protein . These compounds may therefore be useful for killing drug-resistant tumor cells.

Lancet, 1993 Mar 13, 341(8846), 647 - 50
Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis; Telenti A et al.; Control of tuberculosis is threatened by widespread emergence of drug resistance in Mycobacterium tuberculosis . Understanding the molecular basis of resistance might lead to development of novel rapid methods for diagnosing drug resistance . We set out to determine the molecular basis of resistance to rifampicin, a major component of multidrug regimens used for treating tuberculosis . Resistance to rifampicin involves alterations of RNA polymerase . The gene that encodes the RNA polymerase subunit beta (rpoB) was cloned . Sequence information from this gene was used to design primers for direct amplification and sequencing of a 411 bp rpoB fragment from 122 isolates of M tuberculosis . Mutations involving 8 conserved aminoacids were identified in 64 of 66 rifampicin-resistant isolates of diverse geographical origin, but in none of 56 sensitive isolates . All mutations were clustered within a region of 23 aminoacids . Thus, substitution of a limited number of highly conserved aminoacids encoded by the rpoB gene appears to be the molecular mechanism responsible for "single step" high-level resistance to rifampicin in M tuberculosis . This information was used to develop a strategy (polymerase chain reaction-single-strand conformation polymorphism) that allowed efficient detection of all known rifampicin-resistant mutants . These findings provide the basis for rapid detection of rifampicin resistance, a marker of multidrug-resistant tuberculosis.

Nucleic Acids Res, 1993 Mar 11, 21(5), 1061 - 6
High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles; Huber CG et al.; DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns . Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C . This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs . PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml . Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated . Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells.

J Biol Chem, 1993 Mar 5, 268(7), 4592 - 5
Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C; Chambers TC et al.; Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function . The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing . Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and {gamma-32P}ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity . Phosphorylation occurred exclusively on serine residues . Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides . Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation . These sites are clustered in the linker region located between the two homologous halves of Pgp . Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator . These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.

Anticancer Res, 1993 Mar-Apr, 13(2), 487 - 90
mdr 1 gene-expression and villin synthesis in a colon cancer cell line differentiated by sodium butyrate; Petit JM et al.; Sodium butyrate (NaBu) but not dimethylsulfoxide (DMSO) induced the synthesis of villin, a protein of the brush border microvilli cytoskeleton, in a rat colon cancer cell line . Neither NaBu nor DMSO altered mdr 1-mRNA expression or multidrug resistance (MDR)--associated cellular transport of doxorubicin . These results show that mdr 1 gene expression and activity are independent of other brush border proteins induced by differentiating agents at the apical pole of the epithelial cell.

Am J Trop Med Hyg, 1993 Mar, 48(3), 385 - 97
Derivation of highly mefloquine-resistant lines from Plasmodium falciparum in vitro; Peel SA et al.; Serial passage of a multidrug-resistant clone of Plasmodium falciparum in concentrations of mefloquine hydrochloride ranging from 30 to 2,400 ng/ml resulted in the derivation of increasingly resistant parasite lines in vitro . Parasite lines isolated in mefloquine concentrations greater than 300 ng/ml demonstrated increased vacuolization, enhanced pigment production, and increased growth rates as compared with the progenitor clone, W2-mef . Although microdilution incorporation assays demonstrated that the 50% inhibitory concentration (IC50) of mefloquine were similar for all lines, the IC90, IC95, and IC99 levels were significantly increased . Growth rate assays performed in 5% hematocrit suspensions demonstrated different levels of mefloquine resistance among these lines . Under these conditions the most resistant line, Mef 2.4, grew efficiently in approximately 10-fold higher concentrations of mefloquine than the progenitor clone W2-mef . Analysis of drug susceptibility profiles to mefloquine hydrochloride, chloroquine diphosphate, quinine sulfate, and halofantrine hydrochloride indicated that selection for high levels of mefloquine resistance had resulted in significant increases in resistance to halofantrine and increased sensitivity to chloroquine . The phenotypic changes demonstrated in the most resistant line, Mef 2.4, reflect a multidrug resistant-like phenotype, and appear to mimic changes recently reported in drug susceptibility profiles of recrudescent isolates following mefloquine treatment failures in Thailand.

Cell Growth Differ, 1993 Mar, 4(3), 147 - 57
Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation; Uchiumi T et al.; The multidrug resistance 1 (MDR1) gene encodes a M(r) 170,000 membrane glycoprotein termed P-glycoprotein, which catalyzes the energy-dependent efflux of multiple anticancer agents . We investigated the activation of the MDR1 gene promoter by UV light irradiation in human cancer KB cells after both transient and stable transfection assays of the MDR1 promoter fused to the chloramphenicol acetyltransferase (CAT) gene . Following exposure to UV irradiation, CAT gene expression was about 20-fold increased . A series of promoter dissection analyses showed that two elements extending from -136 to -76 of the 5' flanking sequence and from +1 to +121 of the sequence downstream from the initiation site were required for the stress induction of MDR1 promoter activity . Gel shift assays showed that the specific DNA binding activities of the transacting protein to the MDR1 promoter were augmented in nuclear extracts from the cells treated with UV irradiation . A DNA sequence, an inverted CCAAT box, was identified that specifically bound to this protein, and mutation of this sequence abolished the binding of this protein . Two guanines in the inverted CCAAT box were found to be critical, as methylation of these guanines abrogated the binding . Nuclear run-on assay demonstrated that the transcription level was increased about 5-fold . These results suggest that the activation of the MDR1 promoter may result from transcriptional rather than posttranscriptional events . These studies will provide the basis for understanding the regulatory mechanism for appearance of the drug-resistant phenotype during cancer chemotherapy.

Yonsei Med J, 1993 Mar, 34(1), 35 - 44
Combined use of tamoxifen, cyclosporin A, and verapamil for modulating multidrug resistance in human hepatocellular carcinoma cell lines; Kim JH et al.; The intensive use of chemotherapeutic agents for the treatment of cancer has resulted in the cure or improved survival of many patients . But unfortunately, many cancers including human hepatocellular carcinoma (HCC) don't respond to chemotherapy . One of the major mechanisms for the drug resistance in the HCC is an elevated MDR1 RNA expression which makes cells become multidrug resistant . To overcome the multidrug resistance (MDR) phenotype, a high dose of verapamil is required both clinically and experimentally . Accordingly we have examined the MDR modulating effects with combinations of tamoxifen, cyclosporin A, and verapamil in vitro with the physiologically achievable concentrations of each agent, i.e., 2.0 microM/L for tamoxifen, 1.6 microM/L for cyclosporin A, and 2.5 microM/L for verapamil respectively in HCC lines . As expected, verapamil alone with the physiologically achievable concentration at which we tested didn't enhance the doxorubicin cytotoxicity in the HCC lines . Furthermore, any verapamil combination with cyclosporin A or tamoxifen was not effective in overcoming the doxorubicin resistance in the high MDR1 expressor (Hep-G2) line . However tamoxifen reduced the IC50 of doxorubicin by a factor of 1.9 in the low MDR1 expressor (SK-Hep1) and 1.1 in the high MDR1 expressor line (p < 10(-5) respectively) . Of interest, combinations of tamoxifen and cyclosporin A showed a significant reduction in the IC50 of doxorubicin in both HCC lines . The IC50 of doxorubicin was reduced by a factor of 3.9 and 1.3, i.e., from 0.023943 micrograms/ml to 0.006157 micrograms/ml (p < 10(-5)) in the SK-Hep1 cell line, and 0.068819 micrograms/ml to 0.052442 micrograms/ml (p < 10(-5)) in Hep-G2 respectively when tamoxifen and cyclosporin A were administered together . Both the estrogen and progesterone receptors in the SK-Hep1 and Hep-G2 lines were less than 0.01 fmol/mg of cytosol protein, respectively . It is therefore suggested that the reversal of doxorubicin resistance is unrelated to their anti-estrogenic activity in the HCC lines . Three modulator combinations of tamoxifen, cyclosporin A, and verapamil were not more effective than the combination of tamoxifen and cyclosporin A on the sensitivity to doxorubicin . MDR modulators of tamoxifen, cyclosporin A, and verapamil didn't reduce the IC50 of cisplatin to the clinically achievable concentration range in HCC lines . In summary, the combination of tamoxifen and cyclosporin A at the concentrations normally seen after clinical administration of these modulators showed significant synergism on the sensitivity to doxorubicin in both low and high MDR1 expressor HCC lines . These data indicate the need for in vivo trials.

Antimicrob Agents Chemother, 1993 Mar, 37(3), 407 - 13
Powerful bactericidal activity of sparfloxacin (AT-4140) against Mycobacterium tuberculosis in mice; Lalande V et al.; The bactericidal activities of various monotherapies and combined regimens were compared in mice at different stages after infection with Mycobacterium tuberculosis . These therapies mimicked the initial and continuation phases of chemotherapy for human tuberculosis . As monotherapy, the bactericidal activity of sparfloxacin (SPFX) was dose related; the activity of SPFX at 100 mg/kg of body weight was comparable to that of rifampin (RMP) and was significantly greater than those of isoniazid (INH), pyrazinamide (PZA), or ofloxacin (OFLO) during both the initial and continuation phases of chemotherapy . During the initial phase, the addition of SPFX did not enhance or diminish the activities of the combinations INH-RMP-PZA or RMP-PZA; the combinations SPFX-PZA-streptomycin (SM) and SPFX-PZA-kanamycin (KANA) displayed powerful bactericidal activity . Because the area under the plasma concentration-time curve of SPFX (100 mg/kg) in mice is similar to that of SPFX (400 mg) in humans, the promising bactericidal activity displayed by SPFX in mice might be achieved in humans by administration of the drug in a clinically tolerated dosage . In addition, the combinations SPFX-PZA-SM and SPFX-PZA-KANA may be useful for the treatment of multidrug-resistant tuberculosis.

Mol Pharmacol, 1993 Mar, 43(3), 372 - 9
Reversal of resistance to adriamycin by 8-chloro-cyclic AMP in adriamycin-resistant HL-60 leukemia cells is associated with reduction of type I cyclic AMP-dependent protein kinase and cyclic AMP response element-binding protein DNA-binding activities; Rohlff C et al.; 8-Chloro-cyclic AMP (8-Cl-cAMP) produces growth-inhibitory and differentiating activity in the promyelocytic leukemia cell line HL-60 . Adriamycin (ADR)-resistant HL-60 (HL-60/AR) cells exhibit the multidrug-resistant phenotype but do not express the mdr1 gene product P-glycoprotein . To explore potential signaling processes that may be involved in this atypical form of drug resistance, 8-Cl-cAMP was used as a modulator of the cAMP second messenger signal transduction pathway . Treatment for 48 hr with a 10% inhibitory concentration of 8-Cl-cAMP potentiated ADR cytotoxicity 14-fold in HL-60/AR cells but not in the parental cell line . 8-Cl-cAMP was stable to hydrolysis in the medium after 48 hr and was present intracellularly predominantly as phosphorylated metabolites (70%) and the parent compound (30%) . No difference occurred in ADR accumulation in HL-60/AR cells after treatment with 8-Cl-cAMP . Accompanying the 8-Cl-cAMP-mediated increase in ADR cytotoxicity in HL-60/AR cells was a reduction in the cytosolic type I cAMP-dependent protein kinase (PKA) and disappearance of the nuclear PKA holoenzyme . Coincident with these changes in drug-resistant cells was a marked reduction in the DNA-binding activity of the cAMP response element-binding protein to levels equivalent to those in sensitive cells . This effect appears to result from reduced phosphorylation of the cAMP response element-binding protein . These results suggest that the potentiation by 8-Cl-cAMP of ADR cytotoxicity in HL-60/AR cells occurs through down-regulation of nuclear type I PKA and cAMP response element-binding factors whose activities are regulated by PKA.

Cancer Res, 1993 Mar 1, 53(5), 1064 - 71
Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells; de Jong S et al.; The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes . GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S . De Jong et al., Cancer Res., 50: 304-309, 1990) . In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed . Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug . Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells . Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells . On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA . Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance . Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4 . The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting . No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized . These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.

Hum Cell, 1993 Mar, 6(1), 1 - 6
{Molecular basis for resistance to anticancer agents and reversal of the resistance}; Akiyama S; The development of drug resistance and especially of multidrug resistance (MDR) is a serious problem during treatment of various malignant tumors . Overexpression of P-glycoprotein (P-gp) has been observed in various multidrug resistant cells . P-gp acts as an energy-dependent drug-efflux pump . We have shown that the expression of P-gp is closely related to clinical drug resistance in some type of leukemia . We have found agents that reverse MDR and elucidated the molecular basis for the reversal of MDR . Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism, but little is known about its physiological functions . We purified dThdPase from human placenta, and isolated partial cDNA clones for dThdPase . Amino-acid sequences were deduced from nucleotide sequences of the longest clone (288 base pairs) . This sequence was 100% identical to the sequence of platelet derived endothelial cell growth factor (PD-ECGF) (residues 149-244) . dThdPase is one of the activating enzymes for fluorinated pyrimidines . The sensitivity of KB cells transfected with PD-ECGF cDNA to doxifluridine was considerably higher than that of non-transfected KB cells.

Anticancer Res, 1993 Mar-Apr, 13(2), 323 - 9
Establishment and characterization of multidrug-resistant human osteosarcoma cell lines; Serra M et al.; Multidrug resistant variants of two human osteosarcoma cell lines (U-2 OS and Saos-2) were selected by continuous exposure to doxorubicin . The in vitro and in vivo growth characteristics of these sublines as well as the expression of osteoblastic markers and of some surface antigens were analyzed . Resistant variants showed a higher doubling time and a lower cloning efficiency, and a lower metastatic ability after i.v . injection than corresponding parental cell lines . All the sublines showed overexpression of p-glycoprotein (referred to as p170) . The level of expression of this protein in the different cell lines was directly related to the degree of resistance as shown by the in vitro sensitivity to doxorubicin and other multidrug-related drugs . In sublines showing the highest levels of resistance (over 300-fold), p170 overexpression was associated with mdr 1 gene amplification . These are the first multidrug resistant human osteosarcoma cell lines ever reported . They may be used as a model for further investigations into the mechanisms of drug resistance in osteosarcoma and as a standard for the assessment of chemosensitivity in clinical samples.

Jpn J Cancer Res, 1993 Mar, 84(3), 298 - 303
Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene; Takatori T et al.; NF-R2 is a DNA-binding protein that interacts with the MDR1 gene proximal promoter sequence . We previously reported that NF-R2 binds within the promoter's -126 and -102 regions, which contain the ATTCAGTCA motif . In the present study, we have purified NF-R2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S-300, DEAE-Sepharose, heparin-Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose . NF-R2 runs as a single protein of 75 kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF-R2 binding . These results suggest that NF-R2 may be involved in the positive regulation of the MDR1 gene transcription.

Pharmacotherapy, 1993 Mar-Apr, 13(2), 88 - 109
Molecular targets in oncology: implications of the multidrug resistance gene; Lum BL et al.; The curative potential of chemotherapy for a number of tumor types has been obscured by the fact that many patients initially have striking remissions but later relapse and die . At the time of relapse many patients manifest resistance to a wide array of structurally unrelated antineoplastic agents, hence the term multidrug resistance (MDR) . Other tumor types, such as those arising in the colon, kidneys, liver, and lungs, tend to exhibit poor response to available cytotoxic drugs . The MDR phenomenon includes cross-resistance among the anthracyclines (doxorubicin, daunorubicin), the epipodophyllotoxins (etoposide, teniposide), the vinca alkaloids (vinblastine, vincristine), taxol, and other compounds . In vitro studies in cell culture indicate that this form of resistance is associated with amplification or overexpression of the mdr1 gene . The mdr1 gene codes for the expression of a cell surface protein, P-glycoprotein (P-gp), which acts as an energy-dependent efflux pump that transports drugs associated with MDR out of the cell before cytotoxic effects occur . The protein is expressed in normal human tissues such as the gastrointestinal tract, liver, and kidneys, where it is thought to serve as an excretory pathway for xenobiotic drugs and toxins . Preliminary studies demonstrated the presence of P-gp in tumor samples from patients with acute leukemia, multiple myeloma, lymphomas, and a variety of solid tumors . A number of drugs are able to reverse MDR, including calcium-channel blockers, phenothiazines, quinidine, antimalarial agents, antiestrogenic and other steroids, and cyclosporine . Limited results from clinical trials with small numbers of patients suggest that the addition of verapamil, diltiazem, quinine, trifluoperazine, or cyclosporine to chemotherapeutic regimens has the potential to reverse MDR; however, toxicities limit their clinical usefulness . A number of trials are under way to identify more active and less toxic modulators of MDR.

Cancer Res, 1993 Mar 1, 53(5), 977 - 84
Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex; Piwnica-Worms D et al.; The multidrug-resistant P-glycoprotein (Pgp), a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR1), appears to function as an energy-dependent efflux pump . Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH . We tested the hypothesis that the synthetic gamma-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ({99mTc}SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype . The cellular pharmacological profile of {99mTc}SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively . Steady-state contents of {99mTc}SESTAMIBI in V79, 77A, and LZ cells were 10.0 +/- 0.5 (SEM) (n = 9), 3.6 +/- 0.5 (n = 8), and 0.4 +/- 0.02 (n = 9) fmol.(mg protein)-1 (nMo)-1, respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells . Maximal doses (> 100 microM) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced {99mTc}SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively . The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 microM, and those for cyclosporin A were 0.9, 3, and > 25 microM, respectively . Pgp-mediated {99mTc}SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent Km of 50 microM . Carrier-added {99Tc}SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by {125I}iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess {99Tc}SESTAMIBI . Exploiting the favorable gamma emission properties of 99mTc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of {99mTc}SESTAMIBI . Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo, target reversal agents in vivo, and ultimately provide a means to direct patients to specific cancer therapies.

Br J Cancer, 1993 Mar, 67(3), 471 - 9
Selective modulation of vinblastine sensitivity by 1,9-dideoxyforskolin and related diterpenes in multidrug resistant tumour cells; Shalinsky DR et al.; The ability of 1,9-dideoxyforskolin (DDF), 1-deoxyforskolin (DF) and forskolin to modulate cellular sensitivity to vinblastine (VBL) was examined in drug-sensitive parental KB-3-1 cells and a multidrug-resistant subline, KB-GRC1, derived by transfection of mdr1 . Fifty microM DF and forskolin enhanced the 1 h uptake of VBL by 8.0 +/- 0.7 (s.d.) and 4.7 +/- 2.5-fold, respectively, with 50 microM DDF producing a 13.6 +/- 1.9-fold increase . The greater effect of DDF relative to forskolin indicated that the effect was independent of activation of cAMP, and this was supported by a lack of effect of dibutyryl cAMP on the uptake . The effect of these agents on uptake were < or = 1.4-fold in KB-3-1 cells . DDF selectively inhibited initial efflux in cells expressing a functional P-glycoprotein (PGP), but both forskolin and DDF inhibited the terminal phase of efflux irrespective of PGP expression . Neither agent affected membrane permeability of polarisation and forskolin did not enhance the uptake of VBL in protein-free liposomes . At a non-toxic concentration of 20 microM, DDF and forskolin decreased the IC50 of VBL from 18.9 to 2.7 and 13 nM in KB-GRC1 cells, respectively, and DDF acted synergistically with VBL as shown by median effect analysis {combination index = 0.20 +/- 0.05 (s.d.)} . In contrast, these diterpenes did not affect VBL sensitivity in KB-3-1 cells . These results indicate that the diterpenes modulate VBL sensitivity predominantly by inhibiting PGP-mediated efflux activity.

Arkh Patol, 1993 Mar-Apr, 55(2), 18 - 23
{The differentiation of tumor cells of the human large intestine during the acquisition of multiple drug resistance}; Raikhlin NT et al.; By selection in the medium containing increasing actinomycin D concentrations two sublines with acquired multidrug resistance (MDR) caused by P-glycoprotein (P170) overproduction were isolated . The obtained cell lines as well as parent cells grow in vitro as morphologically organized aggregates, so-called organoids . Comparative electron microscopic study of sensitive and drug resistant organoids has shown that the development of MDR was accompanied by the enhancement of the tumour cell differentiation: the percentage of differentiated cells, the extent of their maturity, and the quantity of lumens were higher in MDR organoids than in parent cell line . The size of glandular structures in resistant organoids was also enlarged . Possible mechanisms of observed phenomenon are discussed.

Zhonghua Zhong Liu Za Zhi, 1993 Mar, 15(2), 101 - 3
{Establishment of the multidrug-resistant cell line K562/A02 and its drug-resistant properties}; Luan FJ; A multidrug-resistant cell line K562/A02 was obtained from its parent K562 by long-term adriamycin (ADM) induction and cloning selection . The K562/A02 has high expression of mdr-1 P-glycoprotein (P170), strong resistance to its induction drug ADM and some other anticancer drugs such as daunorubicin, VP16, homoharringtonine and amsacrine . Its intracellular concentration of daunorubicin is much lower than that in its parent K562, which can be partially reversed by cyclosporin-A and verapamil.

Anticancer Res, 1993 Mar-Apr, 13(2), 317 - 21
Peripheral blood mononuclear cells express antigens associated with multidrug resistance in a small cell lung cancer cell line; Krebes KA et al.; H69AR is a multidrug resistant small cell lung cancer (SCLC) cell line that does not overexpress P-glycoprotein, the plasma membrane drug efflux pump usually associated with this type of resistance . Monoclonal antibodies (MAbs) were previously raised against H69AR cells, and three of these MAbs, 2.54, 3.50, and 3.186, cross-reacted with peripheral blood mononuclear cells . T cells (CD3+), B cells (CD19+), NK cells (CD16+) and monocytes (CD14+) expressed each of the three antigens, but to differing degrees . Immunoprecipitation and partial proteolytic mapping experiments demonstrated that the antigens detected by MAbs 2.54 and 3.186 are identical in H69AR cells and PBMCs . SCLC cells are known to express many hematopoietic antigens; however, this is the first report of SCLC multidrug resistance-associated antigens being expressed on hematopoietic cells.

Drugs, 1993 Mar, 45(3), 430 - 75
Mefloquine . A review of its antimalarial activity, pharmacokinetic properties and therapeutic efficacy; Palmer KJ et al.; Mefloquine is an orally administered blood schizontocide . Initial dose-finding and comparative studies performed between 1977 and 1989 demonstrated efficacy of mefloquine as prophylaxis in nonimmune individuals and in the suppression and treatment of malaria in adults and children caused by multidrug-resistant Plasmodium falciparum . It was also effective against P . vivax infection, while data concerning the treatment of P . ovale and P . malariae infections were limited . In an attempt to delay the emergence of resistance to this promising antimalarial agent, mefloquine was combined with sulfadoxine and pyrimethamine . Although initial clinical trials indicated that this regimen was effective in preventing and treating falciparum malaria, recent treatment failures, the potential for severe dermatological reactions and lack of therapeutic advantage over mefloquine alone has prompted the World Health Organization to recommended that the combination be no longer used for treatment or prophylaxis of malaria . Mefloquine is generally well tolerated in both adults and children, with nausea, vomiting, diarrhoea, headache, dizziness, rash, pruritus and abdominal pain being the most common adverse effects, although it is difficult to distinguish between disease- and treatment-related events . The incidence of these adverse effects is similar to or lower than those observed with other antimalarial agents . Cardiovascular changes, such as bradycardia, occasionally occur . The most notable adverse effects associated with mefloquine are neuropsychiatric disturbances; precipitation of such events should be closely monitored and requires termination of prophylaxis or therapy . The eventual emergence of resistance to mefloquine, as with many other antimalarial agents, was inevitable . Mefloquine resistance is established in certain areas of Thailand and may be becoming a growing problem in other regions of the world . In order to preserve the efficacy of mefloquine in non-resistant areas, this useful agent should be used with care and only prescribed for prophylaxis in travellers and treatment in areas of multidrug-resistant plasmodia . Future options to combat mefloquine resistance may include the combination of mefloquine with other antimalarial agents such as qinghaosu derivatives . Thus, with cautious use and possible combination with other agents, mefloquine is likely to remain an important treatment option for falciparum malaria, a widespread parasitic disease for which an increasing number of drugs have proved inadequate.

Blood, 1993 Mar 1, 81(5), 1342 - 6
Expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells of patients with granular lymphocyte-proliferative disorders; Yamamoto T et al.; Some patients with granular lymphocyte-proliferative disorders (GLPD) have been reported to have an aggressive clinical course with a poor prognosis and to be refractory to chemotherapy . In this study, expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells (PBMC) of 11 patients with GLPD was examined by staining with MRK 16, a monoclonal antibody that binds to an external epitope of P-glycoprotein, and with the dye rhodamine, a known substance to be excreted from the cells through P-glycoprotein . Among those tested, the PBMC of six of eight patients with T-cell-type GLPD as well as those of three of three patients with natural killer cell-type GLPD expressed P-glycoprotein significantly . Furthermore, PBMC of two of two patients were also poorly stained with the dye rhodamine, and the treatment of PBMC with either verapamil or quinidine, multidrug resistance-reversing agents, led to their increased staining, suggesting that these PBMC actively release chemotherapeutic agents.

N Engl J Med, 1993 Feb 25, 328(8), 521 - 6
The emergence of drug-resistant tuberculosis in New York City; Frieden TR et al.; BACKGROUND . In the past decade the incidence of tuberculosis has increased nationwide and more than doubled in New York City, where there have been recent nosocomial outbreaks of multidrug-resistant tuberculosis . METHODS . We collected information on every patient in New York City with a positive culture for Mycobacterium tuberculosis during April 1991 . Drug-susceptibility testing was performed at the Centers for Disease Control and Prevention . RESULTS . Of the 518 patients with positive cultures, 466 (90 percent) had isolates available for testing . Overall, 33 percent of these patients had isolates resistant to one or more antituberculosis drugs, 26 percent had isolates resistant to at least isoniazid, and 19 percent had isolates resistant to both isoniazid and rifampin . Of the 239 patients who had received antituberculosis therapy, 44 percent had isolates resistant to one or more drugs and 30 percent had isolates resistant to both isoniazid and rifampin . Among the patients who had never been treated, the proportion with resistance to one or more drugs increased from 10 percent in 1982 through 1984 to 23 percent in 1991 (P = 0.003) . Patients who had never been treated and who were infected with the human immunodeficiency virus (HIV) or reported injection-drug use were more likely to have resistant isolates . Among patients with the acquired immunodeficiency syndrome, those with resistant isolates were more likely to die during follow-up through January 1992 (80 percent vs . 47 percent, P = 0.02) . A history of antituberculosis therapy was the strongest predictor of the presence of resistant organisms (odds ratio, 2.7; P < 0.001) . CONCLUSIONS . There has been a marked increase in drug-resistant tuberculosis in New York City . Previously treated patients, those infected with HIV, and injection-drug users are at increased risk for drug resistance . Measures to control and prevent drug-resistant tuberculosis are urgently needed.

J Biol Chem, 1993 Feb 25, 268(6), 4197 - 206
Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein; al-Shawi MK et al.; A multidrug-resistant Chinese hamster ovary cell line (CR1R12) was obtained which constitutively expresses P-glycoprotein, up to 32% by weight of plasma membrane protein . CR1R12 plasma membranes had high, drug-activated ATPase activity referable to P-glycoprotein . The specific ATPase activity in the presence of verapamil was calculated to be approximately 9 mumol/min/mg (identical to 21 s-1) at 37 degrees C, pH 7.4 . KM ATP was 1.4 mM, and ADP and 5'-adenylyl imidodiphosphate were competitive inhibitors with Ki values 0.35 and 0.44 mM, respectively . 2'-dATP was a good substrate, GTP and ITP were real but poor substrates, and ADP and AMP were not hydrolyzed . Optimal pH for ATP hydrolysis was 7.3 . MgATP was the preferred substrate, and CaATP was hydrolyzed very weakly . 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) covalently labeled the P-glycoprotein, and incorporation of 1.1 mol of NBD-Cl/mol of P-glycoprotein gave 100% inactivation . ATP protected against NBD-Cl inactivation . N-Ethylmaleimide was a potent inhibitor in the absence of ATP, and in its presence significant protection from inhibition could be achieved . Vanadate and fluoroaluminate were also strong inhibitors . The plasma membranes from CR1R12 cells should provide material for purification and reconstitution of P-glycoprotein and for screening of potential "multidrug-reversal" reagents by enzymic assay.

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 138 - 46
Identification of a functional initiator sequence in the human MDR1 promoter; van Groenigen M et al.; The sequence requirements for proper transcriptional initiation of the downstream human multidrug resistance MDR1 (P1) promoter were determined using a transient expression system in HeLa cells . The MDR1 promoter has no TATA box and the transcription start site has a strong homology with the initiator (Inr) sequence identified in the murine terminal deoxynucleotidyltransferase (TdT) gene . A deletion analysis showed that sequences from -6 to +11 relative to the P1 transcription start site were sufficient for proper transcriptional initiation, whereas deletion of sequences downstream of +11 resulted in a strong reduction of properly initiated transcripts . In this transient assay system, both the MDR1 and TdT initiator require in Hela cells the presence of an upstream activating sequence such as the SV40 enhancer . This is in contrast to the transcription in in vitro systems, in which the initiator sequence is able to direct transcription in the absence of an enhancer . Analysis of mutations in the initiator sequence from -8 to +10 showed that the A and T nucleotides at position +1 and +3, respectively, were essential, whereas other substitutions in this region had little effect on promoter activity.

Nature, 1993 Feb 18, 361(6413), 650 - 4
Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy; Chow YK et al.; Wild-type reverse transcriptase has evolved for the survival of human immunodeficiency virus type 1 (HIV-1) by natural selection . In contrast, therapy relying on inhibitors of reverse transcriptase by nucleosides like zidovudine (AZT) or dideoxyinosine (ddI), and by non-nucleosides like pyridinones or nevirapine, may exert different selection pressures on this enzyme . Therefore the acquisition of resistance to reverse transcriptase inhibitors by selection of mutations in the pol gene may require compromises in enzyme function that affect viral replication . As single mutations are unlikely to confer broad resistance when combinations of reverse transcriptase inhibitors are used, multiple mutations may occur that result in further compromises . Certain drug combinations may prevent the co-existence of adequate reverse transcription function and multi-drug resistance (MDR) . Unlike bacterial or eukaryotic drug resistance, retroviral drug resistance is conferred only by mutations in its own genome and is limited by genome size . Combining drugs directed against the same essential viral protein may thus prevent HIV-1 MDR, whereas the conventional approach of targeting different HIV-1 proteins for combination therapy may not, because genomes with resistance mutations in different HIV-1 genes might recombine to develop MDR . Here we show that several mutations in the HIV-1 reverse transcriptase gene that confer resistance to inhibitors of this enzyme can attenuate viral replication . We tested whether combinations of mutations giving rise to single-agent resistance might further compromise or even abolish viral replication, and if multidrug-resistant viruses could be constructed . Certain combinations of mutations conferring resistance to AZT, ddI and pyridinone are incompatible with viral replication . These results indicate that evolutionary limitations exist to restrict development of MDR . Furthermore, a therapeutic strategy exploiting these limitations by using selected multidrug regimens directed against the same target may prevent development of MDR . This approach, which we call convergent combination therapy, eliminated HIV-1 replication and virus breakthrough in vitro, and may be applicable to other viral targets . Moreover, elimination of reverse transcription by convergent combination therapy may also limit MDR.

J Natl Cancer Inst, 1993 Feb 17, 85(4), 311 - 6
Enhancement of cellular accumulation of cyclosporine by anti-P-glycoprotein monoclonal antibody MRK-16 and synergistic modulation of multidrug resistance; Naito M et al.; BACKGROUND: Drug resistance is a major obstacle to successful cancer chemotherapy . P-glycoprotein, which transports certain antitumor agents out of resistant tumor cells, is known to be a major factor in some types of multidrug resistance . Studies have shown that verapamil and the immunosuppressors cyclosporine and FK-506 can reverse multidrug resistance in vitro and in vivo and that the P-glycoprotein monoclonal antibody MRK-16 increases drug toxicity in multidrug-resistant tumors . PURPOSE: The purpose of this in vitro study was to establish effective treatment modalities for overcoming multidrug resistance . We assessed the synergistic effects of verapamil, cyclosporine, or FK-506 in combination with MRK-16 and antitumor agents . METHODS: Human myelogenous leukemia K562 cells and multidrug-resistant K562/ADM cells were treated with vincristine or doxorubicin combined with MRK-16 and cyclosporine alone or together; MRK-16 and verapamil alone or together; or MRK-16 and FK-506 . The effects of MRK-16 and cyclosporine or verapamil on the accumulation of vincristine and doxorubicin were examined in K562/ADM cells, and the mechanisms of action were analyzed . RESULTS: MRK-16 and cyclosporine synergistically enhanced the antitumor effects of vincristine and of doxorubicin in K562/ADM cells . However, the combined use of MRK-16 with verapamil or FK-506 did not show such synergistic effects in these cells . Studies of the effect of MRK-16 on cellular accumulation of cyclosporine and verapamil revealed that MRK-16 substantially increased accumulation of cyclosporine in K562/ADM cells, but did not increase accumulation of verapamil . CONCLUSIONS: MRK-16 and cyclosporine synergistically enhanced the antitumor effects of vincristine and doxorubicin because MRK-16 increased cellular accumulation of cyclosporine . IMPLICATIONS: These results, together with our previous finding that intravenous administration of MRK-16 induced regression of multidrug-resistant subcutaneous tumors in athymic mice, support the hypothesis that the combined use of MRK-16 and cyclosporine might increase the efficacy of antitumor agents against multidrug-resistant tumors expressing P-glycoprotein . Clinical phase I trials of MRK-16 in the treatment of multidrug-resistant tumors are under consideration.

Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 952 - 60
Overcoming multidrug resistance in human tumor cells using free and liposomally encapsulated antisense oligodeoxynucleotides; Thierry AR et al.; Antisense oligonucleotides offer a molecular targeting tool for overcoming cellular multidrug resistance . In order to improve the in vitro and the in vivo transport of oligodeoxynucleotides, we developed a new liposomal delivery system, using the minimal volume entrapment (MVE) technique . We have demonstrated that cellular uptake and intracellular release of oligodeoxynucleotides were facilitated by delivery in liposomes . 15 mers cap phosphorothioate oligodeoxynucleotides complementary to the 5' end of the coding region or to a loop-forming site in the mdr-1 mRNA were encapsulated in liposomes by the MVE method . P-glycoprotein synthesis and doxorubicin resistance were greatly reduced by exposure of the multidrug resistant SKVLB cells to 5 microM liposomally encapsulated oligonucleotide . A lower effect was observed when free oligodeoxynucleotides were used . Oligomers antisense to the loop-forming site appeared to be more effective and more specific in modulating multidrug resistance than oligomers with antisense sequence to the 5' end coding region.

Leuk Lymphoma, 1993 Feb, 9(3), 247 - 53
Resistance to lipophilic cationic compounds in multidrug resistant leukemia cells; Ramu A et al.; Previously we have shown that multidrug resistant cells are cross-resistant to certain permanently charged cationic lipophilic compounds . In the present study we extend these observations to additional cationic lipophilic compounds with unrelated chemical structures . Study of the growth inhibitory activity of series of triphenylalkyl-phosphonium and alkyl-ammonium compounds revealed that cross-resistance to these compounds in multidrug resistant P-388 murine leukemia cells, was related to the presence of cationic charge but not to the molecular size/degree of lipophilicity.

Anticancer Drugs, 1993 Feb, 4(1), 37 - 48
Removal of the basic center from doxorubicin partially overcomes multidrug resistance and decreases cardiotoxicity; Priebe W et al.; Hydroxyrubicin, a synthetic doxorubicin analog in which the basic amino group at C-3' is replaced by a hydroxyl group, was used as a prototype compound to study the effects of basicity of the sugar moiety on the toxicity and antitumor activity of anthracycline antibiotics . Compared with doxorubicin, hydroxyrubicin showed similar or superior in vitro cytotoxicity against P388, L1210, and M5076 cells, as determined by an MTT assay, and against 8226 and CEM cells, as determined by a growth inhibition assay . Hydroxyrubicin was 5 and 13 times more effective than doxorubicin in inhibiting the growth of multidrug-resistant CEM (CEMvbl) and 8226 (8226R) cells, respectively . Hydroxyrubicin was not cross-resistant with doxorubicin in a cytotoxicity assay against KB 3-1 and KB V1 cells (resistance index 1.1 for hydroxyrubicin versus > 15.6 for doxorubicin) . Cellular uptake and retention of hydroxyrubicin were studied by flow cytometry in parent and multidrug-resistant 8226 cells, and compared with those of doxorubicin . In 8226 sensitive cells, 2 h uptake and retention of doxorubicin were similar or higher than those of hydroxyrubicin . In 8226R cells, uptake and retention of hydroxyrubicin were about 3-fold higher than those of doxorubicin . In mice, the acute LD50 of hydroxyrubicin was about 3-fold higher than that of doxorubicin (79.1 versus 25.7 mg/kg) . At equitoxic doses, hydroxyrubicin was as myelosuppressive as doxorubicin but less cardiotoxic, as assessed by the Bertazzoli test . In contrast to doxorubicin, hydroxyrubicin, due to the lack of basic amine function, showed no selective interaction with negatively-charged cardiolipin (CL) . The observed decrease of affinity to CL might be responsible for the reduced cardiotoxicity of hydroxyrubicin . In in vivo antitumor activity studies, hydroxyrubicin at the optimal dose (37.5 mg/kg, i.p., on day 1) had significant activity against intraperitoneal P388 leukemia resistant to doxorubicin, whereas doxorubicin (10 mg/kg, i.p., on day 1) was inactive (%T/C 163-200 versus 118-120) . These studies indicate that: (i) the amino group at position 3' is not essential for doxorubicin to exert its biological activity, (ii) removal of the basic center (deamination at the C-3') results in an increased cellular uptake and retention, (iii) the increased cellular uptake and retention of hydroxyrubicin in multidrug-resistant cells correlate with a partial or total lack of cross-resistance of this analog with the parent compound, doxorubicin, and (iv) deamination at position 3' confers a reduced cardiotoxicity and diminished affinity for CL.

Parasitology, 1993 Feb, 106 ( Pt 2), 107 - 15
Transcripts of the multidrug resistance genes in chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum; Ekong RM et al.; Homologues of the mammalian multidrug resistance gene have been identified in isolates and clones of Plasmodium falciparum and designated pfmdr1 and pfmdr2 . Mutations in pfmdr1 have been associated with chloroquine resistance but confirmation could not be obtained in a genetic cross . We have examined the copy number and expression of pfmdr1 and pfmdr2 in chloroquine-sensitive and -resistant P . falciparum and have found no relationship between the copy number of either gene and chloroquine resistance . However, a marked correlation was seen between levels of mRNA transcribed for each gene and chloroquine resistance . Two transcripts of pfmdr1 were detected, and in the asexual blood cycle an 8 kb transcript appeared first, followed by the appearance of a 7 kb species.

Bioengineering, Anthrax, Bioreactor, Bacteria, Antibiotic, Bacteriology, Rhizobia, Microorganism, Growth Medium, Microbes, Molecular Microbiology, Microorganisms, Listeria Monocytogenes, Bacterium, o, Cell Biology, d, Microbiology, n, Biotechnology, j, Microbe, a, Dna, Fermentation, Escherichia coli, b, Yeasts, p, Antibiotics, b, Staphylococcus, s, P. aeruginosa, c, Biodegradation, g, Halophilic bacterium, p, Bacillus subtilis, e, Beta lactamase, l, Antimicrobial, c, Cryptococci, s, Microbial, c, Staphylococcus aureus, m, Yeasts, s, Escherichia coli, b, Enterobacter, a, Streptococcus, e, Microorganism, s, Bacteremia, e, Antibiotics, m, Bacterial, s, P. aeruginosa, p, Eubacterium, a, Staphylococcus, m, Phage, m, S. cerevisiae, c, Streptococcal, m, Campylobacter, e, Escherichia coli, E. coli,

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

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

Last modified: May 25, 2005