Multidrug resistant

Leuk Res, 1995 Apr, 19(4), 275 - 82
Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line; Davey RA et al.; A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin . The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein . Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide . Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis) . Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Leuk Res, 1995 Apr, 19(4), 257 - 61
Evaluation of resistance index of several anticancer agents on parental and resistant P-388 cell lines; Testi R et al.; Multidrug resistance is frequently detected in haematological malignancies and in acute leukaemias with a poor prognosis . In the last few years, several reports seem to suggest that the new anthracycline derivative idarubicin and the anthraquinone mitoxantrone have some advantages in the management of untreated or relapsed acute leukaemias compared with older anthracyclines . This could be due to a different interaction of these drugs with multidrug resistance . To evaluate this possibility, we compared the activity of doxorubicin (DOXO), epirubicin (EPI), idarubicin (IDA) and mitoxantrone (MITO) on a murine, multidrug resistant, leukaemic cell line (P-388/Dx) cultured in vitro . ID50 of IDA and MITO was in the ng range whereas that of DOXO and EPI was in the microgram(s) range . Moreover, IDA has a resistance index of 50 whereas DOXO has one of 250 . Verapamil is able to almost completely abolish the resistance to IDA . Efflux experiments confirm that verapamil increases IDA intracellular concentration . IDA and MITO appear to be less involved in multidrug resistance than older anthracyclines.

Br J Dermatol, 1995 Apr, 132(4), 551 - 5
P-glycoprotein expression in primary and metastatic malignant melanoma; Schadendorf D et al.; Metastatic malignant melanoma is notoriously resistant to chemotherapeutic agents, but the exact mechanisms involved in this drug resistance are unknown . One recently defined major mechanism of multidrug resistance involves the overexpression of P-glycoprotein on cell membranes . In order to evaluate the significance of this putative drug efflux pump for chemoresistance of malignant melanoma, five different antibodies were employed to examine P-glycoprotein expression on tissue from 33 primary malignant melanomas and 35 metastases, before and after chemotherapy, using immunohistological techniques . The expression of P-glycoprotein was low on primary cutaneous melanomas (three of 33), and on metastases (one of 35) . Normal tissue in and around the melanoma showed reactivity of endothelial cells, stromal cells and eccrine sweat glands with several antibodies tested . Chemotherapy with drugs commonly used in metastatic melanoma, including agents known to induce P-glycoprotein expression in other tumours (vindesine, cisplatin) had no effect on P-glycoprotein expression in human melanoma metastases . The high chemoresistance of human melanoma cells in vitro and in vivo is probably not mediated via P-glycoprotein, and other possible mechanisms involved will have to be explored in future studies.

Photochem Photobiol, 1995 Apr, 61(4), 390 - 6
Inhibition of the ATPase activity of P-glycoprotein by porphyrin photosensitization of multidrug-resistant cells in vitro; Gibson SL et al.; The effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied . Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of 14C-polyhematoporphyrin and porphyrin photosensitization . Phototoxicity of Photofrin was similar in both cell lines, and no major differences in uptake or efflux of 14C-polyhematoporphyrin were observed . Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity . Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity . The hydrophobic "picket-fence" porphyrin, meso-tetrakis-(o-propionamidophenyl)porphyrin, alpha,alpha,alpha,beta-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin.

Am J Clin Pathol, 1995 Apr, 103(4), 443 - 8
CD31 quantitative immunocytochemical assays in breast carcinomas . Correlation with current prognostic factors; Charpin C et al.; The distribution of PECAM-1/CD31 molecule was investigated in 133 breast carcinomas using monoclonal antibody and frozen sections . Anti-CD31 labels endothelial cells and reflects stromal angiogenesis . The CD31 immunoreactivity was evaluated by computer-assisted analysis of digitized microscopic images . The automatic screening of the whole preparation and the measurements of the mean CD31 immunostained surface was performed in each case . A similar procedure was achieved for p53, cathepsin D, P-gp, pHER-2/neu, Ki67, pS2 estrogen and progesterone antigenic sites immunodetection . The image analysis of positive CD31 surface was variable, ranging from 4% to 33% (mean 14.7%, SD = 5.43) . The CD31 positive surface correlated (P < .01) with the Nottingham prognostic index, but not with the tumor size, the node status, the tumor grade, nor with the patient age . Also the CD31 immunoreactivity was independent of the pHER-2/neu, Ki67 antigen, p53, ER, PR and pS2 immunodetectable expression in tumors, but correlates with that of cathepsin D (P = .024) and P-gp (P = .028), which reflects the multi-drug resistance capacity of tumor cells . In conclusion, CD31 positive vessels assessed on frozen sections by image analysis constitute an excellent method of evaluating tumor stromal angiogenesis, and can be further used for clinical purposes . The results also suggest that the CD31/PECAM molecule may be involved in the spread of tumor by interacting with extracellular matrix lysis that results from the tumor cell proteasic activity and with multidrug resistance.

Mutat Res, 1995 Apr, 342(3-4), 113 - 23
Increased genotoxicity of acetylaminofluorene by modulators of multixenobiotic resistance mechanism: studies with the fresh water clam Corbicula fluminea; Waldmann P et al.; The presence of a 'multixenobiotic resistance' {MXR} mechanism in gills of the freshwater clam Corbicula fluminea was investigated . Western blot analyses of membrane vesicles from gills, applying antibodies to vertebrate P170 multidrug resistance (MDR) protein, revealed a 135 kDa immunoreactive protein . Verapamil caused a reduction of 3H-vincristine (3H-VCR) binding onto vesicles from clam . Exposure of clams to 3H-VCR in the presence of verapamil or staurosporine (STP) enhanced the accumulation of 3H-VCR over control values . Furthermore, clams were exposed instead to VCR, to a model carcinogen, 2-acetylaminofluorene (AAF), to determine the verapamil- and STP-dependent increase of single-strand breaks (SSBs) in DNA from gills of this organism . Verapamil caused no or little increase of SSBs induced by exposure to 0.01 or 0.10 microM AAF, respectively, as measured by the alkaline elution technique . In contrast, in the presence of STP a highly significant and dose-dependent enhancement of AAF-mediated SSBs was measured already at exposure to 0.01 microM AAF . These data indicate (i) that the clam C . fluminea is provided with a P-glycoprotein-like element of the MDR-mechanism, (ii) that this system can be poisoned by chemosensitizers such as verapamil and STP, (iii) the role of protein kinase C in the regulation of MXR function and (iv) the importance of the MXR modulators for the assessment of ecotoxicological effects of pollutants.

Br J Cancer, 1995 Apr, 71(4), 877 - 81
Continuous-infusion verapamil with etoposide in relapsed or resistant paediatric cancers; Cowie FJ et al.; This study evaluates the use of a multidrug resistance (MDR) modulator (verapamil) in combination with a standard dose of single-agent etoposide in relapsed or refractory paediatric malignancy . A total of 20 patients (median age 6.5 years) were treated with an infusion of verapamil (loading dose 0.1 mg kg-1, followed by continuous infusion 0.15 mg kg-1 h-1) for 72 h . Etoposide was given daily (150 mg m-2 day-1) for three doses (each over 1 h); the first dose was given 12 h into the verapamil infusion . Cardiovascular toxicity was monitored by ECG and 2 hourly blood pressure and pulse recordings . Verapamil and norverapamil plasma concentrations were measured daily . Disease response was assessed after two courses . A total of 29/35 treatment courses were given at the desired verapamil dose; five courses required a dose reduction owing to cardiovascular toxicity . No patient required intensive monitoring . All patients who developed cardiovascular toxicity were over 14 years old . There was no correlation between plasma verapamil or norverapamil concentrations and toxicity . There were six partial responses (three rhabdomyosarcoma, three neuroblastoma) after two courses, but because of variation in the dose and schedule of etoposide these cannot be unequivocally contributed to MDR reversal . In conclusion, a regimen using a continuous infusion of verapamil combined with divided-dose etoposide is tolerable in children, and this strategy may be effective in refractory neuroblastoma and rhabdomyosarcoma.

Br J Cancer, 1995 Apr, 71(4), 738 - 43
Buthionine sulphoximine-mediated sensitisation of etoposide-resistant human breast cancer MCF7 cells overexpressing the multidrug resistance-associated protein involves increased drug accumulation; Schneider E et al.; Preincubation of etoposide-resistant human MCF7 breast cancer cells (MCF7/VP) with buthionine sulphoximine (BSO) resulted in their sensitisation to etoposide and vincristine . Chemosensitisation was accompanied by elevated intracellular drug levels . In contrast, simultaneous exposure to BSO did not result in increased drug accumulation . Similar, but quantitatively smaller, effects were also observed when sensitive wild-type MCF7/WT cells were treated with BSO . In agreement with its effect on drug accumulation, BSO pretreatment also increased VP-16-stimulated cleavable complex formation between DNA topoisomerase II and cellular DNA . BSO treatment also led to a significant increase in acid-precipitable VP-16 levels in MCF7/VP, but not MCF7/WT cells . In contrast, no clear effects of BSO on drug efflux were observed and drug retention was only minimally increased after BSO treatment of both MCF7/WT and MCF7/VP cells and no difference between the two cell lines was detected . Thus, chemosensitisation by BSO appeared to be mediated through increased intracellular drug concentrations and/or protein binding.

Br J Cancer, 1995 Apr, 71(4), 670 - 5
Inhibition of N-linked glycosylation of P-glycoprotein by tunicamycin results in a reduced multidrug resistance phenotype; Kramer R et al.; Characterisation of altered glycosylation of P-glycoprotein (P-gp) found associated with the absence of a multidrug resistance (MDR) phenotype in cell lines prompted an investigation to assess the role of post-translational processing in establishing P-gp efflux pump functionally . The clone A cell line used in this study displays a strong MDR phenotype mediated by high constitutive levels of expression of P-gp . Incubation of clone A cells with tunicamycin for different periods resulted in a time-dependent increase in daunorubicin accumulation, reflecting a reduction in P-gp function . Parallel experiments conducted with verapamil resulted in no loss of P-gp functionality in clone A cells . Reduction in surface-associated P-gp following exposure to tunicamycin was established by FACS analysis, Western blot analysis and immunoprecipitation of surface-iodinated P-gp . In addition, immunoprecipitation of P-gp from 32P-orthophosphate-labelled cells demonstrated reduced phosphorylation of P-gp associated with tunicamycin exposure . From these studies we conclude that glycosylation of P-gp is required to establish the cellular MDR phenotype.

Med Pediatr Oncol, 1995 Apr, 24(4), 235 - 40
Vincristine disposition in children with acute lymphoblastic leukemia; de Graaf SS et al.; Vincristine (VCR) has been widely used to treat childhood malignancies for over thirty years, but its plasma disposition has not yet been well-defined . Therefore, we conducted a pharmacokinetic study of VCR in 17 children with acute lymphoblastic leukemia (ALL) receiving the first dose of VCR . A new high-performance liquid chromatographic assay was used for the measurement of VCR in plasma . A two-compartment pharmacokinetic model was fit to the data by nonlinear least-squares regression . Estimated pharmacokinetic parameters were highly variable; mean (S.D.) volume of distribution at steady-state was 360 (176) L.m-2; total body clearance was 431 (238) ml.min-1.m-2, and elimination half-life was 823 (390) min . These results were compared to data from eight adults with lung cancer . Mean volume of distribution in adults and children were similar, but VCR clearance was significantly larger in children (P = 0.01), resulting in a significantly longer elimination half-life in the adults (P < 0.01) . We conclude that administration of a standard dosage of VCR to children with ALL results in a highly variable systemic drug exposure, which may have implications for the oncolytic effect and/or toxicity in individual patients . Comparison of data from children and adults suggests that VCR elimination rate is a function of age; this could account for more severe neurotoxicity in older patients . However, it cannot be excluded that differences between the children and adults may be due to other variables than age . Future studies should focus on the possible influence of multidrug resistance modulating agents on VCR pharmacokinetics and on pharmacokinetic-pharmacodynamic relationships in individual patients.

Singapore Med J, 1995 Apr, 36(2), 209 - 11
Tuberculosis--fighting a losing battle?
Tan KK.
Tuberculosis has resurfaced as a "global emergency" in recent years not only in terms of increase in number of cases world-wide but also the emergence of the deadly multidrug-resistant tuberculosis . World Health Organisation (WHO) has issued a call for the global community to step up its vigilance against the disease . Chemotherapy is the most powerful tool in the fight against tuberculosis and should be used with utmost care and under stringent conditions . It is not enough just to prescribe the correct medication, but more importantly, the patient must be closely monitored for compliance and progress . Any facility which provides for the treatment of tuberculosis must have a good working mechanism to detect treatment defaulter and take immediate remedial action . Only then can we maintain a high standard of control of the disease and prevent the emergence of drug-resistant organisms.

J Chemother, 1995 Apr, 7(2), 157 - 9
Role of protein kinase beta isozyme in multidrug resistance in murine leukemia P388/ADR cells; Gollapudi S et al.; To define a role of protein kinase C (PKC) in multidrug resistance (MDR), we examined the influence of PKC isozyme specific antibodies delivered intracellularly, on drug sensitivity and drug accumulation in P388/ADR cells . Drug sensitive (P388) and drug resistant (P388/ADR) cells were permeabilized at 4 degrees C with L-lysolecithin and were incubated with rabbit anti-PKC, alpha, beta antibodies, or normal rabbit serum for 10 minutes at 37 degrees C . Daunorubicin (DNR) accumulation and drug sensitivity were studied by flow cytometry and MTT assay, respectively . Anti-PKC beta antibody partially corrected drug accumulation defect and completely reversed resistance to DNR . Anti-PKC alpha antibody had no effect on either parameter of MDR . These results suggest that PKC beta plays an important role in MDR in P388/ADR cells . Furthermore, the technique of intracellular delivery of antibodies provides a new approach to discern the role of PKC isoforms in multidrug resistance in various tumor cells.

Minerva Pediatr, 1995 Apr, 47(4), 147 - 51
{Treatment of metastatic osteosarcoma with verapamil, cyclosporine and chemotherapy . A case report}; Brach Del Prever A et al.; Studies on the use of revertants to overcome MDR have aroused a great interest even if they failed to prove their actual usefulness . A case of 10 years and 6 months old boy is described . He suffered from osteoblastic osteosarcoma and underwent chemotherapy following CNR-NEO3 protocol, wide surgical resection and postoperative chemotherapy . Nineteen months after diagnosis he underwent the removal of a little subpleural nodule in the right lung . Forty-five days later, in the same site, a large local metastasis was observed together with many others secondaries localizations spred in both lungs . Because of the rapid evolution they were considered not suitable for surgical treatment . A cyclosporine and verapamil treatment in association with adryamicin and etoposide was begun with the aim overcoming multidrug resistance . Five treatments were provided . ECG monitoring during verapamil infusion did not show any trouble; mielotoxicity was mild, with no need of transfusions . A lung CT scan at the end of the therapy demonstrated an important decrease of the subpleural metastasis and the vanishing of lung nodules . Another surgical intervention was provided together with 2 postoperative chemotherapy treatments . Twenty-six months later no sign of the disease was observed . Association of verapamil and cyclosporine with chemotherapy allowed to get a good clinical response with a very low toxicity, in a critical situation in which chemotherapy alone did not seem to offer any real possibility.

Hematol Oncol Clin North Am, 1995 Apr, 9(2), 363 - 82
Clinical studies with modulators of multidrug resistance; Fisher GA et al.; Improved understanding of the mechanisms underlying chemotherapeutic failure has led to new strategies to circumvent drug resistance . Expression of the multidrug transporter, P-glycoprotein (P-gp), is likely to be a significant mechanism contributing to the clinical resistance of some cancers to chemotherapy . Phase I trials of currently available MDR modulators have yielded important pharmacologic principles pertaining to normal tissue P-gp function and its influence on the disposition of MDR-related anticancer drugs . Currently available P-glycoprotein inhibitors lack the potency to completely reverse the MDR phenotype at clinically achievable concentrations . Despite this, encouraging clinical results have been obtained in the hematolymphoid malignancies . As these more potent modulators become available, careful characterization of pharmacologic interactions with MDR-related cytotoxins will be critical to the rational design of Phase II and III studies that will ultimately test the efficacy of MDR modulation.

Hematol Oncol Clin North Am, 1995 Apr, 9(2), 337 - 61
Modulators of multidrug resistance . Preclinical studies; Ford JM; The study of the cellular, biochemical, and molecular biology and pharmacology of MDR has provided one of the most active and exciting areas within cancer research for translation into potential clinical benefit . Although convincing evidence for the functional role of P-gp in mediating clinical drug resistance in humans remains scant, studies of the clinical expression of P-gp and trials of chemosensitizers with cancer chemotherapy suggest "resistance modification" strategies may be effective in some tumors with intrinsic or acquired drug resistance . However, even if P-gp-associated MDR proves to be a relevant and reversible cause of clinical drug resistance, numerous problems remain to be solved before effective clinical chemosensitization may be achieved . Such factors as absorption, distribution, and metabolism, the effect of chemosensitizers on chemotherapeutic drug clearance, toxicity to normal tissues expressing P-gp, and the most efficacious modulator regimens all remain to be defined in vivo . Clearly, the identification of more specific, more potent, and less clinically toxic chemosensitizers for clinical use remains critical to the possible success of this approach . However, the finding that a number of pharmacologic agents can antagonize a well-characterized form of experimental drug resistance provides promise for potential clinical applications . Further study of chemosensitizers in humans and the rational design of novel chemosensitizers with improved activity should define the importance of MDR to clinically resistant cancer.

Hematol Oncol Clin North Am, 1995 Apr, 9(2), 275 - 318
Multidrug resistance in pediatric malignancies; Chan HS et al.; PURPOSE: Increased expression of P-glycoprotein is an important cause of multidrug resistance in tumor cell lines in vitro . Whether this mechanism is equally relevant as a cause of clinical chemoresistance has not been established and is currently being investigated . This review has examined the immunohistochemical and molecular biologic tools suitable for assessing P-glycoprotein expression in patient samples and methodologic issues important for evaluating the results of clinical studies . Current evidence that supports a role for P-glycoprotein in limiting the efficacy of cancer chemotherapy has been reviewed . DESIGN: Malignancies that have been successfully treated by chemotherapeutic substrates of P-glycoprotein, in which a proportion of patients still fail therapy, may be the most useful models for determining whether this drug efflux transporter is a clinically relevant cause of chemoresistance . RESULTS: Studies of acute myelogenous leukemia, lymphoma, and myeloma in adults have so far provided the best evidence for a relevant role for P-glycoprotein as a cause of clinical multidrug resistance . A similar strong association has been observed between the expression of P-glycoprotein and outcome of treatment in certain malignancies in children, such as neuroblastoma, rhabdomyosarcoma, and acute lymphoblastic leukemia . Some apparent controversies related to this issue of clinical relevance may be explained by the differences in P-glycoprotein detection techniques, methodology, and experimental designs used in different studies . Because several clinical trials have already been initiated to determine whether pharmacologic chemosensitization improves the outcome of chemotherapy in certain malignancies, the successful verification of multidrug resistance limiting the cure rates of these tumors becomes a more critical issue, and identification of those patients with lower levels of P-glycoprotein expression early in the course of their disease, when they are most likely to benefit from multidrug resistance reversal, has assumed an even greater relevance . CONCLUSION: The clinical relevance of the multidrug resistance P-glycoprotein may ultimately be confirmed by the successful prevention of chemotherapy failure by chemosensitizers that specifically reverse this drug efflux mechanism.

Hematol Oncol Clin North Am, 1995 Apr, 9(2), 251 - 73
P-glycoprotein in adult solid tumors . Expression and prognostic significance; Leighton JC Jr et al.; Several potential mechanisms of chemotherapy resistance have been identified in adult solid tumors . The multidrug resistance (MDR) phenotype is one mechanism by which tumors may simultaneously develop resistance to multiple chemotherapeutic agents and is associated with P-glycoprotein expression . In this article, the authors examine the literature and summarize the various techniques used to measure MDR1 gene expression, patterns of expression in adult solid tumors.

J Nat Prod, 1995 Apr, 58(4), 598 - 604
(-)-Roemerine, an aporphine alkaloid from Annona senegalensis that reverses the multidrug-resistance phenotype with cultured cells; You M et al.; A known aporphine alkaloid, (-)-roemerine {1}, isolated from the leaves of Annona senegalensis, was found to enhance the cytotoxic response mediated by vinblastine with multidrug-resistant KB-V1 cells . In the absence of vinblastine, no significant cytotoxicity was observed with KB-3 or KB-V1 cells (ED50 > 20 micrograms/ml), and several other human tumor cell lines were also relatively insensitive . As indicated by its ability to inhibit ATP-dependent {3H}vinblastine binding to multidrug-resistant KB-V1 cell membrane vesicles, (-)-roemerine appears to function by interacting with P-glycoprotein . In addition to alkaloid 1, three inactive compounds {the aporphine alkaloid(-)-isocorydine (reported in the levo-configuration for the first time), and the lignans (+/-)-8,8'-bisdihydrosiringenin {2} (a new natural product), and (+)-syringaresinol} were also isolated.

Clin Microbiol Rev, 1995 Apr, 8(2), 180 - 99
Tuberculosis in the AIDS era; Sepkowitz KA et al.; A resurgence of tuberculosis has occurred in recent years in the United States and abroad . Deteriorating public health services, increasing numbers of immigrants from countries of endemicity, and coinfection with the human immunodeficiency virus (HIV) have contributed to the rise in the number of cases diagnosed in the United States . Outbreaks of resistant tuberculosis, which responds poorly to therapy, have occurred in hospitals and other settings, affecting patients and health care workers . This review covers the pathogenesis, epidemiology, clinical presentation, laboratory diagnosis, and treatment of Mycobacterium tuberculosis infection and disease . In addition, public health and hospital infection control strategies are detailed . Newer approaches to epidemiologic investigation, including use of restriction fragment length polymorphism analysis, are discussed . Detailed consideration of the interaction between HIV infection and tuberculosis is given . We also review the latest techniques in laboratory evaluation, including the radiometric culture system, DNA probes, and PCR . Current recommendations for therapy of tuberculosis, including multidrug-resistant tuberculosis, are given . Finally, the special problem of prophylaxis of persons exposed to multidrug-resistant tuberculosis is considered.

Int J Cancer, 1995 Mar 29, 61(1), 142 - 7
P-glycoprotein epitope mapping . II . The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain; Cianfriglia M et al.; A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein . In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding . The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

Biochemistry, 1995 Mar 21, 34(11), 3858 - 72
Kinetics of transport of dialkyloxacarbocyanines in multidrug-resistant cell lines overexpressing P-glycoprotein: interrelationship of dye alkyl chain length, cellular flux, and drug resistance; Wadkins RM et al.; The membrane transport properties of a series of dialkyloxacarbocyanine {DiOCn(3)} dyes in multidrug-resistant KB cell lines were investigated to determine the influence of alkyl chain length on the ability of p-glycoprotein (i) to protect cells from the toxicity of the dyes and (ii) to affect the plasma membrane flux of the dyes . Cytotoxicity assays revealed that increased levels of p-glycoprotein led to increased resistance to the toxicity of the DiOCn(3) relative to the sensitive KB-3-1 parent line . This resistance could be fully or partially reversed by 10 microM verapamil . Monitoring of DiOCn(3) fluorescence changes allowed the measurement of accumulation and efflux rates for the dyes in the parent and two resistant cell lines at 1.5-s resolution . The flux of DiOCn(3) into and out of the KB85 and KBV1 cell lines was shown to be dramatically different from the parental KB-3-1 line when n < 5, while the transport properties of n = 7 were identical in the three cell lines examined . The membrane transport properties were shown not to be correlated with the 7-day toxicity of DiOCn(3) . Verapamil affected the kinetic processes of DiOC2-5(3) involving redistribution of the dyes within the cells once they had initially passed the plasma membrane . Fluorescence microscopy was used to show no alteration in the subcellular distribution of the DiOCn(3), in response to neither chain length nor cell line . Our results indicate that an alkyl chain length of 5 carbons is the critical length necessary for p-glycoprotein to affect membrane transport of DiOCn(3) but not to protect the cells from the cytotoxicity of the dyes.

Eur J Biochem, 1995 Mar 15, 228(3), 1020 - 9
Potentiation of anticancer-drug cytotoxicity by multidrug-resistance chemosensitizers involves alterations in membrane fluidity leading to increased membrane permeability; Drori S et al.; We are studying the mechanism underlying chemosensitization of anticancer-drug cytotoxicity in wild-type and multidrug-resistant (MDR) mammalian cells . We show here that the chemosensitizers, reserpine and verapamil, display a dramatic potentiation of taxol, anthracycline and Vinca alkaloids cytotoxicity in P-glycoprotein-(P-gp)-deficient hamster and human nasopharyngeal carcinoma cells . We have therefore utilized this phenomenon to probe for the putative P-gp-independent component of cytotoxicity chemosensitization . These chemosensitizers yielded a marked increase in the accumulation of taxol in parental hamster and human carcinoma cells that are devoid of P-gp . These chemosensitizers and non-ionic detergents brought about a pronounced increase in the accumulation of structurally and mechanistically diverse lipophilic chromophores in parental and MDR hamster cells . Furthermore, non-toxic concentrations of these non-ionic detergents yielded a marked potentiation of taxol cytotoxicity in parental cells . These findings were consistent with a chemosensitizer-mediated, P-gp-independent increase in membrane permeability . Thus, several aspects of chemosensitizers' interaction with lipid bilayers and biomembranes were studied . In this respect, like various mild detergents, chemosensitizers induced a dose-dependent leakage of carboxyfluorescein encapsulated in liposomes . Like specialized membrane fluidizers, various chemosensitizers induced a dose-dependent membrane fluidization (and sometimes rigidification) in both liposomes and various wild-type and MDR animal and human cells, as revealed by diphenylhexatriene fluorescence polarization . Furthermore, a favorable correlation was observed between the ability of chemosensitizers to permeabilize lipid bilayers and their capacity to potentiate anticancer-drug cytotoxicity . Thus, we propose that chemosensitizer-mediated changes in the physical properties of biomembranes, including altered fluidity and increased permeability, may be important factors in achieving potentiation of anticancer-drug cytotoxicity in wild-type and MDR mammalian cells . This study offers a basis for the chemosensitizer-mediated potentiation of drug toxicity to healthy tissues, thus emphasizing the importance of a prior evaluation of the potential untoward toxicity when simultaneously using MDR chemosensitizers and cytotoxic agents in the clinic.

Biochem Pharmacol, 1995 Mar 15, 49(6), 755 - 62
Characterization of an anthracycline-resistant human promyelocyte leukemia (HL-60) cell line with an elevated MDR-1 gene expression; Jonsson K et al.; Multidrug resistance to a variety of cytotoxic drugs is due to decreased drug accumulation at the intracellular site of drug action . When due to increased energy-dependent drug efflux, this transport change is often associated with increased expression of an efflux pump for various lipophilic compounds, for example the P-glycoprotein which is the product of the MDR-1 gene . However, previously described HL-60 human promyelocytic leukemia cell lines resistant to the cytotoxic effect of anthracyclines have been reported not to express P-glycoprotein . We have isolated, by drug selection, an anthracycline-resistant HL-60 cell line that, in comparison to parental drug sensitive cells, exhibits a multidrug resistant phenotype including diminished intracellular drug retention, cross-resistance to multiple cytotoxic drugs, increased expression of a monoclonal antibody C219-reactive 180 kDa P-glycoprotein detected by Western blot analysis as well as increased expression of MDR-1 mRNA as determined by Northern blot and solution hybridization/RNAse protection analyses . Evidence is presented that the anthracycline-resistant HL-60 cells have amplified the MDR-1 gene.

Biochemistry, 1995 Mar 14, 34(10), 3338 - 43
Phosphorylation of the multidrug resistance associated protein gene encoded protein P190; Ma L et al.; Recent studies suggest that multidrug resistance of HL60/ADR cells is related to an overexpression of the MRP (multidrug resistance associated protein) gene which encodes a 190-kDa ATP-binding membrane glycoprotein . In the present study we have further characterized P190 and have examined phosphorylation properties of the protein . The results demonstrate that P190 is highly phosphorylated and that the phosphate groups are metabolically active and undergo cycles of phosphorylation and dephosphorylation in the cell . Serine is the single amino acid phosphorylated in P190 and the phosphate groups are contained in nine tryptic peptides . Experiments have also been conducted to analyze the effect of various protein kinase inhibitors on phosphorylation levels of P190 . The results show that H-7, staurosporine, and chelerythrine can reduce the phosphorylation of this protein . In the presence of both H-7 (200 microM) and staurosporine (200 nM) the phosphorylation of P190 is completely blocked . It has also been found that in the presence of these agents there is a major increase in drug accumulation and concomitant inhibition in drug efflux of resistant cells . These results therefore suggest the possibility that certain phosphate groups of protein P190 play an important role in modulating drug accumulation in resistant cells.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 345 - 52
The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance; Gekeler V et al.; The multidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the multidrug resistance associated protein MRP compared to their drug sensitive parental counterparts . This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach . Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD4 receptor antagonist MK571 . A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 microM (HL60/AR) or 50 microM (GLC4/ADR) which by itself did not disturb cellular proliferation . The drug resistance of a mdr1/P-gp overexpressing multidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571 . Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO) . Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated multidrug resistance.

Int J Cancer, 1995 Mar 3, 60(5), 676 - 84
Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon; Eijdems EW et al.; Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP) . In all cell lines reported thus far, over-expression is associated with gene amplification . We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon . In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification . Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation . In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA . Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR) . By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length . Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification . Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.

Spec Care Dentist, 1995 Mar-Apr, 15(2), 50 - 5
Tuberculosis risk in the hospital dental practice; Harlow RF et al.; Tuberculosis has re-emerged as a serious public health concern . Multidrug-resistant strains and an increase in the number of high-risk groups are posing a difficult problem for health care providers . The risk of TB transmission in hospital dental practices is potentially increasing . A 20-question survey was mailed to the membership of the American Association of Hospital Dentists, addressing various issues relating to tuberculosis . One hundred thirty-two surveys were analyzed . Twelve per cent of respondents reported at least one TB skin test conversion by a dental provider within the past year at their institution . Five respondents reported one dental provider contracting TB through patient contact . Oral TB was reported in 21 cases . Over 34% reported that active TB patients are not isolated to negative-pressure isolation rooms, 45% reported that patients are allowed to frequent public areas, and only 59% believed that drug compliance monitoring was adequate . Over 86% support TB screening in the Hospital Dental Practice . It was concluded that Hospital Dental Practice personnel may be at increased risk for exposure to TB . Dental providers must exercise strict TB prevention and employ meticulous referral and follow-up procedures for high-risk patients.

Southeast Asian J Trop Med Public Health, 1995 Mar, 26(1), 154 - 63
Integration of control measures for malaria vectors in endemic areas of Thailand; Kanda T et al.; Various vector control measures were applied in different endemic areas in two provinces, Saraburi and Chanthaburi, with comparison among different control measures . Application of IGR (insect growth regurator, pyriproxyfen) was introduced at Wat Tam Pra Pothisat, Tab-Kwang District, Saraburi Province . Some integration measures were performed at villages 6 and 8, Patavee, Makham District, Chanthaburi Province . In Tab-Kwang District with low malaria endemicity at the study site predators were not able to be released due to rapid velocity of running water . IGR could effectively control malaria compared to the basin released predators . Another endemic areas villagers 6 and 8, Patavee, Makham, Chanthaburi Province was chosen . Highly endemic multidrug resistant malaria has been prevalent for many years in this area . Integration of Kanda's trapping system, application of IGR, use of both residual spraying and impregnated bed-net methods with etofenprox successfully interrupted malaria infection . The application of these methods as an integrated control system could be adjusted to environmental conditions . The results of this study suggest rapid effective vector control.

Biochem Pharmacol, 1995 Mar 1, 49(5), 603 - 9
Mechanism of action of dexniguldipine-HCl (B8509-035), a new potent modulator of multidrug resistance; Hofmann J et al.; It has previously been shown that dexniguldipine-HCl (B8509-035) is a potent chemosensitizer in multidrug resistant cells {Hofmann et al., J Cancer Res Clin Oncol 118: 361-366, 1992} . It is shown here that dexniguldipine-HCl causes a dose-dependent reduction of the labeling of the P-glycoprotein by azidopine, indicating a competition of dexniguldipine-HCl with the photoaffinity label for the multidrug resistance gene 1 (MDR-1) product . Exposure to dexniguldipine-HCl results in a dose-dependent accumulation of rhodamine 123 in MDR-1 overexpressing cells . In the presence of 1 microM dexniguldipine-HCl, rhodamine 123 accumulated in multidrug resistant cells to similar levels as in the sensitive parental cell lines . At this concentration, dexniguldipine-HCl enhances the cytotoxicities of Adriamycin and vincristine . The resistance modulating factors (RMF), i.e . IC50 drug/IC50 drug + modulator, were found to be proportional to the expression of MDR-1, ranging from 8 to 42 for Adriamycin and from 16 to 63 for vincristine . Transfection with the MDR-1 gene was found to be sufficient to sensitize cells to the modulation by dexniguldipine-HCl . The compound does not affect the expression of the MDR-1 gene . Dexniguldipine-HCl has no effect on a multidrug resistant phenotype caused by a mutation of topoisomerase II . It is concluded that dexniguldipine-HCl modulates multidrug resistance by direct interaction with the P-glycoprotein.

Leukemia, 1995 Mar, 9(3), 513 - 6
MDR-related P170-glycoprotein modulates cytotoxic activity of homoharringtonine; Russo D et al.; Homoharringtonine (HHT) is a new drug with antileukemic activity which is currently tested for treatment of acute and chronic leukemias, either alone or in combination with other agents . Since HHT showed a low efficacy in refractory and relapsed acute leukemia and in the blastic phase of chronic myeloid leukemia (CML) which are frequently characterized by an overexpresion of the multidrug resistance (MDR)-related P170-glycoprotein, we postulated a relationship between the poor antileukemic effect of HHT in these leukemias and the expression of P170-glycoprotein . For this reason, sensitive (LOVO109 and CEM) and MDR (LOVO DX and CEM VLB) cell lines were exposed to HHT with or without some MDR modifiers, namely, Cyclosporine A (CyA), the Cyclosporine derivative SDZ PSC 833 (PSC), and the D-isomer of Verapamil (DVRP) . It was found that MDR cells were about 15 times more resistant to HHT than non-MDR cells, and that resistance to HHT was significantly decreased by all the MDR modifiers that were tested . This in vitro study showed that HHT belongs to the category of MDR-related drugs, like anthracyclines, vinca alkaloids, epipodophylline derivatives, and taxol.

Br J Cancer, 1995 Mar, 71(3), 556 - 61
Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line; Nooter K et al.; Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis . Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2 . Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis . Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo . Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar . Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis . In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation . Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar . No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity . In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment.

Br J Cancer, 1995 Mar, 71(3), 505 - 11
Different vimentin expression in two clones derived from a human colocarcinoma cell line (LoVo) showing different sensitivity to doxorubicin; Conforti G et al.; We selected two clones, isolated from the human colocarcinoma cell line LoVo, showing a sensitivity to doxorubicin similar to (LoVo clone 5) or three times lower than (LoVo clone 7) the parental cell line . Since vimentin was atypically expressed in a human breast carcinoma cell line made resistant to doxorubicin, we looked at vimentin expression in these two clones with spontaneously different sensitivity to the drug . For comparison we used the parental cell line LoVo WT and LoVo/DX made resistant pharmacologically . mRNA for vimentin was undetectable by Northern blot analysis in LoVo WT and in LoVo clone 5, while expression of this gene was high in LoVo clone 7 and in LoVo/DX . This increase in mRNA levels was not related to an amplification of DNA, as suggested by Southern blot analysis . Immunofluorescence and immunocytochemistry findings confirmed, at protein level, the mRNA data . In LoVo clones 5 and 7, there were respectively 8.6% and 71% vimentin-positive cells, although the two clones showed similar expression of multidrug resistance gene 1 (mdr-1) and accumulated intracellular doxorubicin at similar levels . Similarly, drug efflux was the same for both clones . Our results show for the first time that cells resistant to doxorubicin express vimentin independently of the mdr glycoprotein . However when cells from clone 5 were transfected with human vimentin cDNA, they did not become resistant, indicating that vimentin can be considered as a marker of resistance in these cells but does not give rise to a resistant phenotype by itself.

Br J Cancer, 1995 Mar, 71(3), 489 - 97
Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells; Soues S et al.; We have isolated from Chinese hamster ovary cells, 30 sublines resistant to vincristine, doxorubicin or etoposide and 43 sublines evading treatment with a pair of these drugs . Isolated in one step and under low selective pressure, sublines were 3- to 25-fold more resistant to their selecting drug(s) than the parental cells . Possible P-glycoprotein-associated multidrug resistance was investigated through pgp gene copy number and mRNA expression level . DNA topoisomerase II alteration was evaluated from the ability of nuclear extracts to form cleavable complexes . Vincristine (all sublines) and doxorubicin (6/7 sublines) preferentially selected for pgp gene amplification and mRNA overexpression, whereas selection with etoposide resulted in a decrease of cleavable complex formation in 11 out of 13 sublines . A common pgp gene-mediated resistance was found in the 13 doxorubicin plus vincristine-selected sublines, whereas all but one of the 12 etoposide plus vincristine-resistant sublines displayed both pgp mRNA overexpression and decreased ability to form cleavable complexes . Among the 18 doxorubicin plus etoposide selected sublines, five exhibited a decreased ability to form cleavable complexes only, six exhibited pgp mRNA overexpression only and six exhibited both alterations . Overall, drug resistance could not be attributed to either mechanism in three of the 73 sublines . We conclude that under low selective pressure it is possible to find a combination of drugs which require simultaneous selection of more than one resistance mechanism; such cells emerge with very low frequency.

J Cell Biol, 1995 Mar, 128(5), 793 - 804
A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol; Hanada K et al.; Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process . To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways . We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-{(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino}caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold . Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles . When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification . These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification . A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised . After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4 . Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells . These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.

Cancer Res, 1995 Mar 1, 55(5), 1086 - 91
Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters; Cardarelli CO et al.; Multidrug resistance (MDR) may be associated with the expression of the MDR1 gene which encodes the 170-kDa cell surface P-glycoprotein (PGP) acting as an energy-dependent multidrug efflux pump . This pump can be inhibited by a variety of drugs including cyclosporin A, quinidine, and verapamil . Substrate specificity of the MDR1 gene product can be altered by a point mutation at amino acid residue 185 in which valine is substituted for glycine, but the effect of this mutation on inhibition of PGP is unknown . Multidrug-resistant NIH3T3 cells transfected with the MDR1 retroviral vector pHaMDR-1/A (G185) or pHaMDR1/A (V185) expressing comparable levels of PGP were compared for patterns of drug resistance and inhibition of drug resistance by MDR reversing agents . The NIH-MDR-G185 transfectants were somewhat preferentially resistant to daunorubicin, taxol, and vinblastine . The mutant (V185) conferred increased resistance to colchicine . This MDR phenotype in both NIH-MDR-G185- and NIH-MDR-V185-transfected NIH3T3 cells was overcome by the addition of cyclosporin A, quinidine, or verapamil . Verapamil was the most potent of the three agents affecting wild-type PGP . However, specific inhibitors showed different potency with wild-type or mutant transporters, depending on the cytotoxic drug whose resistance was being reversed . For example, cyclosporin A at a concentration of 1 microgram/ml, was a powerful reverser of taxol and colchicine resistance for the mutant drug transporter, but was much less effective for the wild-type transporter . In contrast, verapamil reversed resistance to vinblastine more efficiently for the wild-type transporter than for the mutant transporter . These results suggest that the sensitivity of a multidrug transporter to a reversing agent will depend on the reversing agent, the cytotoxic drug, and the presence or absence of mutations which alter substrate specificity.

J Neurosurg, 1995 Mar, 82(3), 469 - 74
Combination therapy with cisplatin and nifedipine inducing apoptosis in multidrug-resistant human glioblastoma cells; Kondo S et al.; The authors found that multidrug-resistant human glioblastoma GB-1 cells demonstrated significantly more resistance to cisplatin than did nondrug-resistant human glioblastoma U87-MG cells (p < 0.1) . They therefore attempted to determine whether calcium channel blockers enhance the antitumor activity of cisplatin against GB-1 cells . Nifedipine, a dihydropyridine calcium channel blocker, significantly enhanced the antitumor effect of cisplatin on GB-1 cells (p < 0.05) . In the absence of normal extracellular Ca++, nifedipine enhanced the cytotoxicity of cisplatin . In addition, the antitumor activity of combined cisplatin and nifedipine was inhibited both by actinomycin D and cycloheximide, suggesting that such activity is dependent upon new RNA and protein synthesis . Surprisingly, DNA fragmentation assay demonstrated that synergism between cisplatin and nifedipine resulted in apoptosis (programmed cell death) at a relatively low concentration of cisplatin, which when tested alone did not induce apoptosis . In addition, it was demonstrated that nuclei from GB-1 cells lacked a Ca(++)-dependent endonuclease that degrades chromatin into nucleosomes and that calcium ionophore A 23187 did not decrease the viability of GB-1 cells . The above findings suggest the hypothesis that the noncytotoxic agent nifedipine synergistically enhances the antitumor effect of cisplatin on multidrug-resistant GB-1 cells lacking Ca(++)-dependent endonuclease, and subsequently induces apoptosis via its interaction with an as yet uncharacterized functional site other than the calcium channel on GB-1 cells.

Cell Growth Differ, 1995 Mar, 6(3), 347 - 54
Overexpression of the class II P-glycoprotein gene in primary rat hepatocyte culture: evidence for increased mRNA stability; Lee CH et al.; The overexpression of P-glycoprotein (Pgp) appears to be responsible for multidrug resistance in some human cancers . The molecular basis of this overexpression is not understood . We have used primary monolayer cultures of adult rat hepatocytes as a model system to study the regulation of Pgp gene expression (Lee et al., J . Cell . Physiol., 157: 392-402, 1993) . We observed a dramatic and specific overexpression of class II Pgp as a function of the time in culture . This isoform of Pgp, which is expressed at a very low level in normal liver, has also been shown to be predominantly overexpressed in several models of rat liver carcinogenesis . In the present study, we have used nuclear run-on assays and mRNA decay studies to investigate the mechanism for the overexpression of class II Pgp in cultured hepatocytes . We conclude that an increased mRNA stability is the major factor involved in the increased expression of class II Pgp . Studies using various drugs indicate that the integrity of the cytoskeleton is important for the maintenance of high expression of class II Pgp . Disruption of the cytoskeleton in cultured hepatocytes with cytochalasin D did not affect the transcriptional activity of the class II Pgp gene but rapidly destabilized its mRNA . This raises the possibility that an association between class II Pgp mRNA and cytoskeletal elements may underlie the mechanism that regulates class II Pgp mRNA stability . These findings have important implications for our understanding of the overexpression of class II Pgp during liver carcinogenesis.

Trans R Soc Trop Med Hyg, 1995 Mar-Apr, 89(2), 213 - 5
Artemether-mefloquine combination in multidrug resistant falciparum malaria; Bunnag D et al.; Plasmodium falciparum in Thailand is highly resistant to chloroquine and sulfadoxine/pyrimethamine and there is increasing resistance to the alternative antimalarials, quinine and mefloquine . In eastern Thailand, the cure rates of mefloquine at 750 and 1250 mg were 30% and 55%, respectively . The use of drug combinations may be necessary in areas where drug-resistant parasites exist . 159 male Thai patients in Chantaburi, eastern Thailand, were allocated at random to receive either oral artemether at a single dose of 300 mg on the first day followed by mefloquine 750 mg at 24 h and 500 mg at 30 h (group A), or oral artemether at a single dose of 300 mg on the first day, mefloquine 750 mg at 24 h and placebo at 30 h (group B) . The follow-up was on days 1, 2, 7, 14, 21, 28, 35 and 42 . Most patients in both groups had a rapid initial response to treatment, parasitaemia being cleared within 24 h and fever cleared within 48 h in both groups . The cure rates were 97% and 90%, respectively, for groups A and B . No serious adverse effect was seen in either group; mild and transient nausea, vomiting and loss of appetite were noted . The adverse effects did not differ between the 2 groups . The results suggested that a single oral dose of artemether (300 mg) can markedly improve the cure rate of mefloquine at a dose of 750 or 1250 mg in multiple drug-resistant falciparum malaria.

Hum Immunol, 1995 Mar, 42(3), 245 - 53
Cell surface expression of major histocompatibility class I antigens is modulated by P-glycoprotein transporter; Masci AM et al.; P-glycoprotein (Mdr1), a member of the ABC superfamily, is a pump able to transport several compounds across plasma membranes . It displays a high level of similarity with the MHC-linked transporters TAP1 and TAP2 which are involved in the delivery of immunogenic peptides across the endoplasmic reticulum . In the present study we analyze the P-glycoprotein's ability to interfere with the biosynthetic pathway of the MHC class I molecules . Our results show that P-glycoprotein is involved in the modulation of the MHC class I expression in multidrug-resistant tumor cell lines, COS1 cells transfected with mdr1 gene, and human T lymphocytes . Epitope screening evokes the possibility that P-glycoprotein induces a modulation of the different MHC class I forms expressed on the cell surface . We propose that P-glycoprotein is involved in the transport of antigenic protein fragments from the cytosol into the endoplasmic reticulum . The suggested mechanism could be physiologically relevant in tissues displaying a high Mdr1 activity, where this transporter could contribute to the regulation of locoregional immune responses.

Diagn Mol Pathol, 1995 Mar, 4(1), 59 - 65
Correlation of p-glycoprotein detection by immunohistochemistry with mdr-1 mRNA levels in osteosarcomas . Pilot study; Kandel RA et al.; All the factors that influence prognosis in patients with osteosarcomas have not been fully determined . One reported predictor of poor outcome is increased multi-drug resistant gene (mdr-1) expression, as measured by reverse transcription and polymerase chain reaction (RT-PCR) . We examined whether immunostaining for p-glycoprotein, the protein product of mdr-1, could be used instead of RT-PCR to indicate the presence of the multidrug-resistant phenotype . The sensitivity of the immunostaining was determined using KB cell sublines . For 13 cases of osteosarcoma, samples were immunostained for p-glycoprotein and the levels of mdr-1 expression quantitated with use of RT-PCR . Three osteosarcomas with undetectable levels of mdr-1 expression by RT-PCR were negative immunohistochemically . Ten cases showed mdr-1 expression ranging from approximately 1 to 32 copies of mdr-1 mRNA/cell . Of these cases, five cases contained occasional tumour cells with positive immunostaining . There was no correlation between levels of expression and the presence or number of immunoreactive cells . These results indicate that the presence of p-glycoprotein immunostaining does not reliably correlate with the level of mdr-1 expression . However it may be useful in conjunction with RT-PCR to further define different subgroups of osteosarcomas that may have different prognoses, and this is currently under investigation.

Anticancer Drug Des, 1995 Mar, 10(2), 103 - 18
Cinchonine per os: efficient circumvention of P-glycoprotein-mediated multidrug resistance; Genne P et al.; We have previously suggested that quinine and cinchonine could be good candidates for the clinical circumvention of multidrug resistance (MDR) in haematological malignancies because of their tolerance and their retained efficacy in serum . We have also shown that cinchonine was more efficient than quinine as an anti-MDR agent in vitro, ex vivo and in vivo after parenteral administration . Here, we report that cinchonine administered per os (po) is much more active than quinine po in circumventing MDR in rats bearing resistant colon tumours . The pharmacokinetics of cinchonine and quinine administered po in rat are shown to be very different . Cinchonine demonstrates a greater absolute bioavailability than quinine (44% versus 30%, respectively) . Its serum concentration correlates with the anti-MDR activity measured ex vivo and in vivo . Cinchonine administered po does not significantly modify the pharmacokinetics of intravenous doxorubicin (DXR) . However, cinchonine induces a significant increase of DXR uptake in organs which express the mdr1 gene (liver, kidney, lung) . When associated with VAD (vincristine, adriamycin, dexamethasone) combined therapy in rats, cinchonine does not significantly increase the toxicity of the cytotoxic drugs . Based on these experimental data, a phase I clinical trial is currently in progress to test the tolerance of this potent MDR-reversing agent administered po.

Carcinogenesis, 1995 Mar, 16(3), 637 - 41
Gene amplification and multidrug resistance induced by the phosphatase-inhibitory tumor promoter, okadaic acid; Wang SJ et al.; The mechanism by which tumor promoters contribute to cellular transformation and tumorigenesis is not completely understood . To investigate further the molecular events involved in these processes, we used okadaic acid, a non-phorbol ester type tumor promoter that specifically inhibits certain protein phosphatases . We describe here that the continuous treatment of murine NIH 3T3 fibroblast cell cultures with okadaci acid resulted in a 50-fold amplification of two genes, mdr-1a and mdr-1b, that conferred multidrug resistance . As a consequence, the cells became cross-resistant to the cytotoxic effects of adriamycin, an antineoplastic drug used in the treatment of human tumors . Since genetic changes have been correlated with cell transformation and tumorigenesis, our results suggest that these processes may constitute an additional factor contributing to tumor promotion by okadaic acid.

Cancer Genet Cytogenet, 1995 Mar, 80(1), 47 - 54
Evolution of chromosomal alterations and biologic features in two small cell lung carcinoma cell lines established from one patient during the course of the disease; Goguel AF et al.; Two small cell lung cancer (SCLC) cell lines were established from metastases of a patient during the course of the disease . SCLC 74A was derived from biopsy material obtained at the time of diagnosis and SCLC 74B was from a biopsy specimen of a relapsed tumor obtained after treatment . A transition occurred from SCLC 74A, an intermediate form with 5% large cells to SCLC 74B, a standard mixed form with 20% of large cells, with a decrease in neuroendocrine markers and a substantial increase in P-glycoprotein, a multidrug resistance marker . For both cell lines, R-banding and FISH indicated a del(1)(p35pter) also found in other neural-crest-derived tumors, the loss of regions with suspected tumor suppressor genes at 3p, 5q, and 17p, and a recurrent translocation of the 6q24-6qter region to 10p14 . Further genetic modifications in SCLC 74B affected chromosomes 2, 3, 5, 10, 11, 14, and 15 . The main observations were a der(2)t(2;5)(p16;q?); a der(3;11)(q10;p10) in SCLC 74A which became der(3;14)(q10;p10) and der(11;14)(p10;q10) in SCLC 74B; and the insertion of the 5q13-5q31 region in the der(10)t(6;10) . The finding of the same structural abnormalities in both cell lines suggests a monoclonal origin for both metastases . Hypotetraploid cells were in the same proportion as large cells whose number was a characteristic feature of each cell line . They possessed twice the same chromosomal alterations observed in the hypodiploid cells . This suggests a permanent process of tetraploidization.

Bull Cancer, 1995 Mar, 82(3), 211 - 7
Evaluation of multidrug resistant phenotype by flow cytometry with monoclonal antibodies and functional tests; Lizard G et al.; Multidrug resistant (MDR) phenotype is characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp) . MDR phenotype can be characterized either with monoclonal antibodies raised against P-gp or with functional tests, most often based on the incorporation of fluorescent compounds . In the present study, data obtained with the monoclonal antibodies C219, JSB1 and MRK16 are compared to those of functional tests performed by flow cytometry including uptake of daunorubicin (DNR), Rhodamine 123 (Rh 123) or Hoechst 33342 . Sensitive and resistant cell lines K562S, K562R, KBA1 and KB31, derived either from a human chronic myeloid leukemia or from a human epithelial carcinoma, were used . In resistant cells, P-gp expression was revealed with either the monoclonal antibodies C219, JSB1 or MRK-16 . The most specific results were obtained with MRK-16 . With functional tests, no matter which dyes were used, the fluorescence was always stronger in sensitive than in resistant cells . However, with DNR and Hoechst 33342, an incorporation of these dyes was exhibited in resistant cells . This phenomenon was not observed with Rh 123, which makes it possible to distinguish clearly between sensitive and resistant cells and to detect as few as 1% of resistant cells . Because of its high sensitivity, the functional test involving incorporation of Rh 123 was successfully used in acute myeloid leukemia to detect multichemoresistant cells.

Infect Control Hosp Epidemiol, 1995 Mar, 16(3), 160 - 5
Transmission of multidrug-resistant Mycobacterium tuberculosis among persons exposed in a medical examiner's office, New York; Ussery XT et al.; OBJECTIVE: To determine the prevalence of and risk factors for having a positive tuberculin skin test (TST) result among employees at a medical examiner's office (MEO) . DESIGN: Cohort study, environmental investigation . SETTING: Several employees at a medical examiner's office were found to have positive TST results after autopsies were performed on persons with multidrug-resistant tuberculosis (MDR-TB) . PARTICIPANTS: Employees of the MEO . RESULTS: Of 18 MEO employees, 5 (28%) had a positive TST result; 2 of these 5 had TST conversions . We observed a trend between TST conversion and participation in autopsies on persons with MDR-TB (2 of 2 converters versus 3 of 13 employees with negative TST; relative risk = 4.3; 95% confidence interval 1.61 to 11.69; P = 0.10) . The environmental investigation revealed that the autopsy room was at positive pressure relative to the rest of the MEO and that air from the autopsy room mixed throughout the facility . CONCLUSIONS: A systematic approach to preventing transmission of Mycobacterium tuberculosis in autopsy suites should include effective environmental controls and routine tuberculin skin testing of employees.

Infect Control Hosp Epidemiol, 1995 Mar, 16(3), 152 - 9
Nosocomial tuberculosis: an outbreak of a strain resistant to seven drugs; Ikeda RM et al.; OBJECTIVE: To evaluate nosocomial transmission of multidrug-resistant (MDR) tuberculosis (TB) . DESIGN: Outbreak investigation: review of infection control practices and skin test results of healthcare workers (HCWs); medical records of hospitalized TB patients and mycobacteriology reports; submission of specimens for restriction fragment length polymorphism (RFLP) typing; and an assessment of the air-handling system . SETTING: A teaching hospital in upstate New York . RESULTS: Skin-test conversions occurred among 46 (6.6%) of 696 HCWs tested from August through October 1991 . Rates were highest on two units (29% and 20%); HCWs primarily assigned to these units had a higher risk for conversion compared with HCWs tested following previous incidents of exposure to TB (relative risk {RR} = 53.4, 95% confidence interval {CI95} = 6.9 to 411.1; and RR = 37.4, CI95 = 5.0 to 277.3, respectively) . The likely source patient was the only TB patient hospitalized on both units during the probable exposure period . This patient appeared clinically infectious, was associated with a higher risk of conversion among HCWs providing direct care (RR = 2.37; CI95 = 1.05 to 5.34), and was a prison inmate with TB resistant to seven antituberculosis agents . The MDR-TB strain isolated from this patient also was isolated from other inmate and noninmate patients, and a prison correctional officer exposed in the hospital . Mycobacterium tuberculosis isolates from all of these patients had matching RFLP patterns . Infection control practices closely followed established guidelines; however, several rooms housing TB patients had marginal negative pressure with variable numbers of air changes per hour, and directional airflow was disrupted easily . CONCLUSIONS: These data strongly suggest nosocomial transmission of MDR-TB to HCWs, patients, and a prison correctional officer working in the hospital . Factors contributing to transmission apparently included prolonged infectiousness of the likely source patient and inadequate environmental controls . Continued urgent attention to TB infection control is needed.

Infect Control Hosp Epidemiol, 1995 Mar, 16(3), 141 - 7
Evaluation of infection control measures in preventing the nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis in a New York City hospital; Stroud LA et al.; OBJECTIVE: To evaluate the efficacy of Centers for Disease Control and Prevention (CDC)-recommended infection control measures implemented in response to an outbreak of multidrug-resistant (MDR) tuberculosis (TB) . DESIGN: Retrospective cohort studies of acquired immunodeficiency syndrome (AIDS) patients and healthcare workers . The study period (January 1989 through September 1992) was divided into period I, before changes in infection control; period II, after aggressive use of administrative controls (eg, rapid placement of TB patients or suspected TB patients in single-patient rooms); and period III, while engineering changes were made (eg, improving ventilation in TB isolation rooms) . SETTING: A New York City hospital that was the site of one of the first reported outbreaks of MDR-TB among AIDS patients in the United States . PARTICIPANTS: All AIDS patients admitted during periods I and II . Healthcare workers on nine inpatient units with TB patients and six without TB patients . RESULTS: The epidemic (38 patients) waned during period II and only one MDR-TB patient presented during period III . The MDR-TB attack rate among AIDS patients hospitalized on the same ward on the same days as an infectious MDR-TB patient was 8.8% (19 of 216) during period I, decreasing to 2.6% (5 of 193; P = 0.01) during period II . In a small group of healthcare workers with tuberculin skin test data, conversions during periods II through III were higher on wards with than without TB patients (5 of 29 versus 0 of 15; P = 0.15), although the difference was not statistically significant . CONCLUSIONS: Transmission of MDR-TB among AIDS patients decreased markedly after enforcement of readily implementable administrative measures, ending the outbreak . However, tuberculin skin-test conversions among healthcare workers may not have been prevented by these measures . CDC guidelines for prevention of nosocomial transmission of TB should be implemented fully at all US hospitals.

Curr Probl Cancer, 1995 Mar-Apr, 19(2), 65 - 124
Clinical reversal of drug resistance; Goldstein LJ; Although combination chemotherapy has had a significant impact on survival for malignancies such as Hodgkin's disease, testicular cancer, and childhood acute leukemias, the majority of cancers are either initially resistant to chemotherapy (renal, colon, etc.) or are initially chemosensitive but acquire resistance during treatment, such as lymphoma and breast cancer . Resistance to chemotherapy remains an obstacle to the successful treatment of human cancer and has been the subject of numerous investigations aimed at identifying the molecular mechanisms of resistance in cancer cells . An improved understanding of the mechanisms by which tumor cells develop resistance to chemotherapy may not only enhance the activity of cytotoxic therapy in advanced malignancies but may ultimately improve the impact of adjuvant therapy, potentially resulting in prolonging disease-free intervals and survival . In this review, therefore, we discuss our current understanding of the MDR1 gene, encoding P-glycoprotein, which is responsible for one mechanism of multidrug resistance (MDR) . We also review the evidence supporting the clinical relevance of the MDR1 gene and clinical trials aimed at reversing MDR-mediated resistance . Although MDR-mediated drug resistance has been well characterized in preclinical models, its role in clinical drug resistance is not as well characterized and requires further investigation . Prospective studies are necessary to establish the role of MDR1 gene expression in the clinical resistance . The ability to identify tumors with increased MDR1 gene expression has several potential applications (for example, the prediction of response to chemotherapy and the design of studies aimed at reversal of resistance with agents that inhibit MDR-mediated drug efflux) . The initial goal of such trials is to demonstrate the ability to reverse MDR1-mediated drug resistance in the appropriate advanced refractory malignancies . Ultimately, it will be important to incorporate these reversal strategies in the treatment of early-stage disease, at which time the tumor burden is smaller and fewer mechanisms of resistance may be present . Prospective phase I, II, and III clinical trials using reversing agents in conjunction with chemotherapy in malignancies that express the MDR1 gene, such as the hematologic malignancies and breast cancer, are necessary before routine use of agents such as verapamil, quinidine, and cyclosporine, which carry innate toxicities . MDR is a mechanism of drug resistance that provides the potential for an alteration in drug efflux, which may have a significant impact on response and possibly result in improved survival for some cancer patients.

In Vivo, 1995 Mar-Apr, 9(2), 133 - 8
P-170 glycoprotein expression in gastric and colorectal carcinomas and normal mucosa . An immunocytochemical study; Caruso ML et al.; During the past few years it has become apparent that simultaneous resistance of tumour cells to a number of heterocyclic drugs (multidrug resistance) is often correlated with overexpression of a P-glycoprotein (P-gp or P-170) . P-gp expression can be studied by molecular biology and immunohistochemical techniques . The latter provide a rapid, sensitive and specific screening method suitable for testing even a relatively small number of tumour cells like those obtained at biopsy . The aim of this study was to detect and localize the immunohistochemical expression of P-gp in normal and neoplastic gastrointestinal tissue using the Mab JSB-1 in formalin-fixed paraffin-embedded specimens . A particularly striking finding of our study was the consistent, prevalent higher expression of P-gp in the stomach than in the colon, with a higher percentage of immunostaining in normal than in neoplastic tissue . This is in agreement with the fact that not only is the prognosis known to be worse for stomach cancer, but also the response to treatment is lower . Further studies should be carried out to verify the possibility of making routine tests of this kind for the evaluation of multidrug resistance, to guide the selection of patients for treatment of cancer with chemotherapeutic drugs.

Minerva Endocrinol, 1995 Mar, 20(1), 105 - 9
Cytotoxic chemotherapy for adrenocortical carcinoma; Dogliotti L et al.; The efficacy of mitotane in providing objective tumour responses in patients with adrenocortical carcinoma (ACC), has been recently questioned . Experience with non specific chemotherapy is limited . Tumour responses have been reported with cisplatin administered as a single agent or in combination . Other reports however failed to show benefit from cytotoxic chemotherapy . The very low number of patients included in each study, mostly of them previously treated with mitotane, may account for these controversial results . The finding that multidrug resistance mediated by MDR-1/P-glycoprotein can be reverted by mitotane provides a rational basis for exploring the use of mitotane in combination with chemotherapeutic agents . In a multicenter cooperative (SWOG) phase II study, a combination of mitotane+cisplatin appeared active in advanced ACC, with 30% response rate in 37 eligible patients . These results prompted us to evaluate the activity of a combination chemotherapy of Eto-poside, Adriamycin and Cisplatin (EAP) in association with mitotane (4 g daily per os) . Up to now we treated 6 patients, obtaining 3 partial responses . Recently, new drugs as suramin and gossypol have been show to have some activity in patients with surgically unresectable ACC, suggesting the need for further investigation . In conclusion, cytotoxic drugs+mitotane and new adrenocorticolytic/cytotoxic agents, should be explored as first line treatments in patients with advanced ACC . However, due to the extreme rarity of the disease, coordinated multicenter investigations should be highly encouraged.

Haematologica, 1995 Mar-Apr, 80(2), 103 - 7
Glutathione-S-transferase activity and multidrug resistance phenotype in chronic lymphocytic leukemia: do they have any clinical relevance?
Di Simone D, Testi R, Caracciolo F, Capochiani E, Ambrogi F, Grassi B, Petrini M.
BACKGROUND . Lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL) have frequently been shown to be positive for the multidrug resistance (MDR) phenotype . However, this phenotype does not seem to be responsible for the resistance to alkylating agents usually employed in the management of CLL . METHODS . Lymphocytes from 42 patients were evaluated by flow cytometry for P-170 expression and by spectrophotometry for glutathione-S-transferase (GST) activity . RESULTS . Our findings show that GST is not related to any clinical parameter but is increased in treated patients . Conversely, 85% of patients were positive for P-170 and this was related to the percentage of CD5/CD19-positive lymphocytes . CD5/CD19-negative patients were also negative for P-170 . MDR was not related to any clinical parameter evaluated nor to GST activity in lymphocytes . CONCLUSIONS . MDR is constitutively expressed in B-cell chronic lymphocytic leukemia and seems to be related to a CD5/CD19 B-CLL phenotype . The increase of GST activity in treated patients is statistically significant (p < 0.005).

Blood Rev, 1995 Mar, 9(1), 47 - 52
Modulation of multidrug resistance in de novo adult acute myeloid leukemia: variable efficacy of reverting agents in vitro . Eastern Cooperative Oncology Group; Paietta E et al.; The efficacy of verapamil and cyclosporine A as modulators of P-glycoprotein, the multidrug resistance (MDR1) gene product, was studied in leukemic blast cells from 56 patients with de novo acute myeloid leukemia (AML) in vitro . Rhodamine123 dye-efflux was measured flow cytometrically as a cellular parameter reflecting P-glycoprotein activity . While dye-efflux was measurable in 3/4 of the cases, the capacity of the P-glycoprotein inhibitors varied substantially among patients . In 23 patients, P-glycoprotein function was completely inhibited by the resistance modulators, whereas in 17 patients neither verapamil nor cyclosporine had any reverting effect on dye-efflux at concentrations even 10-times higher than achievable in vivo . Cells with a drug-sensitive rhodamine123-pump effluxed more efficiently (p = 0.0016) and contained significantly higher levels of MDR1 specific RNA transcripts (p = 0.0002), as determined by quantitative PCR, than cells exhibiting an efflux process that could not be inhibited . However, flow cytometric evaluation of the staining of gated blast cells with the anti-P-glycoprotein antibody, 4E3.16, revealed no difference in P-glycoprotein expression between modulator-sensitive and -insensitive cases (p = 0.86), indicating disproportionate translation of MDR1 mRNA . In leukemic cell populations with increased P-glycoprotein function that could be inhibited, significantly more blasts expressed the progenitor cell antigen, CD34 (median 83%), than was the case in leukemias with P-glycoprotein activity that could not be inhibited (median 7%) (p = 0.0001) . The present study demonstrates that a substantial fraction of AML patients constitutively display a drug-efflux mechanism suggestive of P-glycoprotein activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosurgery, 1995 Mar, 36(3), 565 - 71; discussion 572
Effects of protein kinase C modulators on multidrug resistance in human glioma cells; Matsumoto T et al.; To identify the role of protein kinase C (PKC) in multidrug resistance, the effects of phorbol-12-myristate-13-acetate (PMA), a PKC activator, or calphostin C, a PKC inhibitor, on intracellular vincristine accumulation and expression of P-glycoprotein phosphorylation were studied in one multidrug-resistant and three multidrug-sensitive human glioma cell lines . Basal PKC activities and immunoreactivities of PKC-alpha and -zeta were higher in multidrug-resistant cells than in multidrug-sensitive cells . There was no significant difference in the immunoreactivity of PKC-delta between multidrug-resistant and -sensitive cells, and immunoreactive PKC-beta, -gamma, and -epsilon were not detected in either multidrug-resistant or -sensitive cells . The treatment of multidrug-resistant cells with 100 nM PMA for 2 hours resulted in the activation not of PKC-zeta but of PKC-alpha, with concomitant decrease in vincristine accumulation and increase in P-glycoprotein phosphorylation . The exposure of multidrug-resistant cells to 100 nM PMA for 24 hours induced down-regulation not of PKC-zeta but of PKC-alpha, with concurrent decrease in vincristine accumulation, and reduced but still increased P-glycoprotein phosphorylation . The treatment of multidrug-resistant cells with 100 nM calphostin C for 2 hours decreased immunoreactive PKC-zeta and not immunoreactive PKC-alpha, inducing increase in vincristine accumulation, with concomitant decrease in P-glycoprotein phosphorylation . There was no evidence of significant change in vincristine accumulation in multidrug-sensitive cells treated with PMA or calphostin C . This may suggest that at least two isozymes of PKC, PKC-alpha and -zeta, are involved in P-glycoprotein phosphorylation and that vincristine efflux function in multidrug-resistant human glioma cells is closely associated with P-glycoprotein phosphorylation and is decreased by PKC inhibitor.

FEBS Lett, 1995 Feb 27, 360(2), 165 - 8
Tamoxifen inhibits uptake and metabolism of ethanolamine and choline in multidrug-resistant, but not in drug-sensitive, MCF-7 human breast carcinoma cells; Kiss Z et al.; Tamoxifen (TAM), a widely used agent in the hormonal therapy of breast cancer, is also an antagonist of P-glycoprotein (P-gp), a cell surface protein which confers drug resistance to cells . Here we report that in an estrogen receptor-deficient multidrug-resistant subline of MCF-7 human breast carcinoma cells (MCF-7/MDR), but not in the parent drug-sensitive cells (MCF-7/WT), clinically relevant concentrations (1-5 microM) of TAM inhibited the uptake and phosphorylation of ethanolamine and choline . These inhibitory effects resulted in decreased synthesis of the corresponding phospholipids . In view of the known dependence of P-gp function on phosphatidylethanolamine (PtdEtn), inhibition of PtdEtn synthesis may represent an additional mechanism by which TAM inhibits P-gp-mediated drug efflux.

Biochem Biophys Res Commun, 1995 Feb 27, 207(3), 1003 - 8
Two distinct K(+)-ATPase activities in rabbit distal colon; Abrahamse SI et al.; The distribution of K(+)-ATPase activity in surface and crypt cells from rabbit distal colon was studied . Separation of surface and crypt cells was validated using the multidrug resistance gene (mdr 1) product, P-glycoprotein, as marker for differentiated surface epithelial cells . Western blot analysis revealed a 6-fold higher expression level of P-glycoprotein in colonic surface cells . K(+)-stimulated ouabain-insensitive ATPase activity was present in surface and in crypt cells . In surface cells, this K(+)-ATPase activity was only partly inhibitable by 10 microM SCH 28080, while in crypt cells K(+)-ATPase activity equalled SCH 28080-sensitive ATPase activity . These results strongly suggest the presence of two distinct K(+)-ATPases in colonic epithelial cells.

Biochim Biophys Acta, 1995 Feb 21, 1260(3), 285 - 93
Isolation and molecular cloning of human sorcin a calcium-binding protein in vincristine-resistant HOB1 lymphoma cells; Wang SL et al.; A vincristine-resistant lymphoma cell line (HOB1/VCR1.0) that is resistant to 1.0 microM of vincristine has been established from a human immunoblastic B lymphoma cell line, HOB1 . HOB1/VCR1.0 cells demonstrated the typical multidrug resistant phenotypes . Using two-dimensional gel electrophoresis, we discovered one protein with a molecular mass of 22 kDa and pI 5.7 that was overexpressed in HOB1/VCR1.0 cells . This protein was purified to the degree of apparent homogeneity by preparative isoelectric focusing and sodium dodecylsulfate-polyacrylamide gel electrophoresis . The identification of this protein with sorcin was revealed by comparing the internal amino acid sequence of three Lys-C digested peptides from the purified protein with the sequence previously determined for hamster sorcin . The complete primary structure of the human sorcin was deduced from nucleotide sequence analysis of its cDNA clones . It is composed of 198 amino acid residues with a calculated molecular weight of 21,676, and its sequence is highly similar to that of hamster sorcin (95%) . Direct-binding assay with calcium showed that human sorcin is a calcium-binding protein with four 'E-F hand' structures typical of calcium-binding sites . Like the sorcin of hamster, two of the calcium-binding sites of human sorcin contain putative recognition sites for cAMP-dependent protein kinase . Southern and Northern blot analyses showed that the human sorcin gene was greatly amplified and overexpressed in resistant HOB1/VCR1.0 cells but not detected in the parental HOB1 cells . The overproduction of this protein in resistant cells implies that sorcin plays a role in expression of the resistant phenotype.

Experientia, 1995 Feb 15, 51(2), 137 - 40
Expression of multidrug resistance (mdr) gene(s) in primary lymphoid organs of chicken immune system during embryonic development; Petrini M et al.; The presence of a multidrug resistance (MDR) related protein, P-170, in normal and pathological lymphoid cells has been described . The present report evaluates the expression of the mdr 1 gene by using the reverse Polymerase Chain Reaction (PCR) on cells obtained from the thymus and bursa of chicken embryos starting from day 12 until hatching . Results show that the thymic cells are positive from day 12 to the end of the observation period . In contrast, mdr mRNA was detected in the bursa from day 14 to day 17 of embryonic life . Possible relationships between the expression of mdr and the development of T and B lymphocytes are discussed.

Gene, 1995 Feb 14, 153(2), 299 - 300
The human mdr1 (multidrug-resistance) gene harbours a long homopyrimidine.homopurine sequence next to a cluster of Alu repeated sequences in intron 14; Pauly M et al.; In order to identify specific DNA sequences useful as 'genetic landmarks' in the construction of a complete map of the human mdr1 (multidrug-resistance) gene, we investigated the introns in the central region . In intron 14, we identified a long stretch of a homopyrimidine.homopurine sequence most probably adopting an unconventional DNA conformation, followed by a cluster of three Alu repeated sequences in an inverted orientation . Here, we describe the structure, formation and nucleotide sequence of these DNA elements.

Gan To Kagaku Ryoho, 1995 Feb, 22(3), 365 - 70
{Development of drugs to overcome drug resistance--basic approaches}; Tsuruo T; P-glycoprotein plays a key role in the mechanisms of multidrug resistance in experimental tumors as well as in clinical tumors both in acquired and intrinsic-type resistance . Thus the therapeutic approaches targeting P-glycoprotein would provide benefits in eradication of drug-resistant tumor cells, although some potential problems concerning side effects still remain to be studied . Practical approaches to overcoming multidrug resistance by targeting the P-glycoprotein would be (1) to use antibodies against P-glycoprotein; and (2) to use agents, including calcium channel blocker-related agents and membrane-modifying agents, that interact with P-glycoprotein . These therapeutic approaches will be discussed in the symposium.

Leukemia, 1995 Feb, 9(2), 350 - 6
Low frequency of activity of P-glycoprotein (P-170) in acute lymphoblastic leukemia compared to acute myeloid leukemia; Ludescher C et al.; The purpose of our investigations was to measure P-glycoprotein (P-170) activity in blast cells of 35 adults with acute myeloid leukemia (AML), and 24 children and adults with acute lymphoblastic leukemia (ALL) at time of diagnosis . Studies were based on a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123 (Rh123), which is transported from the cell by the P-170 pump . Dual-fluorescence staining with Rh123 and phycoerythrin-labeled monoclonal antibodies allowed selective measurement of Rh123 efflux in blast cells . Samples were scored positive when the fraction of blast cells showing Rh123 efflux exceeded 10% after a 120-min incubation . Activity of P-170 was observed in 19 (54%) of the 35 AML cases and was completely blocked in the presence of multidrug resistance inhibitors . Efflux activity was significantly higher in CD34-positive AML samples (p < 0.02) . All AML patients with the FAB-subtype M5 (n = 5) lacked Rh123 pumping activity (p < 0.03) . The complete remission rate in response to induction chemotherapy was significantly higher for Rh123-negative (11/13, 85%) than for Rh 123-positive AML patients (4/15, 27%) (p < 0.007) . At a median follow-up of 9 months overall survival was significantly shorter for Rh123-positive than for Rh123-negative patients (p < 0.05) . In contrast to AML, we could detect Rh123 efflux in only two (8%) out of 24 ALL cases . The immunological subtypes of these two positive cases was of B-ALL and pre-T-ALL . Bone marrow cryostat sections from 13 AML and five ALL patients were further analyzed for staining with monoclonal antibodies MM4.17 and JSB1 . Ten of 13 AML and two of five ALL cases expressed the MDR protein . Our results indicate that there is a rather low frequency of P-170 pumping activity in ALL compared with AML . Further, functional activity of P-170 contributes to chemoresistance in de novo AML.

Am J Pathol, 1995 Feb, 146(2), 398 - 408
Relationship between P-glycoprotein expression and cyclosporin A in kidney . An immunohistological and cell culture study; Garcia del Moral R et al.; P-glycoprotein (P-gp), encoded in humans by the mdr-1 gene, acts physiologically as an efflux pump to expel hydrophobic substances from cells . This glycoprotein is closely related to multidrug resistance in tumor cells and can be modulated by cyclosporin A (CsA) . We investigated the relationship between CsA and P-gp in 52 renal allograft biopsies and in cultures of Madin-Darby canine kidney (MDCK) renal tubule cells to determine whether the intrarenal accumulation of CsA or chronic stimulation with the drug modified the expression of P-gp . Expression of P-gp and CsA was analyzed by immunohistochemistry . Immunostaining was evaluated semiquantitatively . Modulation of P-gp in MDCK cells after chronic stimulation with CsA for 7, 30, and 60 days was analyzed by flow cytometry . P-gp and CsA immunostaining in renal post-transplant biopsies showed considerable overlap in all cases (Spearman's test, r = 0.577, P < 0.001) . After 7 days in vitro, the number of cells expressing P-gp increased progressively; a further increase in mean fluorescence was found after 60 days (P < 0.001, Student's t-test) . Our findings suggest that in non-neoplastic cells, CsA may stimulate P-gp as a mechanism of detoxification . Individual differences in the adaptive responses to glycoprotein may be responsible for the appearance of nephrotoxicity or a CsA-resistant rejection reaction in cases of overexpression on lymphocytes and macrophages.

Emerg Med Clin North Am, 1995 Feb, 13(1), 179 - 98
Tuberculosis in the HIV-infected patient; Waxman S et al.; After decades of decline, tuberculosis has emerged as a global health challenge . In the setting of HIV immunocompromise, TB occurs frequently, early, and often atypically . New infections can take an accelerated course . The usual tests for diagnosing Mycobacterium tuberculosis infection are less sensitive when CD4+ counts are low . Increased prevalence of treatment failure, drug-resistant strains, and nosocomial transmission of multidrug-resistant TB are discussed as are new diagnostic tests that will accelerate the time to diagnosis and allow better epidemiologic tracking . Early recognition, isolation, appropriate therapy, and environmental controls that will protect staff and patients from the risk of exposure are also described.

Br J Cancer, 1995 Feb, 71(2), 306 - 10
Selective photodynamic inactivation of a multidrug transporter by a cationic photosensitising agent; Kessel D et al.; We have characterised sites of photodamage catalysed by the cationic photosensitiser tetrabromorhodamine 123, using P388 murine leukaemia cells and a subline (P388/ADR) which has a multidrug resistance phenotype and hyperexpresses mdr1 mRNA for P-glycoprotein . Fluorescence emission spectra were consistent with sensitiser localisation in hydrophobic regions of the P388 cell, and in more aqueous loci in P388/ADR . Subsequent irradiation resulted in photodamage to the P388 cells, resulting in loss of viability . In contrast, P388/ADR cells were unaffected except for an irreversible inhibition of P-glycoprotein, leading to enhanced accumulation of daunorubicin and rhodamine 123 and a corresponding increase in daunorubicin cytotoxicity . These results are consistent with the premise that substrates for P-glycoprotein are confined to membrane loci associated with the transporter, and indicate a very limited migration of cytotoxic photo-products in a cellular environment.

Br J Cancer, 1995 Feb, 71(2), 294 - 9
Interaction of tamoxifen with the multidrug resistance P-glycoprotein; Callaghan R et al.; Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer . Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown . In this study we demonstrate that tamoxifen interacts directly with P-gp . Plasma membranes from P-gp-expressing cells bound {3H}tamoxifen in a specific and saturable fashion . A 180 kDa membrane protein in these membranes, labelled by the affinity analogue tamoxifen aziridine and azidopine, was shown to be P-gp . Tamoxifen reduced the binding of vinblastine and azidopine to P-gp, and tamoxifen increased {3H}vinblastine accumulation in P-gp-expressing cells to levels approaching those in non-P-gp-expressing cells . However, the cellular accumulation of {3H}tamoxifen itself was not influenced by the presence of P-gp . Thus, tamoxifen appears to reverse multidrug resistance by binding to P-gp and inhibiting the transport of cytotoxic drugs, but does not itself appear to be transported by the protein.

Cancer Res, 1995 Feb 1, 55(3), 603 - 9
In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues; Emerson DL et al.; The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs . We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity . We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin . These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay . The compounds, GI147211 {7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin}, and GI149893 {7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin}, were compared to topotecan, a known water-soluble inhibitor of topoisomerase I . Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble . Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein . The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models . In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60% . The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts . Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds . Further clinical development of this class of compounds is ongoing.

Cancer Res, 1995 Feb 1, 55(3), 590 - 6
Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum(II) by anti-interleukin 6 monoclonal antibody or anti-interleukin 6 receptor monoclonal antibody; Mizutani Y et al.; Cytotoxic chemotherapy has shown little antitumor activity against renal cell carcinoma (RCC) . It has been demonstrated that RCC cells secrete interleukin 6 (IL-6) and express IL-6 receptors (IL-6Rs) . IL-6 inhibits apoptosis and enhances manganese superoxide dismutase expression . Several anticancer chemotherapeutic agents exert their cytotoxic activity in part through the induction of apoptosis and the production of free radicals . Thus, the resistance of RCC cells to the anticancer agents might correlate with IL-6 expression . The present study tested this hypothesis by examining the effect of anti-IL-6 mAb and anti-IL-6R mAb on the sensitivity of human RCC cells to anticancer chemotherapeutic agents . Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with cis-diamminedichloroplatinum(II) (CDDP) or mitomycin C overcame their resistance to CDDP or mitomycin C . However, treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with Adriamycin, vinblastine or 5-fluorouracil did not overcome their resistance to these anticancer agents . Treatment of CDDP-resistant Caki-1 cells (Caki-1/DDP), two other RCC cell lines (ACHN and A704), and three freshly derived RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb reversed the resistance to CDDP in all these tumors . We then studied the effectiveness of other platinum derivatives . Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb enhanced their sensitivity to carboplatin, but not to trans-diamminedichloroplatinum(II) . Several experiments investigated the mechanism of the antibody-mediated sensitization of RCC cells to CDDP . Incubation of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb did not change the intracellular accumulation of CDDP . The expressions of the multidrug resistant phenotype (gp170) and c-myc oncogene were not affected by the antibody-mediated sensitization . Treatment of Caki-1 cells with the anti-IL-6 mAb or anti-IL-6R mAb down-regulated the expression of glutathione S-transferase pi mRNA . This study demonstrates that treatment of RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb can overcome their CDDP-resistance and that the down-regulation of glutathione S-transferase pi expression by anti-IL-6 mAb or anti-IL-6R mAb might play a role in the enhanced cytotoxicity obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer Res, 1995 Feb 1, 55(3), 459 - 62
Sequential coexpression of the multidrug resistance genes MRP and mdr1 and their products in VP-16 (etoposide)-selected H69 small cell lung cancer cells; Brock I et al.; Resistance to drugs included in the multidrug-resistance phenotype has been attributed to overexpression of either mdr1 or MRP genes and their products in numerous cell lines, while coexpression, to our knowledge, has not previously been reported in the same cells . Human small cell lung cancer H69/VP cells were developed by continuous incubation in increasing doses of VP-16 . In reverse transcription-PCR assays we found over-expression of both mdr1 and multidrug-resistance protein (MRP) genes, and immunoblots showed both elevated P-glycoprotein and MRP in H69/VP cells . Double immunocytochemical staining demonstrated the expression of both MRP and P-glycoprotein in the same cells, indicating that the observations do not result from the selection of two independent clones . Examination of early passages of H69/VP cells showed that overexpression of MRP mRNA occurred prior to mdr1 . Thus, cell lines and clinical samples in the future should be tested for both mdr1/P-glycoprotein and MRP since a positive result for one of the phenotypes does not preclude the existence of the other.

Cancer, 1995 Feb 1, 75(3), 815 - 20
A phase III randomized study of oral verapamil as a chemosensitizer to reverse drug resistance in patients with refractory myeloma . A Southwest Oncology Group study; Dalton WS et al.; BACKGROUND . Multiple myeloma is considered to be a drug responsive disease; however, there is no cure for this disease and virtually all patients will develop drug resistance . One form of drug resistance that has been documented is the multidrug resistance phenotype or MDR . METHODS . A randomized trial of the combination of vincristine, doxorubicin, and dexamethasone (VAD) and VAD plus oral verapamil (VAD/v) in drug refractory multiple myeloma patients was performed by the Southwestern Oncology Group . Verapamil was used as a chemosensitizing agent to attempt to overcome or prevent MDR and improve the therapeutic outcome . RESULTS . Response rates between the two treatment arms were similar with an overall response rate of 41% for the VAD alone arm and 36% for the VAD/v arm . Overall survival of patients was also similar with a median survival of 10 months for the VAD arm and 13 months for the VAD/v arm . The toxicity profile was also similar for both treatments, with myelosuppression being the dose-limiting toxicity . No significant correlation was observed between expression of P-glycoprotein, serum verapamil levels, and response to therapy . CONCLUSIONS . No beneficial effect was observed from the addition of oral verapamil to the VAD chemotherapy regimen for the treatment of drug-resistant myeloma patients . More effective and less toxic chemosensitizers are needed to study the role of chemosensitizers in reversing MDR in the clinic.

Eur Respir J, 1995 Feb, 8(2), 278 - 84
Drug-resistant pulmonary tuberculosis in Berlin, Germany, 1987-1993; Schaberg T et al.; Resistance of Mycobacterium tuberculosis (M.tb) strains is an increasing problem worldwide . Since no public health data are available for urban populations in Germany, we investigated resistance in our hospitalized patients (n = 1,011) over the last 7 yrs . We therefore evaluated clinical data and results of susceptibility tests (breakpoint technique/proportion method) for isoniazid, streptomycin, rifampin, pyrazinamide, protionamide and ethambutol . Since 1987, there has been a relatively constant rate of 5.9% (3.9%-7.8%) for single-drug resistance (SDR), but an increasing rate of multidrug-resistant (MDR) strains (> or = 2 first-line drugs) from 1.7% in 1987 to 5.8% in 1993 . Sixty nine percent of patients with MDR strains showed resistance to two drugs, and 31% to three or more drugs . Risk factors for SDR and MDR tuberculosis revealed previous therapy (odds ratio (OR) (95% confidence interval (95% CI)); SDR 2.2 (1.7-4.0); MDR 4.5 (2.3-8.8)); and foreign-born status (SDR 2.2 (1.3-3.6); MDR 3.5 (1.8-6.8)) to be the most important factors associated with resistance . Both primary and acquired resistance were higher in foreign-born than in German-born patients . We conclude that there was a considerable increase in multidrug-resistant tuberculosis in our hospital from 1987 to 1993 . Since previously treated patients and patients born in countries with a high level of primary resistance had an increased risk of drug-resistant tuberculosis, we would advise a four drug regimen as initial therapy in those patients.

Eur J Nucl Med, 1995 Feb, 22(2), 177 - 80
Sequential functional imaging with technetium-99m hexakis-2-methoxyisobutylisonitrile and indium-111 octreotide: can we predict the response to chemotherapy in small cell lung cancer?
Moretti JL, Caglar M, Boaziz C, Caillat-Vigneron N, Morere JF.
A case of small cell lung carcinoma (SCLC) demonstrating uptake on functional indium-111 octreotide scintigraphy is presented . Technetium-99m hexakis-2-methoxyisobutylisonitrile (MIBI) scintigraphy clearly delineated an absence of radionuclide uptake at the tumour site . This suggested the presence of multidrug resistance-mediated P glycoprotein (Pgp) on tumour cells, which recognizes certain chemotherapeutic agents as well as MIBI as a substrate and avoids radionuclide concentration . Following three courses of chemotherapy, the patient failed to improve and eventually died . This case demonstrates the importance of functional images, which have the potential to predict the outcome in response to chemotherapy.

Anticancer Drugs, 1995 Feb, 6(1), 135 - 46
Cyclosporin A, verapamil and S9788 reverse doxorubicin resistance in a human medullary thyroid carcinoma cell line; Massart C et al.; Multidrug resistance was investigated in TT cells, a human medullary thyroid carcinoma (MTC) cell line and in normal thyrocytes . MDR1 mRNA was revealed by polymerase chain reaction (PCR) analysis both in normal and neoplastic cells despite the absence of glycoprotein P (Pgp) by immunohistochemistry using JSB-1 monoclonal antibody . Glutathione-S-transferase mRNA was undetectable by Northern blotting in TT cells . Doxorubicin-induced cytotoxicity was evaluated in TT cells with MTT, lacticodehydrogenase (LDH), glutathione (GSH) assays and neutral red uptake . IC50 values obtained for MTT assays were higher than those obtained with the three other tests . Cyclosporin A (CSA) (3 microM), verapamil (10 microM) and S9788 (5 microM) partially reversed the resistance to doxorubicin after a 48 h co-incubation (followed by a 24 h post-incubation for the S9788) . Under these conditions, GSH levels were altered by verapamil and S9788, whereas CSA decreased LDH activity . CSA and verapamil had no effect on MTT assay . In conclusion this MTC cell line exhibited over-expression of the MDR1 gene and its resistance to doxorubicin can be partially reversed by CSA, verapamil and S9788.

Cytometry, 1995 Feb 1, 19(2), 126 - 33
Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P-glycoprotein activity?
Lahmy S, Viallet P, Salmon JM.
Although bisbenzimidazole-DNA interactions have been studied in solution, little information has been available in living cells . The reduced accumulation of the nuclear dye Hoechst 33342 (H342) in cells with multidrug resistant (MDR) phenotype suggested its possible use in a functional test for detection of these cells . We performed experiments to elucidate the mechanisms involved in the H342-exclusion from resistant cells . As contradictory results have been reported in literature, we compared the entire fluorescence spectra of H342 in solution and in intact living cells under different experimental conditions . The study was performed by fluorescence image cytometry . This technique allow accurate quantification of the amount of H342 bound to DNA in living cells . The dye uptake was followed in sensitive and resistant cells, a lymphoblastoid cell line, CCRF-CEM, and its resistant variant selected with vinblastine CEM/VLB100 under conditions that could modulate H342-cell binding . Competition experiments with sodium azide, verapamil, and vinblastine indicated that resistant cells did not differ in the number of possible binding sites for H342 . The obtained results ruled out the possibility of discriminating cells on the basis of a spectral shift . Two modes of binding, differing in their affinity for the dye, seem to co-exist in intact cells . Although it clearly appeared that the P-glycoprotein expressed in MDR cells was mainly responsible for the H342-exclusion, other mechanisms might also be involved.

Cell Biol Int, 1995 Feb, 19(2), 113 - 9
Colchicine-resistance and enhancement of P-glycoprotein activity after co-cultivation of drug-sensitive cells with multidrug resistant variants; Eliseenkova AV et al.; The role of cellular interactions in the resistance of Djungurian hamster cells to colchicine (CH) and in the efficiency of P-glycoprotein function was studied . Mixtures of CH-resistant and CH-sensitive cells as well as control unmixed cells were propagated for 3 days and the sensitivity of the cells to CH was measured by colony forming assay . Identification of individual subpopulations was possible due to genetic marker (6TG-resistance) . The data show that the survival of CH-sensitive cells in CH-supplemented medium increased after co-cultivation with CH-resistant counterparts . To measure Pgp activity the fluorescent dye RH123 and FACScan analysis were used . Pgp-mediated RH123 efflux increased after co-cultivation of CH-sensitive and CH-resistant cells.

Calcif Tissue Int, 1995 Feb, 56(2), 170 - 4
P-glycoprotein is expressed in parathyroid epithelium and is regulated by calcium; Axiotis CA et al.; P-glycoprotein (Pgp), the multidrug resistance (mdr) gene product, has been described in normal tissues with diverse physiologic functions . A broad role as a transporter protein for toxins, hormones, and physiologic metabolites has been provisionally deduced, based on structural analysis and immunoanatomic localization . Recently, significant levels of Pgp have been demonstrated in endocrine and hormonally responsive tissues and tumors . We examined calcium-regulated, clonal parathyroid epithelial (PT-r) and endothelial cells (BPE-1) and frozen parathyroid tissue from normal human parathyroid, parathyroid hyperplasia, parathyroid adenoma, and parathyroid carcinoma for expression of the multidrug resistance gene (Mdr1) and Pgp utilizing Northern and Western analysis and immunohistochemistry . We also investigated the effect of extracellular calcium (eCa) on Pgp expression in PT-r cells at the molecular/cellular level . Immunohistochemistry, utilizing three murine monoclonal antibodies (MAbs)--C494, JSB-1, and C219--which recognize spatially distinct cytoplasmic epitopes of Pgp, revealed strong immunoreactivity in PT-r cells, normal parathyroid, and parathyroid hyperplasia, and weak immunostaining in parathyroid adenomas . BPE-1 cells, endothelial cells, and parathyroid carcinoma were negative . PT-r cells showed a single 130 kDa band (120 KDa after glycosidase treatment) on Western blot and a 4.6 kb transcript on Northern analysis, consistent with Pgp . Western and Northern blot analysis of PTr cells cultured in different eCa concentrations showed that eCa up-regulated Pgp expression.

Dev Dyn, 1995 Feb, 202(2), 172 - 80
Mouse multidrug resistance 1a/3 gene is the earliest known endothelial cell differentiation marker during blood-brain barrier development; Qin Y et al.; Molecular mechanisms of endothelial cell differentiation during blood-brain barrier (BBB) development is not well understood due to the lack of specific molecular markers . Here we describe that expression of the mouse multidrug resistance 1a/3 (mdr1a/3) gene can be detected specifically in subsets of vascular endothelial cells associated with neural tissues at as early as embryonic day 10.5 (E10.5) . This onset of mdr1a/3 gene expression coincides with the previously described first appearance of morphologically distinct endothelial cells in neural tissues during BBB development . To our knowledge, the mdr1a/3 gene is the earliest endothelial cell differentiation marker gene during BBB development described thus far . In addition, we have found that neither the level nor pattern of mdr1a/3 gene expression in BBB endothelial cells is affected by aberrant cortical neuronal layers in mutant mouse reeler.

Curr Opin Pediatr, 1995 Feb, 7(1), 6 - 12
Tuberculosis in children; Costello AM et al.; Tuberculosis in children is a growing problem, both globally and in many industrialized countries . With a rising incidence, clinicians will again face the challenge of the myriad presentations of tuberculous disease . Multidrug resistance and HIV coinfection present additional complications . Renewed interest in tuberculosis among scientists has led to progress in our understanding of antimycobacterial effector mechanisms, the regulation of Th1 and Th2 immune responses, virulence factors, and the potential for immunotherapeutic agents to reduce the duration of antituberculous therapy.

J Antibiot (Tokyo), 1995 Feb, 48(2), 113 - 8
A novel bioactive delta lactone FD-211 . Taxonomy, isolation and characterization; Nozawa O et al.; During our screening program for natural product drugs effective against multidrug-resistant mammalian cells, we have discovered a new delta lactone FD-211 from the fermantation broth of Myceliophthora lutea TF-0409 . FD-211 had a broad spectrum activity against cultured tumor cell lines, including adriamycin-resistant HL-60 cells.

Mol Carcinog, 1995 Feb, 12(2), 61 - 5
Phenotypic variability in induction of P-glycoprotein mRNA by aromatic hydrocarbons in primary human hepatocytes; Schuetz EG et al.; To determine whether human liver responds to treatment with aromatic hydrocarbons (AHs) with induction of the multidrug resistance (mdr) gene product P-glycoprotein and whether AH induction of mdr involves the Ah receptor, we compared induction of mdr mRNA with induction of cytochrome P450 (CYP)1A1 mRNA in AH-treated cultures of primary human hepatocytes . Hepatocytes from all 15 individuals tested responded to treatment with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with induction of CYP1A1 mRNA . However, only 62% and 55% of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA . Indeed, in some individuals mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction . These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway . The dramatic variability in AH induction of mdr may be a predictive risk factor that will help to identify an individual's risk of AH-associated toxicities.

J Bioenerg Biomembr, 1995 Feb, 27(1), 77 - 84
Multidrug resistance and P-glycoproteins in parasitic protozoa; Ullman B; Drug resistance has emerged as a devasting impediment to the treatment and control of diseases of parasitic origin . The underlying mechanisms that contribute to this drug resistance in field isolates, however, are poorly understood . Members of the P-glycoprotein gene (pgp) family have been identified, cloned, and sequenced in Plasmodia, Leishmania, and Entamoeba, and variations in pgp copy number and/or expression have been implicated as a basis for drug resistance in each of these genera . The spectrum of drugs to which parasitic protozoa containing amplified pgp genes and/or transcripts are refractory range from a phenotype similar to that observed with multidrug-resistant mammalian cells to those that are completely distinct . The availability of molecular probes to pgp genes provides valuable reagents to dissect the role of pgp gene amplification and overexpression in mediating drug resistance in parasitic protozoa and to determine the physiological function of P-glycoproteins in this clinically consequential group of human pathogens.

J Bioenerg Biomembr, 1995 Feb, 27(1), 71 - 6
Yeast multidrug resistance: the PDR network; Balzi E et al.; This minireview describes a network of genes involved in multiple drug resistance of the yeast S . cerevisiae . The transcription regulators, PDR1, PDR3, PDR7, and PDR9 control the expression of the gene PDR5, encoding a membrane protein of the ATP-binding-cassette superfamily and functioning as a drug extrusion pump . Next to PDR5, several other target genes, encoding membrane pumps of the ABC type, such as SNQ2, STE6, PDR10, PDR11, YOR1, but also other membrane-associated (such as GAS1, D4405) or soluble proteins (such as G3PD), involved or not in multidrug resistance, are found to be controlled by PDR1 . On another side, the PDR3 regulator participates with its homolog PDR1 to co- and auto-regulation circuits of yeast multidrug resistance.

J Bioenerg Biomembr, 1995 Feb, 27(1), 7 - 13
Using purified P-glycoprotein to understand multidrug resistance; Shapiro AB et al.; Since P-glycoprotein was discovered almost 20 years ago, its causative role in multidrug resistance has been established, but central problems of its biochemistry have not been definitively resolved . Recently, major advances have been made in P-glycoprotein biochemistry with the use of purified and reconstituted P-glycoprotein, as well as membranes from nonmammalian cells containing heterologously expressed P-glycoprotein . In this review we describe recent findings using these systems which are elucidating the molecular mechanism of P-glycoprotein-mediated drug transport.

J Bioenerg Biomembr, 1995 Feb, 27(1), 53 - 61
Effects of phosphorylation of P-glycoprotein on multidrug resistance; Germann UA et al.; Cells expressing elevated levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug resistance phenotype . Studies involving protein kinase activators and inhibitors have implied that covalent modification of P-glycoprotein by phosphorylation may modulate its biological activity as a multidrug transporter . Most of these reagents, however, have additional mechanisms of action and may alter drug accumulation within multidrug resistant cells independent of, or in addition to, their effects on the state of phosphorylation of P-glycoprotein . The protein kinase(s) responsible for P-glycoprotein phosphorylation has(ve) not been unambiguously identified, although several possible candidates have been suggested . Recent biochemical analyses demonstrate that the major sites of phosphorylation are clustered within the linker region that connects the two homologous halves of P-glycoprotein . Mutational analyses have been initiated to confirm this finding . Preliminary data obtained from phosphorylation- and dephosphorylation-defective mutants suggest that phosphorylation of P-glycoprotein is not essential to confer multidrug resistance.

J Bioenerg Biomembr, 1995 Feb, 27(1), 43 - 52
Heterologous expression systems for P-glycoprotein: E . coli, yeast, and baculovirus; Evans GL et al.; Chemotherapy, though it remains one of the front-line weapons used to treat human cancer, is often ineffective due to drug resistance mechanisms manifest in tumor cells . One common pattern of drug resistance, characterized by simultaneous resistance to multiple amphipathic, but otherwise structurally dissimilar anticancer drugs, is termed multidrug resistance . Multidrug resistance in various model systems, covering the phylogenetic range from bacteria to man, can be conferred by mammalian P-glycoproteins (PGPs), often termed multidrug transporters . PGPs are 170-kD polytopic membrane proteins, predicted to consist of two homologous halves, each with six membrane spanning regions and one ATP binding site . They are members of the ATP-binding cassette (ABC) superfamily of transporters, and are known to function biochemically as energy-dependent drug efflux pumps . However, much remains to be learned about PGP structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of drug transport . Much of the recent progress in the study of the human and mouse PGPs has come from heterologous expression systems which offer the benefits of ease of genetic selection and manipulation, and/or short generation times of the organism in which PGPs are expressed, and/or high-level expression of recombinant PGP . Here we review recent studies of PGP in E . coli, baculovirus, and yeast systems and evaluate their utility for the study of PGPs, as well as other higher eukaryotic membrane proteins.

J Bioenerg Biomembr, 1995 Feb, 27(1), 37 - 41
Drug-stimulated ATPase activity of the human P-glycoprotein; Scarborough GA; The human multidrug resistance protein, or P-glycoprotein (Pgp), exhibits a high-capacity drug-dependent ATP hydrolytic activity that is a direct reflection of its drug transport capability . This activity is readily measured in membranes isolated from cultured insect cells infected with a baculovirus carrying the human mdr1 cDNA . The drug-stimulated ATPase activity is a useful alternative to conventional screening systems for identifying high-affinity drug substrates of the Pgp with potential clinical value as chemosensitizers for tumor cells that have become drug resistant . Using this assay system, a variety of drugs have been directly shown to interact with the Pgp . Many of the drugs stimulate the Pgp ATPase activity, but certain drugs bind tightly to the drug-binding site of the Pgp without eliciting ATP hydrolysis . Either class of drugs may be useful as chemosensitizing agents . The baculovirus/insect cell Pgp ATPase assay system may also facilitate future studies of the molecular structure and mechanism of the Pgp.

J Bioenerg Biomembr, 1995 Feb, 27(1), 31 - 6
ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells; Senior AE et al.; ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied . Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein . Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5 . A high-affinity binding site involved in catalysis was not apparent . Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP . DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory . Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.

J Bioenerg Biomembr, 1995 Feb, 27(1), 23 - 9
Purification and reconstitution of functional human P-glycoprotein; Ambudkar SV; The overexpression of the P-glycoprotein, the MDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors . P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds . We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport . P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column . Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent {3H}vinblastine accumulation . Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate . In addition, the vinblastine transport was inhibited by verapamil and daunorubicin . These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter . The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.

J Bioenerg Biomembr, 1995 Feb, 27(1), 15 - 22
Characterization and functional reconstitution of the multidrug transporter; Sharom FJ; P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CHRC5 cells using a selective two-step detergent extraction procedure . The partially purified protein displays a high level of ATPase activity, which has a high KM for ATP, is stimulated by drugs, and can be distinguished from that of other membrane ATPases by its unique inhibition profile . Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures . P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity . Proteoliposomes containing P-glycoprotein display osmotically sensitive ATP-dependent accumulation of 3H-colchicine in the vesicle lumen . Drug transport is active, generating a stable 5.6-fold concentration gradient, and can be blocked by compounds in the multidrug resistance spectrum . Reconstituted P-glycoprotein also exhibits a high level of ATPase activity which is further stimulated by various drugs . P-Glycoprotein therefore functions as an active drug transporter with constitutive ATPase activity.

J Bioenerg Biomembr, 1995 Feb, 27(1), 63 - 70
P-glycoprotein and cell volume-activated chloride channels; Higgins CF; The multidrug resistance P-glycoprotein (P-gp) is an active drug transporter which can expel hydrophobic compounds from cells . Expression of P-gp has many effects on cells and tissues and the physiological function, or functions, of P-gp are still unclear . Recently, expression of P-gp has been associated with altered activity of chloride channels which play a role in regulating cell volume of response to osmotic shock or nutrient uptake . The nature and physiological role of this association has been a subject of some debate . In this article, mechanisms by which P-gp might influence cell volume-activated chloride currents is discussed, and the potential physiological role of this regulation considered.

Leuk Lymphoma, 1995 Feb, 16(5-6), 419 - 24
Expression of MDR1 by normal bone marrow cells and its implication for leukemic hematopoiesis; Drach J et al.; Expression of MDR1 is a well-characterized mechanism leading to resistance of tumor cells to drugs like vinca-alkaloids, anthracyclines, and epipodophyllotoxins . In hematopoiesis, recent data indicate that not only leukemic cells, but also some populations of normal hematopoietic cells, particularly CD34+ progenitor cells as well as peripheral blood lymphocytes, express a functional multidrug-resistant phenotype . Among CD34+ cells, we found evidence that myeloid committed precursor cells (CD34+/CD33+) have lower levels of MDR1 expression than earlier CD34+ cell populations, but there was no difference in MDR1 expression between CD34+/HLA-DR- and CD34+/HLA-DR+ subpopulations . During normal myeloid differentiation, MDR1 expression is down-regulated, which is similar to our observations in acute myelogenous leukemia (AML): MDR1 expression was only rarely detected in acute promyelocytic leukemia, which was in contrast to other subtypes of AML; also, within leukemic subpopulations of the same patient, higher MDR1 levels were correlated with a more immature immunophenotype . Regarding regulation of MDR1 expression, we did not observe changes of MDR1 expression in normal CD34+ cells in response to various cytokines . However, in 2 patients with AML treated with interleukin-3 and granulocyte-colony stimulating factor, respectively, a significant down-regulation of MDR1 expression was found after 24 hours . In conclusion, there is evidence that the pattern of MDR1 expression observed in leukemias reflects the distribution of MDR1 in normal hematopoiesis . In contrast to normal CD34+ cells, leukemic cells from some AML patients can respond to cytokines with a down-regulation of MDR1, which may contribute to response to cytokine/chemotherapy combinations.

J Clin Oncol, 1995 Feb, 13(2), 381 - 6
Phase II study of paclitaxel in relapsed non-Hodgkin's lymphomas; Wilson WH et al.; PURPOSE: To assess the efficacy and toxicity of paclitaxel administered as a 96-hour infusion to patients with relapsed non-Hodgkin's lymphomas (NHLs) . PATIENTS AND METHODS: Eligible patients had relapsed NHL and measurable disease and were considered incurable . Paclitaxel was infused at a dose of 140 mg/m2 every 3 weeks . Premedications to prevent paclitaxel hypersensitivity reactions were not administered and no patients received corticosteroids . Expression of the multidrug resistance (mdr-1) gene was determined in tumor from 17 patients by mRNA quantitative polymerase chain reaction (PCR) . RESULTS: Thirty-one patients received a total of 99 cycles of paclitaxel . Two patients were not assessable for response . The median age was 50 years, 71% had stage IV disease, and intermediate/high-grade histology was present in 65% of patients . Patients had received a median of three prior chemotherapy regimens, and 68% of patients had responded to the previous chemotherapy (chemotherapy-sensitive) . Of 29 assessable patients, five (17%) achieved a partial response (PR) . With a median potential follow-up time of 17 months, the median event-free and overall survival durations were 1.6 and 7.5 months, respectively . No correlation was found between response to paclitaxel and extent of prior treatment or response . The mdr-1 gene was easily detectable in 14 of 17 tumor biopsies, but was low in all but one sample . The most serious toxicity was grade 4 neutropenia, which occurred during 14% of cycles . CONCLUSION: Paclitaxel was well tolerated, but had a low response rate in patients with relapsed NHLs . There was no clear association between response to paclitaxel and extent of our response to prior treatment . Most patients had chemotherapy-sensitive disease, which suggests that the low response rate to paclitaxel was probably not due to general chemotherapy resistance . Paclitaxel provided good palliation in a minority of patients and is a reasonable agent to consider for use in patients who have failed to respond to standard chemotherapy.

Cancer Lett, 1995 Jan 27, 88(2), 171 - 8
An M(r) 7-kDa membrane protein overexpressed in human multidrug-resistant ovarian cancer cells; Yang X et al.; We have developed a monoclonal antibody (designated 1D7) which recognizes an M(r) 7-kDa plasma membrane protein overexpressed in ovarian MDR cancer cells . The expression of the M(r) 7-kDa protein in various human multidrug-resistant and drug-sensitive cell lines was analysed by Western blot and flow cytometry methods . The small molecular weight protein was overexpressed in the human ovarian carcinoma cell line, SKVLB which was selected for vinblastine resistance from SKOV3 cells and in OVCAR 4/ADR100 and OVCAR 4/VBL200 which were generated from NIH:OVCAR4 by stepwise selection against adriamycin and vinblastine, respectively . Only a minor amount of the M(r) 7-kDa protein was found in the parent cell line, SKOV3 . It was not found in other drug-resistant human cell lines such as the vinblastine-resistant CEM cells (CEM/VLB300), the intrinsic MDR colon cell line HCT15 and the human MDR breast cancer cell line, MCF7/AdrVp . 1D7 specifically inhibited the proliferation of the resistant cells . Our results suggest that the expression of the M(r) 7-kDa protein on the plasma membrane of ovarian MDR cancer cells may be involved in a mechanism related to the proliferation of the drug resistant cancer cells.

J Biol Chem, 1995 Jan 27, 270(4), 1894 - 8
Wild type p53 stimulates expression from the human multidrug resistance promoter in a p53-negative cell line; Goldsmith ME et al.; The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line . Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells . Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression . Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels . MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1 . This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence . Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.

J Biol Chem, 1995 Jan 27, 270(4), 1742 - 6
Topological determinants of internal transmembrane segments in P-glycoprotein sequences; Zhang JT et al.; P-glycoprotein (Pgp) is a polytopic membrane protein responsible for multidrug resistance in cancer cells . Previously, we have used a coupled cell-free translation/translocation system to investigate the membrane orientation of Pgp sequences and have made the unexpected observation that predicted transmembrane (TM) segments from both the NH2-terminal and COOH-terminal halves inserted in microsomal membranes in two different orientations (Zhang, J.-T., Duthie, M., and Ling, V . (1993) J . Biol . Chem . 268, 15101-15110) . How these topological forms of Pgp are regulated is not known . In the present study, we have used site-directed mutagenesis to investigate if the amino acids surrounding the internal TM segments of Pgp may affect their orientation . We discovered that the charged amino acids flanking TM4 are important in determining the membrane orientation of the NH2-terminal half molecule of Pgp . This is a novel observation demonstrating the existence of internal topogenic sequences in a mammalian polytopic membrane protein . These findings thus suggest A) that the topological structure of a mammalian polytopic membrane protein does not integrate into the membrane simply by following the lead of the first inserted TM segment but that internal TMs may have independent topogenic information and B) that the TM segments in a multi-spanning membrane protein may be more dynamic than have been previously anticipated, i.e . mutations in the amino acids surrounding internal TMs could drastically change the overall topology of the molecule.

J Natl Cancer Inst, 1995 Jan 18, 87(2), 94 - 104
Enhancement by recombinant human interferon alfa of the reversal of multidrug resistance by MRK-16 monoclonal antibody; Fogler WE et al.; BACKGROUND: The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance . Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance . PURPOSE: Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp) . METHODS: Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma . The parental cell lines HT-29par and MCF-7 were used as controls . The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression . The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice . RESULTS: For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells . Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or {3H}vincristine efflux . rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells . Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days) . Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice . In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001) . Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001) . CONCLUSIONS: MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.

J Natl Cancer Inst, 1995 Jan 18, 87(2), 123 - 8
Reversal of multidrug resistance in human colon cancer cells expressing the human MDR1 gene by liposomes in combination with monoclonal antibody or verapamil; Sela S et al.; BACKGROUND: Colorectal cancer is a major cause of cancer-related mortality in the world and the second leading cause of neoplastic death in the United States . A major obstacle in the chemotherapy of this neoplasm is the emergence of multidrug resistance that is frequently associated with the expression of P-glycoprotein (p170) encoded by MDR1 (also known as PGY1) genes . Previously, we demonstrated that liposome-encapsulated doxorubicin is more cytotoxic than free doxorubicin in human promyelocytic leukemia and human breast cancer cells with the multidrug-resistant phenotype . PURPOSE: Our purpose was to investigate modulation of multidrug resistance by liposome-encapsulated vincristine (VCR) in a drug-resistant human colon cancer cell line HT-29mdr1 and the potentiation of this modulation in combination with monoclonal antibody MRK-16 or verapamil . METHODS: HT-29 parental cells and HT-29mdr1 cells were exposed to free VCR or liposome-encapsulated VCR alone or in combination with MRK-16 or verapamil . Cytotoxicity of cells after various treatments was determined by neutral red staining, and cellular content of VCR was measured by using radiolabeled VCR; p170 expression of cells was assessed by azidopine . RESULTS: HT-29mdr1 cells express a high amount of p170, thus conferring sixfold to sevenfold resistance to VCR compared with the parent cell line . Liposome-encapsulated VCR lowers drug resistance in HT-29mdr1 cells fourfold; IC50 values (concentration that causes 50% reduction in cell number) were 12.5 +/- 2.5 ng/mL compared with 42.5 +/- 5.0 ng/mL with free VCR . IC50 values for free VCR with empty liposomes were 25 +/- 1.25 ng/mL . The combination of MRK-16 and free VCR produced a twofold increase in cytotoxicity over free VCR in p170-expressing cells; the combination of MRK-16 and liposome-encapsulated VCR produced a 10-fold potentiation of cytotoxicity . toxicity . Nonspecific monoclonal antibody NR-LU-10 had no effect on cytotoxicity of HT-29mdr1 cells with free VCR or liposome-encapsulated VCR . The combination of 1.5 microM verapamil potentiated the cytotoxicity of free VCR ninefold to 10-fold, IC50 values reduced to 5.0 +/- 1.5 ng/mL, and in combination with liposome-encapsulated VCR, IC50 values reduced to 2.5 +/- 1.0 ng/mL, demonstrating a 15- to 17-fold potentiation of cytotoxicity . There were no significant differences in drug accumulation in HT-29mdr1 cells when treated with liposome-encapsulated VCR or free VCR . Liposomes inhibited the photoaffinity labeling of azidopine to p170 HT-29mdr1 cells . CONCLUSIONS: Liposome encapsulation of VCR effectively modulates multidrug resistance in human colon cancer cells and may become an important modality in treatment for colon cancers.

Ann Intern Med, 1995 Jan 15, 122(2), 90 - 5
Efficacy of control measures in preventing nosocomial transmission of multidrug-resistant tuberculosis to patients and health care workers; Maloney SA et al.; OBJECTIVE: To assess the efficacy of control measures in decreasing nosocomial transmission of multidrug-resistant tuberculosis . DESIGN: Retrospective cohort study . SETTING: A teaching hospital in New York City . POPULATION: 40 patients hospitalized with multidrug-resistant tuberculosis (case-patients) and health care workers receiving tuberculin skin testing . INTERVENTIONS: Centers for Disease Control and Prevention (CDC) 1990 guidelines for preventing transmission of tuberculosis, including 1) prompt isolation and treatment of patients with tuberculosis; 2) rapid diagnostic techniques for processing Mycobacterium tuberculosis specimens; 3) negative-pressure isolation rooms; and 4) molded surgical masks for health care workers . MEASUREMENTS: Proportion of case-patients with nosocomially acquired tuberculosis and rate of tuberculin skin test conversion among health care workers before and after implementation of control measures . RESULTS: The proportion of patients with multidrug-resistant strains of M . tuberculosis decreased after the interventions (10 of 70 {14%} compared with 30 of 95 {32%} patients before the intervention; relative risk {RR}, 0.5; 95% CI, 0.2 to 0.9) . Before onset of multidrug-resistant tuberculosis, case-patients in the intervention period were as likely to be hospitalized on high-risk wards containing patients with tuberculosis (4 of 10 compared with 17 of 30 patients; RR, 0.7; P = 0.5) but were less likely to be exposed to another case-patient with tuberculosis (1 of 10 compared with 20 of 30 patients; RR, 0.2; P = 0.003) . Tuberculin skin test conversion rates for health care workers assigned to wards housing patients with tuberculosis were lower in the intervention period than in the preintervention period (4 of 78 {5%} compared with 15 of 90 {17%} conversions; P = 0.02), decreasing to levels observed for workers assigned to other wards (4 of 78 {5%} compared with 9 of 228 {4%} conversions; P = 0.7) . CONCLUSIONS: Implementing control measures reduced nosocomial transmission of multidrug-resistant strains to patients and health care workers.

Eur J Pharmacol, 1995 Jan 13, 292(2), 119 - 25
Potentiation of growth inhibition due to vincristine by ascorbic acid in a resistant human non-small cell lung cancer cell line; Song EJ et al.; A human cell subline (PC-9/VCR) resistant to vincristine was established from non-small cell lung cancer PC-9 cells by incremental exposure of the cells to vincristine . The resistant cells showed phenotypic resistance to vincristine (10-fold), colchicine (6.9-fold) and cisplatin (1.4-fold) but they showed sensitivity to other chemotherapeutic agents including melphalan and etoposide VP-16 . The characteristics of the vincristine resistance was partially inhibited (5-7-fold) by co-treatment of PC-9/VCR cells with a nontoxic concentration of L-ascorbic acid (25 micrograms/ml) . Co-treatment or 96 h pre-treatment with ascorbic acid resulted in potentiation of the vincristine effect on the resistant, but not on the sensitive, cell line . The growth inhibition due to vincristine treatment after 24 or 96 h growth in ascorbic acid-free medium was decreased in the resistant as well as in the sensitive cell line . In both cell lines, enhanced growth rate has been shown after ascorbic acid treatment . Similarly, cross-resistance of PC-9/VCR cells to colchicine could also be blocked by ascorbic acid . In addition, a nontoxic concentration of verapamil, a known multidrug resistance inhibitor, did not affect the resistant phenotype of PC-9/VCR cells . These findings suggest that an ascorbic acid-sensitive mechanism may be involved in drug resistance per se in the human lung cancer cells, which differs from the classical phosphoglycoprotein-mediated or previously reported non-phosphoglycoprotein-mediated multidrug resistance.

Arch Biochem Biophys, 1995 Jan 10, 316(1), 135 - 40
Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein; Urbatsch IL et al.; Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I . L . Urbatsch, M . K . al-Shawi, and A . E . Senior (1994, Biochemistry 33, 7069-7076) . The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity . Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously . Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E . coli lipids . Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids . Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids . It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.

Biochemistry, 1995 Jan 10, 34(1), 32 - 9
Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators; Tang-Wai DF et al.; Possible functional differences between P-glycoproteins (P-gps) encoded by the human MDR1 and mouse mdr1 and mdr3 genes with respect to drug resistance profiles and sensitivity to known modulators have been investigated . For this, the three genes were introduced and overexpressed in the same cellular background, that of Chinese hamster LR73 ovary cells, and drug-resistant clones expressing comparable amounts of the corresponding P-gps were selected under the same conditions . Analysis of the specific drug resistance profiles encoded by each P-gp for colchicine, adriamycin, vinblastine, and actinomycin D revealed overlapping but distinct patterns of drug resistance for the three isoforms . While all three P-gps conferred levels of resistance to vinblastine that did not vary by more than 2.5-fold, each isoform could be clearly distinguished by its capacity to confer resistance to colchicine and actinomycin D . Likewise, the study of structurally related and unrelated P-gp modulators indicated strong isoform-specific differences in the capacity of individual modulators to abrogate vinblastine resistance in the corresponding mdr transfectants . The study of several disubstituted piperazine analogs indicated that minor chemical modifications of the linker region of this modulator had strong effects on the sensitivity profile of each isoform to the modulator . Together, these results indicate that the three P-gp isoforms analyzed have specific and distinguishable functional characteristics with respect to interactions with drugs and modulators . These findings also suggest that P-gp positive murine transplantable tumors should be used with caution in the design and in vivo testing of novel P-gp modulators to be used to reverse multidrug resistance to tumor cells expressing human MDR1.

Cancer Lett, 1995 Jan 6, 88(1), 37 - 40
Inhibition of protein kinase C by a synthetic peptide corresponding to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 envelope glycoprotein; Ward NE et al.; This report describes the inhibition of protein kinase C (PKC) by a synthetic peptide corresponding to a viral sequence expressed in mammalian cells . The peptide corresponds to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp41), and it inhibits Ca(2+)- and phosphatidylserine (PS)-dependent phosphorylation of synthetic peptide substrates and histone by purified PKC with IC50 values ranging from 9 to 32 microM . Although previously described pKC-inhibitory synthetic peptides corresponding to sequences expressed in mammalian cells are also effective against the phosphorylation of synthetic peptide substrates, they fail to affect PKC-catalysed phosphorylation of potent protein substrates such as histone . This may limit their usefulness as inhibitors of PKC-catalysed protein phosphorylation in cellular systems . PKC activation is a major contributing factor in multidrug resistance (MDR) in cancer . Our observation that the synthetic peptide gp41(828-848) inhibits pKC-catalysed phosphorylation of a protein substrate suggests the potential value of expressing the viral sequence gp41(828-848) in cancer cells as a novel in vitro model system of MDR reversal.

J Biol Chem, 1995 Jan 6, 270(1), 33 - 6
The release of fibroblast growth factor-1 from NIH 3T3 cells in response to temperature involves the function of cysteine residues; Jackson A et al.; Fibroblast growth factor (FGF)-1 is released from NIH 3T3 cells in response to heat shock as a biologically inactive protein that is unable to bind heparin and requires activation by (NH4)2SO4 to generate a biologically active extracellular heparin-binding growth factor (Jackson, A., Friedman, S., Zhan, X., Engleka, K . A., Forough, R., and Maciag, T . (1992) Proc . Natl . Acad . Sci . USA 89, 10691-10695) . To further study the mechanism of FGF-1 release in response to heat shock (42 degrees C), we examined the kinetics of FGF-1 release from FGF-1-transfected NIH 3T3 cells and observed that the cells require at least 1 h of exposure to heat shock conditions for the release of FGF-1 . Interestingly, agents that interfere with the function of the endoplasmic reticulum-Golgi apparatus, exocytosis, and the multidrug resistance pathway (brefelden A, methylamine, and verapamil, respectively) do not inhibit the release of FGF-1 in response to temperature; rather, they exaggerate the release of FGF-1 . Because immunoblot analysis of FGF-1 in the conditioned medium of heat-shocked NIH 3T3 cells revealed the presence of a minor band with an apparent molecular weight of a FGF-1 homodimer and because we have previously shown that FGF-1, but not FGF-2, is able to form a homodimer in response to chemical oxidation by CuCl2 (Engleka, K . A., and Maciag, T . (1992) J . Biol . Chem . 267, 11307-11315), we examined whether reducing agents would substitute for (NH4)2SO4 and activate extracellular FGF-1 . Indeed, dithiothreitol and reduced glutathione are able to individually generate a FGF-1 monomer as a heparin-binding protein from the conditioned medium of heat-shocked NIH 3T3 cell transfectants . To confirm that cysteine residues are involved in the release of FGF-1 in response to temperature, we used mutagenesis to prepare a human FGF-1 Cys-free mutant in which Cys30, Cys97, and Cys131 were converted to serine . Analysis of the release of the FGF-1 Cys-free mutant in NIH 3T3 cells transfected with the FGF-1 Cys-free mutant demonstrated that the FGF-1 Cys-free mutant is not released into the conditioned medium in response to temperature . Interestingly, exposure of the NIH 3T3 cell FGF-1 Cys-free transfectants to brefelden A followed by heat shock also demonstrated the absence of the extracellular FGF-1 Cys-free mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

EMBO J, 1995 Jan 3, 14(1), 68 - 75
Protein kinase C-mediated phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels; Hardy SP et al.; The multidrug resistance P-glycoprotein (P-gp), which transports hydrophobic drugs out of cells, is also associated with volume-activated chloride currents . It is not yet clear whether P-gp is a channel itself, or whether it is a channel regulator . Activation of chloride currents by hypotonicity in cells expressing P-gp was shown to be regulated by protein kinase C (PKC) . HeLa cells exhibited volume-activated chloride currents indistinguishable from those obtained in P-gp-expressing cells except that they were insensitive to PKC . HeLa cells did not express detectable P-gp but, following transient transfection with cDNA encoding P-gp, the volume-activated channels acquired PKC regulation . PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P-gp were replaced with alanine . Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation . These data demonstrate that PKC-mediated phosphorylation of P-gp regulates the activity of an endogenous chloride channel and thus indicate that P-gp is a channel regulator.

Int J Cancer, 1995 Jan 3, 60(1), 108 - 14
Nuclear DNA content and chromatin texture in multidrug-resistant human leukemic cell lines; Dufer J et al.; Nuclear morphological alterations associated with multidrug resistance (MDR) were evaluated by image cytometry in various human leukemic cell sub-lines: 3 cell lines with P-gp-mediated resistance (CEM-VLB, HL60/Vinc, K562-Dox), the non-Pgp-mediated MDR HL60/AR leukemic cell line with over-expression of MRP, and the at-MDR CEM-VMI leukemic cell line with alteration of topoisomerase II . All these MDR cell sub-lines were obtained by drug selection and were compared with their sensitive counterparts and with the hamster LR73-R cell line obtained by transfection of mouse mdrl cDNA . All MDR cell sub-lines obtained by drug selection displayed decreased DNA Feulgen stainability as compared with their respective sensitive parental cell line, a phenomenon not observed in the transfected LR73-R cells . Nuclear texture analysis on G0/G1-selected cell nuclei revealed 2 types of textural phenotype . The first phenotype was characterized by chromatin decondensation with small but compact chromatin clumps, and was observed in drug-selected P-gp-mediated MDR cells (CEM-VLB, HL60-Vinc, K562-Dox) and in the non-P-gp-mediated MDR HL60/AR cell line . The second phenotype was characterized by a condensed and homogeneous chromatin pattern, and was observed in the at-MDR CEM-VMI cell line . LR73-R cells transfected with mdrl cDNA did not display any significant changes in textural phenotype as compared with sensitive LR73 cells, suggesting that P-gp over-expression alone cannot account for the cytological modifications observed in MDR cells . These data suggest that multidrug resistance could be associated with specific nuclear morphological changes which appeared to be a consequence of alterations occurring during selection by cytotoxic drugs rather than of P-gp over-expression.

Prog Liver Dis, 1995, 13, 101 - 23
Regulation and function of the multidrug resistance genes in liver; Silverman JA et al.; The P-glycoproteins are integral membrane proteins that function as ATP-dependent transporters . The multidrug resistance genes which encode P-gp comprise a small gene family, with 2 members in humans and 3 in rodents . The P-gp encoded by the mdr1 gene functions as a drug efflux pump to remove drugs from cells and may serve as a barrier to protect cells from cytotoxic agents . In normal tissues, P-gp is localized on the luminal surface of transporting epithelia in the liver, kidney, small intestine, testes, and blood-brain barrier . Transient exposure to drugs transcriptionally increases the level of expression of the mdr1 genes, however, the cellular pathways critical to this regulation are yet unknown . This observation may have some implications on the level of expression in tumors and response to chemotherapy . Examination of the basal level of MDR expression in tumors may not be a reliable predictor of the effect of P-gp on chemotherapy . Induction of MDR transcription by drugs may further impede the effectiveness of anti-cancer agents . This is most obvious for drugs which are substrates for P-gp transport, however, it also applies significantly to compounds which are not themselves substrates but affect the response to other drugs simultaneously or subsequently administered . A clear understanding of the mechanisms that regulate basal and drug-induced mdr transcription will facilitate development of novel agents which circumvent this obstacle or permit targeted modification of mdr expression . Expressed on the bile canalicular surface of the liver, P-gps represent the first ATP-dependent biliary transporters to be characterized . The P-gp encoded by mdr2 is the major form of P-gp expressed in normal liver and transports phospholipids into bile . A defect in this protein leads to severe liver disease caused by chronic inflammation of the biliary system that results from high concentrations of free bile salts . The cellular origin and molecular basis of the ensuing liver tumors in these mice are unclear . It is possible that the chronic damage to the biliary ductules causes an increased growth rate of the surrounding cells, including putative stem cells in the liver . Thus, these mice may serve as a model for carcinogenesis in which the liver is under constant promotion placing the proliferating cells at increased risk to further genetic alterations or expansion of preexisting, but normally quiescent, mutations . Mdr2-deficient animals may also provide a model for human chronic inflammatory liver disease . Clearly, these exciting results indicate that further characterization of the P-gps as normal physiologic canalicular membrane transporters is necessary.

Cancer Detect Prev, 1995, 19(6), 527 - 34
Flow cytometry evaluation of the multidrug-resistant phenotype with functional tests involving uptake of daunorubicin, Hoechst 33342, or rhodamine 123: a comparative study; Lizard G et al.; Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp) . The MDR phenotype can be characterized either by use of monoclonal antibodies raised against P-gp or with functional tests based on the intracellular accumulation of fluorescent molecules . The aim of the present study was to compare the effectiveness of functional tests performed by flow cytometry including uptake of daunorubicin (DNR) (2 micrograms/ml), Hoechst 33342 (5 micrograms/ml), or rhodamine 123 (RH 123) (0.1 microgram/ml); and to evaluate the effect of cell death induced by heating at 60 degrees C for 2 h on incorporation of DNR and RH 123 . Sensitive and resistant human hematopoietic K 562 cells expressing P-gp were identified by monoclonal antibodies C 219 and MRK-16 . Fluorescence of the dyes was always higher in sensitive than in resistant cells . However, DNR and Hoechst 33342 produced a slight incorporation in resistant cells, while RH 123 showed lack of incorporation in resistant cells . Thus, RH 123 allows sensitive and resistant cells to be clearly distinguished . In case of cell death, accumulation of RH 123 and DNR were different . With RH 123, fluorescence intensity strongly decreased in sensitive cells . With DNR, fluorescence intensity was enhanced in resistant cells . Thus, when the MDR phenotype is defined by uptake of DNR or RH 123, artifactual results due to cell death may be avoided by using a dye such as propidium iodide to eliminate dead cells.

Scand J Infect Dis Suppl, 1995, 98, 17 - 8
Drug-resistant mycobacterium tuberculosis; some data from Sweden, Estonia and Ethiopia; Hoffner SE; Drug-resistant tuberculosis is a threat not only to the estimated 50 million people in the world carrying resistant strains, but also to the success of national TB control programmes . In Sweden, multidrug-resistant (MDR) tuberculosis comprises around 1% of the isolated strains . Isoniazid resistance is found in 7% of strains and resistance to other drugs in < 3% . Of patients with resistant TB 70% are born outside Scandinavia . Preliminary data from Estonia show that close to 20% of the strains are resistant to each of the drugs isoniazid, rifampicin and streptomycin . MDR is seen in around 16% . In a study in Ethiopia isoniazid resistance was found in 46% and MDR in 11% among treatment failures and relapse cases.

Scand J Infect Dis Suppl, 1995, 98, 15 - 6
Mycobacterial infections in Sweden; Romanus V; The incidence of tuberculosis has continued to decline in the Swedish-born population, but there has been an increase in the foreign-born population, especially among young adults and children . Multidrug-resistant tuberculosis is still rare in Sweden . The annual number of patients with culture confirmed atypical mycobacteria, especially of M . avium-intracellulare, has increased . The increased incidence of atypical mycobacteria observed in non-BCG vaccinated Swedish children seems to be related to the restricted BCG policy brought into force in 1975.

Oncol Res, 1995, 7(10-11), 559 - 64
TNF-induced apoptosis in multidrug resistant friend erythroleukemia is not influenced by the P-glycoprotein and glutathione status of the cell line; Flugy A et al.; The effects that TNF-alpha exerts on Friend erythroleukemia (FLC) and on one multidrug resistant variant (FLC-DXR) of the cell line were studied . Resistance to doxorubicin of FLC-DXR entails two mechanisms: overexpression of P-glycoprotein; and increased glutathione-related activities . Both these might also decrease the effects of the cytokine . Nonetheless, TNF caused even greater cytotoxicity and apoptosis, with no induction of differentiation markers, in FLC-DXR . In addition, TNF produced minor changes of the levels of reduced and oxidized glutathione in the cell lines, and its cytotoxic effects were not inluenced by agents that modify the cell glutathione content such as buthionine sulfoximine, ethacrynic acid, or N-acetyl cysteine . We can exclude that the mechanisms of drug resistance of FLC-DXR prevent the response to the cytokine.

Oncol Res, 1995, 7(10-11), 517 - 27
Novel mechanism of resistance to paclitaxel (Taxol) in human K562 leukemia cells by combined selection with PSC 833; Jaffrezou JP et al.; A paclitaxel-resistant cell line, KPTA5, was established by co-selecting the parental erythroleukemic cell line K562 with stepwise increased concentrations of paclitaxel (Taxol) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype . KPTA5 cells are 9-fold resistant to paclitaxel and taxotere, but do not exhibit significant resistance to Vinca alkaloids, etoposide, anthracyclines, antimetabolites, or alkylating agents . Doubling time and morphology were similar to the parental K562 cells . Reverse transcriptase-polymerase chain reaction (rt-PCR) analysis revealed no alterations in the expression of the mdr1 and MRP genes . Cellular paclitaxel accumulation was unchanged . Cell cycle analyses showed that at 20 nM there was a significantly higher proportion of K562 cells blocked in G2/M, in comparison with KPTA5 cells . In both cases, disruption of the mitotic spindles and the presence of multiple mitotic asters were comparable but occurred at lower paclitaxel concentrations in K562 cells than in KPTA5 cells . There was no difference in total tubulin content between K562 and KPTA5 cells as analyzed by immunoblotting with an anti-beta-tubulin monoclonal antibody . However, we found that KPTA5 cells presented a 2-fold increase both in 5 beta-tubulin mRNA expression and in the corresponding tubulin protein Class IV isotype content, as evaluated by rt-PCR and immunostaining . In conclusion, the KPTA5 cell line displays a novel mechanism of resistance to paclitaxel which does not involve altered cellular drug accumulation . The data presented suggest that alterations in expression of the 5 beta-tubulin gene may be involved in paclitaxel resistance.

Acta Biochim Pol, 1995, 42(4), 509 - 16
Mechanisms of resistance to azole antifungals; Marichal P et al.; Until the late eighties, clinical resistance to azole antifungals was a rare phenomenon . Only a few cases of resistance to ketoconazole were found in patients with chronic mucocutaneous candidiasis (CMC) . The spread of AIDS and the widespread prophylactic and therapeutic use of the hydrophilic azole compound fluconazole resulted both in the selection and induction of resistant strains and in a shift in the nature of the infecting organisms . Most azole antifungals such as itraconazole, ketoconazole and fluconazole are active against a variety of fungal diseases . However, the concentration needed to inhibit growth is dependent on the nature of the infecting species . Mucor spp., e.g., are almost insensitive to present available azole compounds and can be regarded as intrinsically resistant to azole treatment . Physiochemical features, such as the hydrophobicity and pKa, of a given azole, define whether or not it will be active or cross-resistant against a given species . Fluconazole is almost inactive against Candida krusei and Aspergillus fumigatus, whereas the lipophilic itraconazole is active against these species . A third type of resistance is acquired or induced resistance . This is the most controversial type because, even within a given species, organisms may differ in their response to the same azole . For these strains, convincing evidence can only be obtained when there is a genotypically related strain, which does not show resistance . In a limited number of biochemical or molecular biological studies the mechanisms of resistance have been investigated at the molecular level . These studies show that resistance can occur when there is an insufficient intracellular content of the azole . This can be due to impermeability problems, inactivated uptake systems or, and more likely, the presence of active multidrug resistance gene products of the P-glycoprotein type . Alteration or overexpression of the target for azole antifungals, the cytochrome P450-dependent 14 alpha-demethylase, also induces resistance . The nature and amount of the accumulating sterols also are of great importance for azole-induced growth inhibition . This may explain why mutations in other enzymes of the ergosterol biosynthesis pathway, e.g . the delta 5-6 desaturase, can contribute to azole resistance.

Bull World Health Organ, 1995, 73(5), 631 - 42
Mefloquine treatment of acute falciparum malaria: a prospective study of non-serious adverse effects in 3673 patients; ter Kuile FO et al.; Between 1990 and 1994, a series of prospective studies were conducted to optimize the treatment of multidrug-resistant falciparum malaria on the borders of Thailand . The tolerance of various treatment regimens containing either mefloquine 15 mg/kg (M15) or 25 mg/kg (M25) was evaluated in 3673 patients aged between 6 months and 88 years . Early vomiting (within 1 hour) is an important determinant of treatment outcome in these areas, despite re-administration of the dose . Overall, 7 % of the patients vomited within an hour . Significant risk factors were age < or = 6 years (relative risk (RR), 3.9) or > or 50 years (RR, 2.7), the higher mefloquine dose (M25) (RRm 2.7), vomiting < 24 hours before enrolment (RR, 2.5), axillary temperature > 38.0 degrees C (RR, 1.6), and parasitaemia > 10,000/microliter (RR, 1.3) . In children < or = 2 years, 30% vomited with M25, and 13% did not tolerate a repeat dose . Vomiting was reduced 40% by splitting the higher dose (RR, 0.6; 95% CI, 0.4-0.8), and 50% by giving mefloquine on the second day in combination with artesunate (RR, 0.5; CI, 0.3-0.9) . Anorexia, nausea, vomiting, dizziness, and sleeping disorders were 1.1-1.4 times more frequent with M25 than M15 in the three days following treatment, but were similar in the single or split-dose M25 groups, despite twofold higher mefloquine concentrations obtained with the latter . There was no evidence that diarrhoea, headache, and abdominal pain were associated with mefloquine use . High-dose mefloquine is well tolerated but should be given as a split dose.

Crit Rev Biochem Mol Biol, 1995, 30(6), 445 - 600
The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance; Hayes JD et al.; The glutathione S-transferases (GST) represent a major group of detoxification enzymes . All eukaryotic species possess multiple cytosolic and membrane-bound GST isoenzymes, each of which displays distinct catalytic as well as noncatalytic binding properties: the cytosolic enzymes are encoded by at least five distantly related gene families (designated class alpha, mu, pi, sigma, and theta GST), whereas the membrane-bound enzymes, microsomal GST and leukotriene C4 synthetase, are encoded by single genes and both have arisen separately from the soluble GST . Evidence suggests that the level of expression of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals . In this article the biochemical functions of GST are described to show how individual isoenzymes contribute to resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress . A description of the mechanisms of transcriptional and posttranscriptional regulation of GST isoenzymes is provided to allow identification of factors that may modulate resistance to specific noxious chemicals . The most abundant mammalian GST are the class alpha, mu, and pi enzymes and their regulation has been studied in detail . The biological control of these families is complex as they exhibit sex-, age-, tissue-, species-, and tumor-specific patterns of expression . In addition, GST are regulated by a structurally diverse range of xenobiotics and, to date, at least 100 chemicals have been identified that induce GST; a significant number of these chemical inducers occur naturally and, as they are found as nonnutrient components in vegetables and citrus fruits, it is apparent that humans are likely to be exposed regularly to such compounds . Many inducers, but not all, effect transcriptional activation of GST genes through either the antioxidant-responsive element (ARE), the xenobiotic-responsive element (XRE), the GST P enhancer 1(GPE), or the glucocorticoid-responsive element (GRE) . Barbiturates may transcriptionally activate GST through a Barbie box element . The involvement of the Ah-receptor, Maf, Nrl, Jun, Fos, and NF-kappa B in GST induction is discussed . Many of the compounds that induce GST are themselves substrates for these enzymes, or are metabolized (by cytochrome P-450 monooxygenases) to compounds that can serve as GST substrates, suggesting that GST induction represents part of an adaptive response mechanism to chemical stress caused by electrophiles . It also appears probable that GST are regulated in vivo by reactive oxygen species (ROS), because not only are some of the most potent inducers capable of generating free radicals by redox-cycling, but H2O2 has been shown to induce GST in plant and mammalian cells: induction of GST by ROS would appear to represent an adaptive response as these enzymes detoxify some of the toxic carbonyl-, peroxide-, and epoxide-containing metabolites produced within the cell by oxidative stress . Class alpha, mu, and pi GST isoenzymes are overexpressed in rat hepatic preneoplastic nodules and the increased levels of these enzymes are believed to contribute to the multidrug-resistant phenotype observed in these lesions . The majority of human tumors and human tumor cell lines express significant amounts of class pi GST . Cell lines selected in vitro for resistance to anticancer drugs frequently overexpress class pi GST, although overexpression of class alpha and mu isoenzymes is also often observed . The mechanisms responsible for overexpression of GST include transcriptional activation, stabilization of either mRNA or protein, and gene amplification . In humans, marked interindividual differences exist in the expression of class alpha, mu, and theta GST . The molecular basis for the variation in class alpha GST is not known . (ABSTRACT TRUNCATED)

Oncol Res, 1995, 7(7-8), 407 - 16
Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth; Hashimoto S et al.; V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges . They showed no amplification of the multidrug-resistant p-glycoprotein . In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively . By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence . In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79 . V511 showed drastically decreased topo II alpha in both log growth and quiescence . In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting . All cell lines demonstrated relatively stable topo II beta throughout the cell cycle . Topo II alpha showed little cell cycle variation in V79 or V513 . However, in V511, it was only detectable at low levels in G2/M phase . When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79 . These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.

Pediatr Neurosurg, 1995, 23(6), 283 - 91; discussion 291-2
Multidrug resistance gene expression in childhood medulloblastoma: correlation with clinical outcome and DNA ploidy in 29 patients; Chou PM et al.; Twenty-nine children treated for medulloblastoma between 1987 and 1991 were reviewed . Thirteen patients with high-risk medulloblastoma characterized by incomplete resection, diploid tumor or subarachnoid dissemination received chemotherapy following radiation therapy . Three received postoperative chemotherapy . Eight patients who had been treated with postoperative radiation therapy also received chemotherapy for recurrent tumors . After a minimum 3-year follow-up period, 16 were alive but 13 had died from recurrent tumors . In order to evaluate the possible participation of P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) in medulloblastoma therapy and its correlation with prognosis, archival specimens were examined by immuno-histochemistry utilizing 3 monoclonal antibodies against Pgp and 6 cases by reverse-transcriptase polymerase chain reaction (RT-PCR) using MDR1-specific primers . Sixteen patients (55%) had MDR expression detected either by 1 of the 3 antibodies or by RT-PCR . DNA ploidy study was also performed on 18 specimens . We correlated patients' outcome with variable factors (extent of surgical resection, chemotherapy, DNA ploidy) and MDR expression . Patients who were treated with radiation therapy and adjuvant chemotherapy had a significantly better (p = 0.036) survival than those with radiation therapy alone, despite the fact that the former group of patients was considered to be high-risk . The extent of surgical resection and DNA ploidy did not correlate with prognosis . However, a statistically significant association was found between MDR expression and outcome (p = 0.007) . Among the patients who received chemotherapy, positive MDR expression significantly correlated with poor outcome (p = 0.036) . Our results showed that Pgp-mediated intrinsic MDR in medulloblastomas seems to correlate with an adverse outcome . This information may be used in designing new therapeutic protocols for medulloblastoma.

Invest New Drugs, 1995, 13(3), 181 - 6
Developing gossypol derivatives with enhanced antitumor activity; Liang XS et al.; Preclinical and clinical studies have pointed to the antitumor potential of the naturally occurring polyphenolic binaphthyl dialdehyde, gossypol, as well as its purified (-,+) enantiomers . To explore further the antitumor properties of this multifunctional agent, we synthesized several reactive derivatives including the (-,+) enantiomers of gossypolone and four different gossypol Schiff's bases (AR1, AR2, AR3, AR4) . The biological activities of these new agents were screened by measuring their in vitro antiproliferative activity against malignant (MCF-7, MCF-7/adr) or immortalized (HBL-100) human breast epithelial cell lines . Racemic gossypolone showed relatively uniform antiproliferative activity against all of the breast epithelial cell lines with 3- to 5-fold less activity than (--)-gossypol against MCF-7 and MCF-7/adr cells . Of interest, the relative antitumor potency of purified gossypolone enantiomers was reverse that of gossypol enantiomers, since (+)-gossypolone showed up to 3-fold greater inhibition of MCF-7 culture growth than (--)-gossypolone . Of the Schiff's base derivatives only AR3 with its isopropyl amine substituent demonstrated cytotoxic activity comparable to that of (--)-gossypol; derivatives with ethyl, propyl, or butyl amine substituents (AR1, AR2, AR4) had little growth inhibitory activity at culture concentrations up to 25 microM . AR3 activity was greatest against HBL-100 and MCF-7 cells {MCF-7 IC50 values: AR3 = 0.9 microM, (--)-gossypol = 2.3 microM}; unlike (--)-gossypol, however, AR3 showed substantially reduced activity against the multidrug-resistant subline, MCF-7/adr . These structure-activity comparisons suggest that isolation of (-,+)-enantiomers of AR3 and additional chemical modifications including the synthesis of an isopropyl amine Schiff's base of gossypolone will likely yield a newer generation of gossypol analogues with enhanced anticancer potential.

Biol Cell, 1995, 84(3), 195 - 204
Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses; Zini N et al.; Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX) . The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein . In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy . Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level . Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost . These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.

Oncol Res, 1995, 7(12), 619 - 24
P-glycoprotein expression in ovarian cancer cell line following treatment with cisplatin; Yang X et al.; Human ovarian cancer cell line SKOV3 was grown during a period of four months in the presence of increasing concentrations of cisplatin (25-100 ng/ml) . In the course of this treatment, the cells exhibited dramatic changes in morphology, including reduction in cell size, loss of cellular projections and clustering . This was accompanied by the appearance of P-glycoprotein (Pgp) on the cell membrane, as detected by flow cytometry and immunochemistry methods using the anti-Pgp monoclonal antibodies MRK16 and C219 . The new cell line, designated SKOV3/CIS, was also resistant to alkylating agents, such as chlorambucil, similarly to the parental SKOV3 cells . In addition, it also acquired resistance to classical multidrug resistance drugs, such as doxorubicin, taxol and actinomycin D . Verapamil enhanced the sensitivity of SKOV3/CIS to doxorubicin (260-fold), in conformity with the proposed mechanism of Pgp in multidrug resistance (MDR), but it did not potentiate cisplatin cytotoxicity in SKOV3/CIS cells . Our results suggest that cisplatin can cause Pgp expression, and that both cisplatin-resistance and Pgp-mediated MDR phenotypes can coexist in some tumor types . Although Pgp does not appear to be responsible for cisplatin resistance, exposure to cisplatin can lead to the development of MDR phenotype, a complication that should be considered in clinical situations, especially in the chemotherapy of ovarian cancer.

Oncol Res, 1995, 7(12), 603 - 10
Analysis of the interactions of SDZ PSC 833 ({3'-keto-Bmt1}-Val2}-Cyclosporine), a multidrug resistance modulator, with P-glycoprotein; Archinal-Mattheis A et al.; Multidrug resistance (MDR) is considered to be an important impediment to the effective treatment of cancer . P-glycoprotein, the drug efflux pump that mediates this resistance, can be inhibited by a wide variety of pharmacological agents, resulting in the circumvention of the MDR phenotype . SDZ PSC 833 ({3'-keto-Bmt1}-Val2}-cyclosporine), a nonimmunosuppressive cyclosporine D derivative, was identified to be a potent MDR modulator (Gaveriaux et al . J . Cell Pharmacol . 2:225-234; 1991) . In this study, the interactions of P-glycoprotein with two cyclosporine derivatives, SDZ PSC 833 and cyclosporine A (CsA, Sandimmune), were analyzed . SDZ PSC 833 enhanced the sensitivity of the MDR cells to anticancer drugs by increasing the accumulation and inhibiting the efflux of cytotoxic agents from resistant cells more efficiently than CsA . The two cyclosporine analogs competed with the labeling of P-glycoprotein by a photoactive cyclosporine derivative . In addition, membrane vesicles derived from resistant cells bound SDZ PSC 833 . However, CsA was transported by P-glycoprotein, whereas SDZ PSC 833 was not actively transported . This resulted in a prolonged inhibitory effect by SDZ PSC 833 . The studies suggest that the binding of SDZ PSC 833 to P-glycoprotein in the absence of its transport from MDR cells mediated its high potency as an MDR reversing agent . In addition, the comparison of the two cyclosporine analogs indicated that limited chemical modifications of MDR reversing agents can affect their potential to inhibit P-glycoprotein function.

J Cancer Res Clin Oncol, 1995, 121 Suppl 3, R3 - 6
Dexverapamil to overcome epirubicin resistance in advanced breast cancer; Thurlimann B et al.; Resistance to cytotoxic chemotherapy is a major problem in the management of patients with metastatic breast cancer . Various data suggest P-glycoprotein-associated multidrug resistance (MDR) to be a relevant resistance mechanism in this tumor . The purpose of this study was to evaluate feasibility and activity of combining oral dexverapamil, a second-generation chemosensitizer currently in clinical development for MDR reversal, with epirubicin in patients with epirubicin-refractory high-risk metastatic breast cancer . Patients first received epirubicin alone at 120 mg/m2 . In cases of clinical refractoriness, epirubicin was continued at the same dose and schedule but supplemented with oral dexverapamil . Dexverapamil was given at 300 mg every 6 h for a total of 13 doses and commenced 2 days prior to epirubicin administration . At the time of this interim analysis, 41 patients had received epirubicin alone and 20 proceeded to treatment with epirubicin plus dexverapamil . Of the 20 patients, 14 were considered evaluable for toxicity and activity . Addition of dexverapamil resulted in a significant decrease in mean heart rate and blood pressure as well as prolongation of PQ time as compared to epirubicin alone . However, these cardiovascular effects of dexverapamil were usually mild, and subjective tolerance of treatment was good . In 7/14 patients, dose escalation of dexverapamil was feasible . Dexverapamil had no effect on epirubicin toxicities and did not require reduction of the epirubicin dose . In 2/14 patients, the addition of dexverapamil to epirubicin was able to convert progressive disease and no changes respectively, into partial responses . In 3 patients with progressive disease, addition of dexverapamil temporarily prevented further tumor progression . Analyses of dexverapamil and nor-dexverapamil plasma levels, of in vitro reversal activity of patient sera containing dexverapamil, and of epirubicin pharmacokinetics without and with dexverapamil are currently in progress . Addition of oral dexverapamil to epirubicin 120 mg/m2 proved to be feasible in a multiinstitutional setting . Patient accrual is continuing to determine whether dexverapamil is capable of overcoming epirubicin refractoriness in a significant number of patients with metastatic breast cancer.

J Cancer Res Clin Oncol, 1995, 121 Suppl 3, R25 - 9
Modulation of multidrug resistance by dexverapamil in EPOCH-refractory lymphomas; Wilson WH et al.; We conducted a controlled trial of dexverapamil, an inhibitor of Pgp, in 45 Hodgkin's (HD) and 154 Non-Hodgkin's (NHL) lymphomas refractory to EPOCH chemotherapy . A total of 154 patients initially received EPOCH alone and (4.2%) with stable disease over two cycles or progressive disease "crossed over" to receive dexverapamil with EPOCH . Dexverapamil was escalated 8 dose levels, from 240 to 1200 mg/m2 per day . When possible, serial biopsies were obtained to measure MDR-1 expression by quantitative polymerase chain reaction . Median age was 44 years, 67% had stage IV disease, and median (range) prior regimens were 2 (1-12) in NHL and 1 (1-4) in HD . The maximum tolerated dose of dexverapamil was 900 mg/m2/day, and median plasma average concentrations of dexverapamil and nor-dexverapamil were 1.2 and 1.4 microM, respectively . There were 3 complete and 2 partial responses (12%) and 5 minor responses in NHL, and 2 of 10 HD patients achieved partial responses . MDR-1 was measured in 44 biopsies from 19 patients . Pre-therapy, MDR-1 was low (median 2.5 U) but increased (median 12.2 U) at cross-over . Among 6 patients with MDR-1 > 15, 3 responded to dexverapamil whereas only 1/8 patients with MDR-1 < 15 responded . EPOCH and dexverapamil were well tolerated . This study suggests that MDR-1 plays a role in clinical drug resistance of lymphomas, but also suggests that non-MDR-1 mechanisms are present in such patients . Earlier intervention with dexverapamil may be more effective and warrants further study.

J Cancer Res Clin Oncol, 1995, 121 Suppl 3, R11 - 6
Dexverapamil to modulate vinblastine resistance in metastatic renal cell carcinoma; Mickisch GH et al.; Multidrug resistance (MDR) in a variety of human tumours such as renal cell carcinoma (RCC) is thought to be caused by expression of the MDR1 gene and may be reversed by applying modern chemosensitisers such as dexverapamil, which inhibit the MDR1 gene product P-glycoprotein . This preliminary report gives information on a clinical study complying with good clinical practice regulations in patients with advanced RCC . The final evaluation is pending . Vinblastine, if anything the most effective chemotherapeutic agent (5-day continuous regimen), was combined with oral dexverapamil (6 times per day) as a chemosensitiser and dexamethasone to increase dexverapamil tolerance . All patients had histologically proven RCC, which was metastatic and progressive at study entry . The statistical design featured a pre-study regimen of two cycles of vinblastine alone followed by evaluation . If no response was documented, with all patients thus serving as their own control, dexverapamil and dexamethasone were added for three cycles of combination therapy . Having obtained institutional permission from the ethical review committee, we enrolled patients of whom 25 qualified for the combined-treatment arm; 13 patients finished the study, 5 patients failed to complete all treatment cycles (1 because of treatment-related toxicity, 3 for personal reasons, not related to treatment, 1 for tumour-related reasons) and 7 patients were at too early a stage for evaluation . Altogether, 61% of all patients tolerated a dose of dexverapamil of at least 2400 mg/day with peak serum levels reaching, in some cases, approximately 8 microM (the sum of dexverapamil plus nordexverapamil levels) . WHO grade 3 and 4 toxicities were mainly myelosuppression (5/18) . The combination of 1.4 mg m-2 day-1 vinblastine plus dexverapamil was generally felt to be safe and well tolerated . One partial response and 7 stable diseases were noted in this heavily pretreated study population . Four-hourly administration of dexverapamil in combination with dexamethasone plus escalation to the individually tolerated doses have permitted increases in serum levels of dexverapamil.

Southeast Asian J Trop Med Public Health, 1995, 26 Suppl 1, 333 - 6
Analysis of RNA polymerase gene mutation in three isolates of rifampicin resistant Mycobacterium tuberculosis; Vattanaviboon P et al.; Drug resistance in tuberculosis (TB) has become a major public health threat, particularly when the disease cannot be 100% controlled by BCG vaccination . In Thailand, resistance to rifampicin, a major component of multidrug regimens of treatment, is the common cause of tuberculosis recurrence . The mechanism of rifampicin resistance involves alterations of the RNA polymerase subunit beta (rpo B) gene . Mutations in rpo B gene were often found to cluster within a region of 23 amino acids starting from amino acid residue 511 to residue 533 . Direct PCR sequencing was utilized to compare base changes in rpo B gene in three rifampicin resistant phenotypes of M . tuberculosis isolated from Thai patients . The sequences showed one base substitution at codon 531 resulting in an amino acid change from serine (TCG) to leucine (TTG) in a multidrug resistant isolate compared to that of a sensitive isolate, whereas a point mutation at codon 516 causing a change from aspartic acid (GAC) to tyrosine (TAC) was detected in a multidrug resistant isolate from a HIV positive patient . In an isolate resistant only to rifampicin a double mutation at codon 531 changing serine (TCG) to phenylalanine (TTT) was found . No mutations were observed in the same region in streptomycin, ethambutol or isoniazid resistant isolates . This finding reports two new types of mutation (GAC to TAC at codon 516 and TCG to TTT at codon 531) and confirms a direct correlation between rpo B gene alteration and rifampicin resistant phenotype in M . tuberculosis.

Invest New Drugs, 1995, 13(2), 125 - 131
Activity of the morpholino anthracycline 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) against human tumor colony-forming units in vitro; Ebrahim el-Zayat AA et al.; In several preclinical systems, the morpholino anthracycline MX2 has greater antitumor activity than doxorubicin, is less cardiotoxic, and is effective against multidrug resistant cancer cells . We used a human tumor soft-agar cloning assay to test the cytotoxicity of MX2 against single cell suspensions from freshly obtained human tumors . Tumor cells were exposed to MX2 at 0.05 and 0.5 micrograms/ml either for 1 hour (2-1 specimens; 77 {38%} assessable) or continuously (231 specimens; 91 {39%} assessable) . Superior antitumor activity was observed with continuous exposure (19% in vitro response at 0.05 micrograms/ml and 69% at 0.5 micrograms/ml) than with 1-hour exposure (1.3% at 0.05 micrograms/ml and 12% at 0.5 micrograms/ml) . Activity was seen against all types of cancer tested including renal (91%), melanoma (88%), ovarian (73%), breast (71%) and non-small-cell lung (67%) cancer at a MX2 concentration of 0.5 micrograms/ml after continuous exposure . If appropriate plasma levels can be achieved in patients, MX2 could have significant clinical activity with those tumors.

Clin Oncol (R Coll Radiol), 1995, 7(5), 300 - 3
Cyclosporin plus doxorubicin, vincristine and etoposide in the treatment of refractory non-Hodgkin's lymphoma: a phase II study; Moore DF et al.; In an attempt to circumvent clinical multidrug resistance, we conducted a Phase II trial of cyclosporin plus combination chemotherapy in patients with relapsed or refractory non-Hodgkin's lymphoma . Thirteen patients, all of whom had been previously treated with a doxorubicin-containing regimen, received doxorubicin 50 mg/m2 intravenous continuous infusion (IVCI) over 96 h (days 1-4), vincristine 2 mg i.v . (day 1), and etoposide 75 mg/m2 i.v . daily for 4 days (days 1-4) . Four days prior to chemotherapy, patients received a loading dose of cyclosporin (0.88 mg/kg i.v . over 2 h), followed by a maintenance dose (1.8 mg/kg per day IVCI for 9 days) . Cyclosporin dose escalation was permitted, conventionally defined therapeutic levels of cyclosporin were achieved; this drug was well tolerated at these doses . The study was closed due to a poor response rate; only one patient achieved a complete remission of 33 weeks' duration . Grade 3 and 4 toxicities included gastrointestinal haemorrhage (one patient), sensory neuropathy (two patients), stomatitis (two patients), and transaminase elevation (one patient) . Asymptomatic grade 1-2 toxicities (elevated creatinine and transaminase levels) occurred in 33% of patients . There were no treatment associated deaths . Prolonged neutropenia and thrombocytopenia were the primary haematological toxicities . Although the addition of cyclosporin at this dose and schedule did not improve response rates in this patient group, future trials using higher doses of cyclosporin with combination chemotherapy may warrant further investigation.

J Urol (Paris), 1995, 101(3), 122 - 4
{Resistance to chemotherapy in cancers of the kidney and therapeutic perspectives}; Oudard S et al.; Usual treatments combining surgery, radiation therapy, chemotherapy and hormonotherapy are poorly effective . The immunotherapy gave and objective response rate of 25% but is associated with many side effects . Multidrug resistance (MDR) can be explained, in part, by an mdr1 gene overexpression in renal carcinoma . The MDR is related to expression of a 170 Kda membrane glycoprotein, the so-called P glycoprotein (Pgp) . This protein is able to extrude from cytoplasm drugs with various structures and mechanisms . Reversal compounds capable of inhibiting Pgp, given with antineoplastic drugs, could be able to increase their intracellular concentrations . Nevertheless, renal cell carcinomas are characterized by their multifactorial resistance and a better knowledge in this field will allow to design new circumvention resistance to chemotherapy.

Oncol Res, 1995, 7(3-4), 191 - 200
Combined effects of buthionine sulfoximine and cepharanthine on cytotoxic activity of doxorubicin to multidrug-resistant cells; Kisara S et al.; We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein . The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line . BSO reduced GSH content of MDR cells compared to that of the sensitive ones . The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance . The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect . Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE . The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX . Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time . The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone . These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.

Drugs, 1995, 49 Suppl 2, 67 - 75
Activity of quinolones against mycobacteria; Jacobs MR; The fluoroquinolones have been shown to be highly active in vitro against many mycobacterial species, including most strains of Mycobacterium tuberculosis and M . fortuitum, and some strains of M . kansasii, M . avium-intracellulare (MAI) complex and M . leprae . Ciprofloxacin, ofloxacin and sparfloxacin are the best studied of this class of drugs to date, and they are among the most active of these against M . tuberculosis and other mycobacteria . The use of ofloxacin in the treatment of patients with multidrug-resistant pulmonary tuberculosis has resulted in the selection of quinolone-resistant mutants in a few patients . Many strains of MAI, however, are resistant to fluoroquinolones, and structure-activity relationship studies have been undertaken to identify the moieties associated with activity and inactivity . The most important features determining activity against MAI were found to be a cyclopropyl ring at the N1 position, fluorine atoms at positions C6 and C8, and a C7 heterocyclic substituent . On the basis of these structural requirements, a series of compounds were tested, and many did indeed show good activity against MAI in vitro . Application of these data to macrophage and animal models is in progress . Clinical evaluation of some of these new fluoroquinolones is also being undertaken in multidrug-resistant tuberculosis and MAI and M . leprae infections . Although the development of resistance and the influence of host factors may limit their use, they have considerable potential if prudently used.

Drugs, 1995, 49 Suppl 2, 132 - 5
The new quinolones in the treatment of diarrhoea and typhoid fever; Waiz A; Diarrhoea and typhoid fever are two important diseases in the developing world, particularly the Asian countries . The management of these conditions is becoming increasingly difficult in the face of emerging pathogen resistance . The new fluoroquinolones demonstrate good in vitro activity against the causative pathogens involved, including those that are multidrug resistant . These agents have been shown to be very effective in the treatment of diarrhoea and typhoid in clinical trials, achieving results equal to, or better than, standard drugs . Importantly, fluoroquinolones also considerably shorten the duration of illness, thereby offering rapid relief to the patient.

Oncol Res, 1995, 7(5), 207 - 12
Fractionation of Cremophor EL delineates components responsible for plasma lipoprotein alterations and multidrug resistance reversal; Kessel D et al.; Treatment of cancer patients with 3-h infusions of taxol formulated with Cremophor EL resulted in a marked decrease in the electrophoretic mobility of all plasma lipoproteins . Cremophor was fractionated by reverse-phase chromatography to determine which components were responsible for this behavior . Effects of different Cremophor fractions on reversal of multidrug resistance, amino acid transport, and cytotoxicity also were evaluated using murine leukemia cells in culture . Lipoprotein alterations were caused by Cremophor components of intermediate hydrophobicity, which also antagonized amino acid transport and decreased viability of murine leukemia cells . Cremophor components responsible for reversal of multidrug resistance were of greater hydrophobicity . The lipoprotein-altering components of Cremophor could be selectively removed without affecting either taxol solubilization or multidrug-resistant reversal.

Wien Klin Wochenschr, 1995, 107(22), 681 - 6
Structure-activity-relationship studies on modulators of the multidrug transporter P-glycoprotein--an overview; Ecker G et al.; Resistance of tumor cells to a wide variety of cytotoxic agents represents a major problem in cancer therapy . In most cases, the cross resistance profile has been shown to be accompanied by a decrease in drug accumulation in the resistant cells . At present it seems to be widely accepted that this decrease in intracellular drug levels is due to active efflux of these drugs caused by P-glycoprotein (PGP) . Within the past decade, several substances have been identified as being capable of inhibiting the active drug efflux caused by P-glycoprotein . Although many excellent reviews on the phenomenon of multidrug resistance (MDR) have been published, little is known about SAR (Structure-Activity-Relationship)- or QSAR (Quantitative-Structure-Activity-Relationship)-studies of modulators of MDR . The aim of this article is to review first results in this field.

Mol Med, 1995 Jan, 1(2), 142 - 52
Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene; Chen J et al.; BACKGROUND: Mdm-2, a zinc finger protein, negatively regulates the p53 tumor suppressor gene product by binding to it and preventing transcriptional activation (16) . MATERIALS AND METHODS: Assays for p53 mediated transcription, repression and activation by mutant and wild-type p53 proteins were used to measure the ability of mdm-2 to block each activity . RESULTS: Mdm-2 was able to inhibit all three functions of the wild-type and mutant p53 activities; transcriptional activation by the wild-type protein, transcriptional activation by the mutant p53 protein, and repression by the wild-type protein . CONCLUSIONS: The mdm protein binds to the amino terminal portion of the p53 protein and, in so doing, blocks the ability of p53 to interact with the transcriptional machinery of the cell (23) . The mdm-2 protein binds to both leucine-tryptophan residues at amino acids 22 and 23, from the amino terminal end of the protein, and in so doing, prevents all p53 functions . The ability of a mutant p53 protein to transactivate a multidrug resistance-1 gene promoter is blocked by mdm-2 and the ability of the wild-type p53 protein to repress transcription of some genes is also blocked by the mdm-2 protein . Thus, all three functions of the p53 protein-transcriptional activation, repression and mutant protein activation-require the p53 amino terminal domain functions and are regulated by the mdm-2 protein in a cell . When mdm-2 is overproduced, resulting in a tumor or transformation of a cell, all of the p53 activities are inactivated.

Oncol Res, 1995, 7(6), 299 - 306
Influence of exogenous ras and p53 on P-glycoprotein function in immortalized rodent fibroblasts; Kopnin BP et al.; The ability of ras oncogenes and mutant p53 to activate reporter gene expression from human and rodent mdr1 gene promoters was described, although functional significance of this finding was unclear . We analyzed the influence of various forms of recombinant human ras and p53 on the mdr1 gene expression and P-glycoprotein (Pgp) function in rodent immortalized fibroblasts . The ras genes, in addition to activation of exogenous human mdr1 gene promoter, caused an increase in (i) expression of endogenous mdr1 mRNA, (ii) Pgp activity as determined by flow cytometry analysis of Rhodamine 123 exclusion, and (iii) resistance of cells to the cytotoxic action of colchicine and some other drugs . To elucidate whether the same signalling pathway is responsible for multidrug resistance induced by various oncogenes and protein kinase C (PKC), we tested the effects of v-mos and the PKC agonist 12-O-tetradecanoylphorbol-13-acetate . Similarly to cells transformed by ras, a Rat1 subline transformed by the v-mos oncogene was characterized by decreased drug sensitivity . On the contrary, Rat1 cells treated with the protein kinase C agonist 12-O-tetradecanoylphorbol-13-acetate showed neither increased mdr1 mRNA expression nor stimulation of Pgp function . Introduction by retrovirus-mediated gene transfer of wild-type p53 into Rat1 cells or into murine p53-deficient 10(1) and 10(3) cells did not change the Pgp function significantly, whereas in Rat1 cells transformed by activated N-ras or v-mos, expression of wild-type p53 caused partial reversion of oncogene-induced drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pathol, 1995 Jan, 175(1), 23 - 9
Differentiation of a human rhabdomyosarcoma cell line after antineoplastic drug treatment; Melguizo C et al.; The feasibility of treating solid tumours with differentiation therapy using antineoplastic drugs is currently being investigated, but the emergence of multidrug resistance remains the major limitation to this therapeutic approach . A rhabdomyosarcoma cell line resistant to actinomycin D (RD-DAC) has been used as an in vitro model to investigate, with light and electron microscopy, the degree of differentiation in multidrug-resistant cells . The parental cell line (RD), derived from a human embryonic-type rhabdomyosarcoma, is undifferentiated, with no evidence of specific ultrastructural markers . Examination of resistant cells by transmission electron microscopy revealed myofilaments arranged parallel to the long axis of the cell, which was considered clear evidence of myogenic differentiation . These observations suggest that actinomycin D, the drug of choice in the treatment of rhabdomyosarcoma, induces differentiation in the cell line RD . It is postulated that multidrug resistance can interfere with cellular differentiation.

J Pathol, 1995 Jan, 175(1), 13 - 22
Expression of mdr1/P-glycoprotein and p110 in neuroblastoma; Ramani P et al.; Overexpression of the multidrug resistance gene, mdr1, and its product, P-glycoprotein (Pgp), has been associated with cross-resistance to structurally unrelated compounds in cell lines and tumours . Recently, a non-Pgp-mediated form of drug resistance has been described, due to the overexpression of p110, a transport protein . Thirty formalin-fixed, paraffin-embedded neuroblastoma samples from 21 cases were examined for overexpression of mdr1 and Pgp using newly established non-radioactive in situ hybridization and sensitive immunocytochemical techniques . Tumours were examined from patients with all the stages of disease and from primary and metastatic sites . Paired tumour samples (pre-chemotherapy and post-chemotherapy) were available from cases with stage 2 (n = 1) and stage 4 disease (n = 8) . Immunoreactivity to p110 was also tested on all the samples . Mdr1 mRNA was expressed in 16/21 cases and in all the stages . Pgp immunoreactivity was detected in all the cases . Weak cytoplasmic immunoreactivity to p110 was seen in the ganglion cells in 12/21 cases . The expression of mdr1, Pgp, and p110 showed a statistically significant (two-sided Fisher exact test, P = 0.04, 0.03, 0.04, respectively) correlation with differentiation (Beckwith and Martin grading) but there was no correlation with survival . Pgp immunoreactivity also showed a significant correlation with favourable clinical variables: age less than 1 year at diagnosis and stages 1, 2, and 4 s (two-sided Fisher exact test, P = 0.01, 0.005, respectively).

Int J Radiat Biol, 1995 Jan, 67(1), 57 - 64
Haematopoietic radioprotection by Cremophor EL: a polyethoxylated castor oil; Bertoncello I et al.; The polyethoxylated castor oil, Cremophor EL (Cremophor) is approved for human use as a vehicle for oral and intravenous administration of water-insoluble compounds . Cremophor has also previously been shown to reverse the multidrug resistance phenotype at clinically acceptable doses . This study demonstrates that doses of Cremophor in the range of 25-50 microliters/kg intravenously (i.v.) administered 1 day prior to near-lethal irradiation protected the regenerative capacity of the marrow, resulting in haematopoietic radioprotection and long-term survival of near-lethally-irradiated mice . In normal mice, Cremophor administration (1) markedly reduced the level of serum haematopoietic inhibitory activity 4-8 h following injection; (2) resulted in a transient decrease in femoral bone marrow cellularity and upregulated B220 (B cells), and 7/4 (neutrophils and activated macrophages), but not Thy-1 (T-cells) surface antigen expression in bone marrow cells within 24 h of injection; and (3) transiently elevated the incidence of both primitive and committed haematopoietic progenitor cells detected in clonal agar culture within 48 h of injection . Bone marrow progenitor cell content, and peripheral blood white cell, platelet and reticulocyte counts were unaffected . This suggests that the haematopoietic radioprotection and recovery observed in irradiated mice pretreated with Cremophor may be the result of accessory cell activation and/or modulation of accessory factors regulating haematopoietic progenitor cells . Our data suggest a potential clinical use of Cremophor as an adjunct to, or as a substitute for, cytokines to minimize myelosuppression following cytotoxic therapy.

Cancer Chemother Pharmacol, 1995, 35(5), 432 - 6
Inhibition of intestinal P-glycoprotein and effects on etoposide absorption; Leu BL et al.; P-glycoprotein (Pgp) actively pumps a number of antineoplastic drugs, such as etoposide, out of cancer cells and causes multidrug resistance . Pgp is also expressed at the brush-border membrane of the small intestine under normal physiological conditions . We hypothesized that inhibition of intestinal Pgp might decrease the efflux of etoposide from the blood into the intestinal lumen, thereby, increasing the bioavailability of etoposide . The absorption of etoposide was studied using everted gut sacs prepared from rat jejunum and ileum . The addition of C219, a monoclonal antibody of Pgp, at 100 ng/ml or of 0.2 M 5'-adenylylimidodiphosphate, a nonhydrolyzable adenosine triphosphate (ATP) analog, increased the absorption of etoposide . Quinidine, an antiarrythmic agent, has been demonstrated to circumvent multidrug resistance in cell lines, possibly by interfering with Pgp function . Adding quinidine at 1 mg/ml to the everted gut sac increased the absorption of etoposide . In vivo absorption of etoposide was also studied by intraluminal perfusion of the drug in the small intestine of anesthetized rats . Intravenous infusion of quinidine at either 1 or 2 mg/h increased the serum level of etoposide in a dose-dependent manner . Intravenous infusion of etoposide at 0.2 mg/h resulted in luminal exsorption of the drug in the small intestine . The intestinal clearance of etoposide was 41.7 +/- 7.2 ml kg-1, which decreased to 18.4 +/- 3.9 ml kg-1 with the infusion of quinidine at 1 mg/h . The present data confirm that intestinal Pgp mediates the efflux of etoposide and that the use of Pgp-inhibiting agents such as quinidine may increase the bioavailability of etoposide.

Cancer Chemother Pharmacol, 1995, 35(5), 403 - 10
Subcellular localisation of the antitumour drug mitoxantrone and the induction of DNA damage in resistant and sensitive human colon carcinoma cells; Fox ME et al.; Cellular uptake and subcellular localisation of the antitumour agent mitoxantrone were studied in a human colon-carcinoma cell line and a mitoxantrone-resistant subline showing features consistent with an atypical multidrug-resistance phenotype involving altered topoisomerase II . Flow cytometry indicated a reduced uptake of mitoxantrone in the resistant line . Confocal microscopy indicated that mitoxantrone-associated fluorescence was primarily found within discrete cytoplasmic inclusions and around the periphery of the nucleus, with low levels being observed within the nucleus . The frequency of cytoplasmic inclusions was reduced in mitoxantrone-resistant cells as compared with parental cells . Fluorescence in cytoplasmic inclusions persisted throughout a 24-h post-treatment period in both cell lines . The results suggest that the persistence of mitoxantrone in cells is a determinant for the continuous induction of DNA damage, perhaps through chronic topoisomerase II trapping, and that modified sequestration may contribute to clinically relevant moderate levels of non-classic multidrug resistance.

Cancer Chemother Pharmacol, 1995, 35(5), 377 - 86
Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells; Lenehan PF et al.; PURPOSE: It has been recognized that enhanced antioxidant defenses can contribute to the resistance of cancer cells displaying multidrug resistance (MDR) that arises in conjunction with the overexpression of P-glycoprotein (Pgp) . The purpose of this study was to determine if the defenses against oxidant stress in MDR human leukemia cells (HL-60/AR) that overexpress multidrug-resistance-associated protein (MRP), but not Pgp, contribute to the mechanism of drug resistance in this cell line . METHODS: HL-60/AR cells were evaluated in comparison with wild-type cells with respect to sensitivity to the oxidants hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BuOOH), the activities and amounts of the antioxidant enzymes catalase and glutathione peroxidase (GSH-Px), and the effects that manipulation of the activities of these enzymes may have on cellular sensitivity to the oxidants and to daunorubicin . We also evaluated the ability of the cells to generate daunorubicin semiquinone free radical as measured by electron spin resonance (ESR) spectroscopy . RESULTS: HL-60/AR cells were > 10-fold resistant to the cytotoxic effects of the H2O2 or t-BuOOH as compared with parental, drug-sensitive HL-60 cells . This phenomenon could be attributed largely to elevated activity and protein levels of catalase in HL-60/AR cells . Furthermore, inhibition of catalase by 3-amino-1,2,4-triazole (AT) diminished the resistance of HL-60/AR to these oxidants by > 80% or > 50%, respectively . Despite these findings, AT was incapable of causing sensitization of HL-60/AR cells to the cytotoxic effects of daunorubicin . We found that the activity and amount of selenium-dependent glutathione peroxidase (GSH-Px) was no greater in HL-60/AR cells than in HL-60 cells . Cultivation of cells in selenium-deficient medium caused a marked reduction in GSH-Px activity in HL-60/AR cells and a profound inhibition of GSH-redox cycling manifested by a decrease in baseline hexose monophosphate shunt activity (HMPS) and markedly blunted stimulation of the HMPS by the oxidant t-BuOOH in both wild-type and resistant cells . These variations in GSH-Px activity and GSH-redox cycling, however, were not associated with an alteration in cellular sensitivity to daunorubicin . The failure of catalase inhibition or selenium manipulation of GSH-Px activity to affect daunorubicin cytotoxicity was not due to the inability of these cells to produce free-radical species of daunorubicin, since ESR studies revealed that the generation of daunorubicin semiquinone free radical by HL-60/AR cells was equal to and, in fact, 3-fold that obtained with HL-60 cells . CONCLUSIONS: In comparison with parental HL-60 cells, MRP-overexpressing HL-60/AR cells have demonstrable alterations in antioxidant defenses that are manifested by cellular resistance to the cytotoxic effects of H2O2 and t-BuOOH and by elevated protein levels and activity of catalase . Whether these alterations are epiphenomena or are related to overexpression of MRP remains to be determined . However, it does appear that the enhanced antioxidant defenses observed in HL-60/AR cells do not contribute to the resistance to daunorubicin manifested by this cell line . Although HL-60/AR cells generate daunorubicin semiquinone free radical to an extent equal to or greater than that observed in HL-60 cells, the failure of alterations in GSH-Px activity or inhibition of catalase to change the sensitivity of HL-60/AR cells to daunorubicin suggests that the cytotoxicity of daunorubicin in these cells in not mediated through H2O2 or other peroxide species detoxified by these enzymes.

Orv Hetil, 1995 Jan 1, 136(1), 19 - 25
{Multidrug resistance of testicular cancers . (Detection of P-glycoprotein and MDR1 gene expression and their clinical connection)}; Bak M et al.; The most frequently reported alteration of multidrug-resistant cells is overexpression of a 170 kD glycoprotein (P-glycoprotein or P-170) encoding by the MDR1 gene family . Expression of the multidrug-resistance gene product P-glycoprotein was screened in 55 untreated human germ cell testicular tumors using monoclonal antibody (C219) and immunoenzyme staining . In samples out of 17 seminomatous germ cell testicular tumors (SGCT) 2 seminomas, and out of 38 non-seminomatous tumors (NSGCT) 20 carcinomas (15 teratomas, 4 embryonal carcinomas, 1 with Yolk sac differentiation and 1 embryonal rhabdomyosarcoma) showed high expression of P-glycoprotein . NSGCT-s, which are more refractory than seminomas to anticancer chemotherapy, frequently expressed P-glycoprotein . These immunohistochemically detected elevated P-170 expressions were correlated by the overexpression of MDR1 mRNA gene sequences . A relationship between clinical resistance and P-glycoprotein expression seems thus to exist in 4 teratomas 3 embryonal carcinomas, and 1 seminomas . A significant correlation (p < 0.02) between P-170 expression and clinical drug resistance in stage II-III germ cell testicular tumors could be demonstrated . The results suggest that a multidrug resistant phenotype may also occur and P-glycoprotein might contribute to drug resistance in testicular tumors.

Mol Pharmacol, 1995 Jan, 47(1), 51 - 6
Evidence for transcriptional control of human mdr1 gene expression by verapamil in multidrug-resistant leukemic cells; Muller C et al.; We investigated the mechanism of verapamil (VRP) effects on mdr1 gene expression in two leukemic multidrug-resistant (MDR) cell lines, K562/ADR and CEM VLB100 . Exposure to VRP for 24 hr resulted in a decrease in mdr1 mRNA levels that was dose related at concentrations between 15 and 50 microM . The maximal decrease of mdr1 mRNA levels was found to be 6-fold in the K562/ADR cells and 3-fold in the CEM VLB100 cells . The effect of VRP on mdr1 mRNA levels was, however, biphasic . At 100 microM VRP, which strongly inhibited cell proliferation, a 2-fold increase of mdr1 mRNA levels was observed in the K562/ADR cells . To determine whether the decrease of mRNA levels resulted from post-transcriptional mechanisms, mRNA stability was studied after blocking of transcription with actinomycin D in VRP-treated cells and in control cells . This study revealed that mdr1 mRNA was stable in both cell lines and no increase in mdr1 mRNA degradation was observed in the 30 microM VRP-treated cells versus control cells (half-lives of 23 hr versus 14 hr for the K562/ADR cells and 15.5 hr versus 10.0 hr for the CEM VLB100 cells) . The suggestion of a transcriptional mechanism was confirmed by nuclear run-on assays . A 4-fold decrease in the mdr1 gene transcription rate was observed in the 30 microM VRP-treated CEM VLB100 cells . The decreased transcription rate could be due to the decrease in mdr1 proximal promoter activity observed in CEM VLB100 cells transiently transfected with the mdr1 promoter fused to the chloramphenicol acetyltransferase gene . Indeed, after exposure to 30 microM VRP, chloramphenicol acetyltransferase activity was decreased by 2-fold . This study reports for the first time a down-regulation of mdr1 gene transcription by a pharmacological agent . These results provide further identification of the regulatory mechanisms involved in the overexpression of mdr1 in MDR cells and may help in the development of new strategies for MDR reversal.

DNA Cell Biol, 1995 Jan, 14(1), 47 - 59
Amplification of the murine mdr2 gene and a reconsideration of the structure of the murine mdr gene locus; Kirschner LS; A common feature of cells selected in vitro for the multidrug resistance (MDR) phenotype is the amplification and concomitant overexpression of the mdr genes . In murine macrophage-like J774.2-derived MDR cell lines, there is a good correlation between levels of amplification and expression for the mdr1b gene, but not for the other two gene family members, mdr1a and mdr2 . To understand this phenomenon better, a study of the amplification and expression of the mdr2 gene was undertaken . Southern blotting of genomic DNAs from a series of six MDR cell lines revealed that five of these lines had 5'-end amplification of mdr2, whereas only three contained 3'-end amplification . The analysis also suggested the involvement of a recombination hot-spot in this phenomenon . Despite the observation that the ratio between the number of copies of the 5' and 3' ends of the gene differs among cell lines, the ratio of 5' to 3' end transcription of mdr2 was approximately 1 in all cell lines . An analysis of promoter methylation in MDR cell lines demonstrated that this mechanism may play a role in regulating the transcription of mdr2, but not of mdr1b . Long-range mapping of the mdr locus in parental and amplified cell lines suggested that the three mdr genes are oriented in the same direction, and also revealed the presence of a number of rearrangement events . Models for the murine mdr gene locus in wild-type cells and in a cell line containing a rearrangement are presented.

Cancer Chemother Pharmacol, 1995, 35(4), 345 - 8
The role of methoxymorpholino anthracycline and cyanomorpholino anthracycline in a sensitive small-cell lung-cancer cell line and its multidrug-resistant but P-glycoprotein-negative and cisplatin-resistant counterparts; van der Graaf WT et al.; The cytotoxic action of two morpholino anthracyclines, methoxymorpholino anthracycline (MRA-MT, FCE 23,762) and cyanomorpholino anthracycline (MRA-CN), was compared with the cytotoxicity of doxorubicin (DOX), the topoisomerase II inhibitor etoposide (VP-16), the topoisomerase I inhibitor camptothecin, methotrexate, and cisplatin in GLC4, a human small-cell lung-cancer cell line, in GLC4-Adr, its P-glycoprotein (Pgp)-negative, multidrug-resistant (MDR; 100-fold DOX-resistant) subline with overexpression of the MDR-associated protein (MRP) and a lowered topoisomerase II activity, and in GLC4-CDDP, its cisplatin-resistant subline . GLC4-Adr was about 2-fold cross-resistant for the morpholino anthracyclines and GLC4-CDDP was, relative to GLC4, more resistant for the morpholino anthracyclines than for DOX . Overall, MRA-CN was about 2.5-fold more cytotoxic than MRA-MT . The cytotoxicity profile of the morpholino anthracyclines in these cell lines mimicked that of camptothecin.

Cancer Chemother Pharmacol, 1995, 35(4), 271 - 7
Reversal of multidrug resistance by a novel quinoline derivative, MS-209; Sato W et al.; MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells . MS-209 at 1-10 microM completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) . MS-209 at 1-10 microM also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells . In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested . MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice . When MS-209 was given p.o . at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 micrograms/kg VCR, a treated/control (T/C) value of 155% was obtained . MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice . The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%-194% for the combined treatment at an MS-209 dose of 200-450 mg/kg . MS-209 inhibited {3H}-azidopine photolabeling of P-glycoprotein efficiently . Furthermore, the accumulation of ADM in K562/ADM cells was increased more efficiently by MS-209 than by verapamil . These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.

J Surg Oncol, 1995 Jan, 58(1), 63 - 9
Clinical significance of multidrug resistance and P-glycoprotein expression in patients with gastric carcinoma; Fujii H et al.; Twenty-four fresh tumors of gastric carcinoma were assessed by flow cytometric detection of P-glycoprotein (P-gp) using monoclonal antibody C219 . Eight patients were P-gp positive . Differentiated gastric carcinomas contained significantly higher concentrations of P-gp positive . Incidence of P-gp positive was high in advanced stage . In 16 cases estimated chemosensitivity was test assessed by thymidine incorporation assay (TIA) . Seven of nine multidrug-resistant cases according to TIA were P-gp positive and all of seven nonmultidrug resistant cases were P-gp negative . Expression of P-gp and multidrug resistance were closely correlated (P < 0.01) . Also, in 89 patients with operable gastric carcinoma, the relation between in vitro chemosensitivity test (TIA) and their clinicopathologic features as well as their survival lengths were studied . Thirty-one of 89 specimens from gastric carcinoma patients were multidrug resistant according to TIA . Patients in the multidrug-resistant group had a significantly poorer cumulative survival rate than those who were not multidrug resistant (P < .05) . The multivariate analysis showed that multidrug resistance is a useful indicator of prognosis (P < 0.1) . We suggest that multidrug-resistant cases or P-gp-positive cases of gastric carcinoma are highly malignant, and these determinations are clinically useful.

Br J Cancer, 1995 Jan, 71(1), 52 - 8
Expression of a 95 kDa membrane protein is associated with low daunorubicin accumulation in leukaemic blast cells; Doyle LA et al.; A 95 kDa membrane protein (P-95) has been previously noted to be overexpressed in a doxorubicin-resistant subline of the MCF-7 breast cancer line and in clinical samples obtained from patients with solid tumours refractory to doxorubicin . We performed Western blotting on blast cell lysates from adults with acute myeloid leukaemia, using antisera to P-95 . Concomitant flow cytometric assays measured daunorubicin accumulation and retention . Blasts from 16/46 patient samples had detectable P-95 and had reduced accumulation of daunorubicin compared with the negative marrows . Experiments with the P-95 positive MCF-7 multidrug-resistant subline demonstrated decreased daunorubicin accumulation and retention relative to the sensitive parent line . AML blast cells positive for P-95 also demonstrated greater overall in vitro survival in the presence of daunorubicin relative to the P-95-negative samples . The expression of P-95 did not correlate with failure to achieve an initial complete remission with daunorubicin and cytarabine induction chemotherapy . We conclude that the P-95 protein may possess an efflux transporter function, and may represent another mechanism responsible for anthracycline resistance in acute myeloid leukaemia.

Br J Cancer, 1995 Jan, 71(1), 40 - 7
Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines; Eijdems EW et al.; Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II . We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573 . Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha . No alterations of topo II beta levels were detected . To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells . The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity . Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels . In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels . Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells . These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents . The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.

South Med J, 1995 Jan, 88(1), 60 - 4
Appearance of drug-resistant tuberculosis in rural Tennessee; Mehta J et al.; Drug-resistant tuberculosis (DRTB) is a growing national health concern in both urban populations and rural areas and is exacerbated by the growing epidemic of human immunodeficiency virus (HIV) infection . Between 1989 and 1992, 7 cases of DRTB (5 with multidrug-resistance) were diagnosed in an eight-county region of East Tennessee . During 1990 and 1991 alone, 5 of 100 patients with tuberculosis had drug-resistant strains (5%) . All 7 patients with DRTB had 100% resistance to isoniazid; 5 also had resistance to streptomycin, 2 to rifampin, and 1 to pyrazinamide and ethambutol . All patients were white, U.S.-born, and without evidence of HIV infection . Contact investigation revealed that more contacts of patients with DRTB (13 of 74, 18%) were infected than were contacts of patients with drug-sensitive tuberculosis (46 of 290, 16%) . Our study demonstrates that DRTB is not confined to geographically distinct areas, but may be a subtle and easily missed diagnosis in presumably low-risk rural populations.

Cancer Chemother Pharmacol, 1995, 35(3), 267 - 9
The structure of P-glycoprotein and the secretion of lysosomal enzymes in multidrug-resistant cells; Warren L et al.; We have previously demonstrated that multidrug-resistant cells have a lower content of lysosomal enzymes, a consequence of an increased rate of secretion . The question was therefore to know whether an intact functional P-glycoprotein was necessary for expression of this property . Control NIH3T3 and mdr1-gene-transfected cells (pHaMDR1) were used together with 2 variants either lacking 23 amino acids at the carboxyl terminal (pHaMDRC 23) or in which 4 extra amino acids are inserted (pHaMDRBL2) . Transfected and variant cells exhibited reduced uptake of {3H}-vinblastine and {3H}-daunomycin, a finding consistent with their drug resistance . By contrast, only pHaMDR1 cells had a reduced level of N-acetyl glucosaminidase that paralleled an increased rate of secretion of the same enzyme . The mutant cells secreted lysosomal enzyme at the same rate and had the same intracellular lysosomal enzyme content as NIH3T3 cells . Abnormal behavior of lysosomal enzymes in multidrug-resistant cells therefore seemed to require an intact P-glycoprotein molecule . Although sequestration in lysosomes and then secretion of drugs may possibly contribute to protection, it would not be an essential component of multidrug resistance.

Cancer Res, 1995 Jan 1, 55(1), 102 - 10
Characterization of the M(r) 190,000 multidrug resistance protein (MRP) in drug-selected and transfected human tumor cell; Almquist KC et al.; Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types . The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated {S . P . C . Cole et al., Science Washington, DC), 258: 1650-1654, 1992} . Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C . E . Grant et al., Cancer Res., 54: 357-361, 1994) . Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio . Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known . We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells . These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000 . No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells . Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein . The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles . The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.

J Infect Dis, 1995 Jan, 171(1), 170 - 6
Temporal trends and transmission patterns during the emergence of multidrug-resistant tuberculosis in New York City: a molecular epidemiologic assessment; Shafer RW et al.; To ascertain the role of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis transmission on multidrug-resistant (MDR) tuberculosis (TB) emergence in New York City, medical records, drug susceptibilities, and restriction fragment length polymorphisms (RFLPs) of TB cases at a city hospital between two 9-month periods (1987-1988 and 1990-1991) were reviewed . The proportion of TB patients with MDRTB increased from 10% (27/267) to 17% (38/222; P = .03) . Among MDRTB patients of known HIV status, the proportion with HIV increased from 16% (3/19) to 58% (22/38; P = .006) . HIV-infected MDRTB patients were more likely than the seronegative ones to have initial MDRTB (88% vs . 56%; P = .03) . Among 56 MDR cases with RFLP results, 12 had unique patterns; 44 belonged to one of six clusters . During 1990-1991, 27 (75%) of 36 MDRTB patients were infected with strains isolated from HIV-seronegative patients during 1987-1988 . The increase in MDRTB caused by transmission from immunocompetent to immunocompromised persons underscores the urgency of TB control in populations with increasing HIV prevalence.

Eur J Cancer, 1995, 31A(3), 389 - 94
Failure of liposomal encapsulation of doxorubicin to circumvent multidrug resistance in an in vitro model of rat glioblastoma cells; Hu YP et al.; We studied the capacity of doxorubicin encapsulation in liposomes of various lipid compositions to circumvent multidrug resistance in several variants of the C6 rat glioblastoma cell line in culture, and to inhibit azidopine binding to membranes isolated from these cells . Three formulations of liposomes were prepared: (a) phosphatidylcholine (PC)/phosphatidylserine (PS)/cholesterol (cho) at a 9/24 ratio; (b) PC/cardiolipin (CL)/cho at 10/1/4 ratio; (c) dipalmitoylphosphatidylcholine (DPPC)/cho at 11/4 ratio . Unloaded liposomes presented no cytotoxicity against sensitive or resistant cells . Doxorubicin encapsulated in PC/PS/cho and PC/CL/cho liposomes had a cytotoxic activity close to that of free doxorubicin, whereas doxorubicin encapsulated in DPPC/cho liposomes was significantly less active than free doxorubicin in sensitive as well as in two of the three multidrug resistant cell lines, and as active as free doxorubicin in the third one . Free doxorubicin was able to decrease 50% of {3H}azidopine photolabelling to P-glycoprotein at a concentration of 40 microM; doxorubicin encapsulated in PC/CL/cho or PC/PS/cho liposomes was able to inhibit {3H}azidopine binding similarly as free drug, whereas doxorubicin encapsulated in DPPC/cho liposomes had no significant effect on this parameter . Unloaded liposomes of either lipid composition had no effect on {3H}azidopine binding . Together with physical studies performed in parallel on doxorubicin trapping in liposomes (J Liposome Res 1993, 3, 753-766), these results suggest that doxorubicin leaked out of fluid liposomes (PC/PS/cho or PC/CL/cho), whereas rigid liposomes (DPPC/cho) were able to sequester the drug more efficiently . In that case, however, no availability of the drug to the cells was possible and only a weak cytotoxicity was exhibited, especially without any favourable effect on multidrug resistance . In conclusion, no reversal of doxorubicin resistance was found to occur through liposomal encapsulation of the drug.

Eur J Cancer, 1995, 31A(3), 380 - 8
KT-5720 reverses multidrug resistance in variant S49 mouse lymphoma cells transduced with the human MDR1 cDNA and in human multidrug-resistant carcinoma cells; Galski H et al.; T-25-Adh cells, cell variants derived from S49 mouse lymphoma, were transduced with a retrovirus containing the human MDR1 cDNA . The resultant cells (HU-1) are cross-resistant to colchicine, doxorubicin, vinblastine and actinomycin D, and their resistance to colchicine is reversed by verapamil . HU-1 cells were used to screen several protein kinase modulators for their ability to reverse multidrug resistance . Among the tested indole carbazole (K-252a) family of protein kinase inhibitors, only the antibiotic alkaloid KT-5720 (9-n-hexyl derivative of K-252a) could overcome the multidrug resistance of HU-1 cells and KB-V1 human carcinoma cells . Since other protein kinase A, C and G modulators did not reverse multidrug resistance in the tested multidrug-resistant cells, the chemosensitising activity of KT-5720 on these cells is apparently independent of its kinase inhibitory effects . Since KT-5720 fully reversed multidrug resistance at non-toxic concentrations, it might be a candidate for clinical chemosensitisation in combination chemotherapy.

Cancer Chemother Pharmacol, 1995, 36(3), 244 - 8
Experimental solid tumour activity of N-{2-(dimethylamino)ethyl}-acridine-4-carboxamide; Baguley BC et al.; N-{2-(Dimethylamino)ethyl}acridine-4-carboxamide (DACA), a DNA intercalator that exerts its antitumour action through the enzyme topoisomerase II, has previously been shown to be curative against the transplantable Lewis lung adenocarcinoma growing as lung tumour nodules in mice . On the basis of this finding as well as its high in vitro activity against multidrug-resistant cell lines, DACA has been chosen for clinical trial under the auspices of the Cancer Research Campaign, United Kingdom . In the present study the activity of DACA was assessed against advanced (5-mm diameter) s.c . colon 38 adenocarcinomas in BDF1 mice using tumour-growth delay as an end point . Its activity was found to be related positively to the total dose given and negatively to the total duration of the dose schedule . Adoption of a split-dose i.p . administration schedule or slow i.v . infusion allowed the administration of large doses without toxicity . The activity of DACA was comparable with that of 5-fluorouracil and superior to that of doxorubicin, cyclophosphamide and the experimental amsacrine analogue CI-921 . Mitoxantrone, amsacrine, etoposide, teniposide and daunorubicin showed minimal activity . DACA also demonstrated significant activity against the NZM3 melanoma human cell line growing as a xenograft in athymic mice.

Cancer Chemother Pharmacol, 1995, 36(3), 223 - 6
Comparative resistance of idarubicin, doxorubicin and their C-13 alcohol metabolites in human MDR1 transfected NIH-3T3 cells; Kuffel MJ et al.; The anthracycline analog idarubicin (ID) is useful in the treatment of leukemias, and is of further interest because of the unique activity of its major circulating metabolite idarubicinol (IDOL) . In vitro studies have shown that ID retains activity against tumor cells made resistant by prolonged exposure to substrates of the p-glycoprotein energy-dependent efflux pump . To selectively investigate multidrug resistance to ID in tumor cells, ID, IDOL, doxorubicin (DX) and doxorubicinol (DXOL) were evaluated for growth inhibitory activity when incubated with NIH-MDR1-G185 (MDR) cells or with the parent NIH-3T3 (3T3) cells . The MDR cells are transfected with the human multidrug gene mdr1, and express a functional p-glycoprotein . ID growth inhibitory activity was much less affected by p-glycoprotein-mediated efflux than was DX . ID IC50 values were only 1.8-fold greater in the MDR cell line than in the parental 3T3 cell line, while the IC50 value for DX was 12.3-fold greater in the transfected cell line . Verapamil (VRP) fully restored drug sensitivity of the MDR cell line to ID and DX . In studies with the alcohol metabolites, IDOL and DXOL IC50 values were 7.8- and 18.9-fold greater, respectively, for the MDR cell line than for the parental cell line . Intracellular concentrations of DX and DXOL, but not ID and IDOL, were substantially increased in the MDR cells when VRP was present in the incubation mixtures . ID and IDOL retain substantial growth inhibitory activity in mdr1-transfected cells, and ID may be of value in clinical settings where multidrug resistance mediated by p-glycoprotein is a potential limitation of therapy.

Cancer Chemother Pharmacol, 1995, 36(3), 195 - 203
In vitro activity of S 9788 on a multidrug-resistant leukemic cell line and on normal hematopoietic cells-reversal of multidrug resistance by sera from phase I-treated patients; Soudon J et al.; The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials . In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype . S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs . At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells . S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors . The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied . Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788 . In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration . In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells . The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM . These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

Virchows Arch, 1995, 426(3), 249 - 56
Intracellular localization, vesicular accumulation and kinetics of daunorubicin in sensitive and multidrug-resistant gastric carcinoma EPG85-257 cells; Seidel A et al.; In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with stepwise increased concentrations of the drug . The new variant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 kDa P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and mdr1-specific RT-PCR . To investigate the intracellular pathway of DAU the subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EPG85-257P . When sensitive cells were exposed to DAU the drug rapidly accumulated in the nucleus until cell death . No redistribution of DAU to the cytoplasm was observed . In resistant cells exposed to the drug DAU also accumulated in the nucleus but to a lesser extent than in parent cells . Following exposure, nuclear fluorescence was observed to decrease over a time period of up to 48 h . Six hours after DAU exposure formation of fluorescent vesicle formation started in the perinuclear region and increased continuously . After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles . During this period the vesicles moved from the region of origin to the cell periphery . A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus . Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation . Disruption of microtubules by nocodazole led to an accumulation of vesicles in the perinuclear region indicating that microtubules are involved in vesicular transport . Treatment of EPG85-257DAU cells with the actin disruptor cytochalasin B led to accumulation of vesicles in the cell periphery indicating that actin may be involved in exocytosis . Uptake and efflux of DAU and rhodamin (RH) were determined in sensitive and resistant cells using a fluorescence activated cell sorter . Uptake of both compounds was distinctly lower in resistant than in sensitive cells . When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating transmembrane transport by P-Gp . In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol . This indicates that in resistant cells intracellular DAU at least in part may be inaccessible for P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis.

Cancer Chemother Pharmacol, 1995, 36(2), 160 - 4
Tolerance, safety, and kinetics of the new antineoplastic compound dexniguldipine-HCl after oral administration: a phase I dose-escalation trial; Ukena D et al.; Dexniguldipine-HCl is a new dihydropyridine compound that exerts selective antiproliferative activity in a variety of tumor models and, in addition, has a high potency in overcoming multidrug resistance . The purpose of this trial was to determine the toxicity and pharmacokinetics of dexniguldipine and to establish a recommended dose for phase II trials . A total of 37 patients with cancer were treated with oral dexniguldipine in increasing doses for up to 7 days . The main parameters evaluated were subjective tolerance and laboratory and cardiovascular parameters (blood pressure and ECG) . Blood samples were drawn for analysis of the drug's pharmacokinetics . Dizziness and nausea were the major adverse events observed in seven patients, but episodes were generally mild and not clearly dose-related . Vomiting occurred in one patient . Hypotensive effects and orthostatic dysregulation were observed in some patients but were not considered to be dose-limiting . Therefore, no dose-limiting toxicity was found and the maximally tolerable dose could not be determined . Pharmacokinetic data showed wide interindividual variation and a dose-dependent increase in steady-state serum concentrations at doses of up to 1,000 mg daily, with no clear further increase being observed at higher doses . Consistently high concentrations were achieved with the 2,500-mg dose . Despite the lack of dose-limiting toxicity, higher doses of dexniguldipine do not appear to be useful for clinical evaluation because of the pharmacokinetic properties of the compound: therefore, 2,500 mg/day is recommended as the daily dose for phase II trials.

Biosci Biotechnol Biochem, 1995 Jan, 59(1), 147 - 9
Functional analysis of the promoter of the Saccharomyces cerevisiae multidrug resistance gene YDR1, which encodes a member of the ATP binding cassette (ABC) superfamily; Miyahara K et al.; The Saccharomyces cerevisiae gene YDR1/PDR5/STS1, which encodes a member of the ATP binding cassette (ABC) superfamily, is important for cross-resistance to apparently unrelated drugs . The expression of YDR1 is induced by various drugs and heat shock {Hirata et al., Curr . Genet., 26, 285-294 (1994)} . By deletion analysis of the 5'-noncoding region of the YDR1 gene, two drug responsive regions (-604 to -485 and -335 to -275) were identified with fluphenazine and cycloheximide . Three conserved palindrome sequences were found in these regions.

Breast Cancer Res Treat, 1995, 33(1), 27 - 37
Current status of paclitaxel in the treatment of breast cancer; O'Shaughnessy JA et al.; Paclitaxel is a highly active single agent as therapy for previously untreated as well as doxorubicin-refractory metastatic breast cancer, with associated response rates of 62% and 20-48%, respectively . Complete responses with paclitaxel occur chiefly in breast cancer patients whose metastatic disease has not been previously treated with chemotherapy . Early data suggest a possible dose-response relationship for paclitaxel in metastatic breast cancer, but the optimal dose has not yet been defined . The optimal duration of infusional paclitaxel treatment is also not yet known . A study of 96-hour infusional paclitaxel in the treatment of doxorubicin- or mitoxantrone-refractory metastatic breast cancer patients has demonstrated a 48% response rate suggesting that prolonged exposures to paclitaxel may offer a therapeutic advantage . Randomized trials of 3- vs 96-hour paclitaxel are ongoing or planned . The relative efficacy of paclitaxel versus standard chemotherapy as front-line or salvage therapy for metastatic breast cancer is currently under study . In addition, two randomized trials are under way in node positive breast cancer patients to study whether treatment with paclitaxel following standard or high dose doxorubicin and cyclophosphamide adjuvant therapy results in improved patient benefit . Combining paclitaxel with other active agents in the treatment of metastatic breast cancer is an area of active investigation . Combined paclitaxel and doxorubicin, administered concurrently or sequentially, is associated with modest complete response rates in metastatic breast cancer patients . Sequential paclitaxel-->doxorubicin administration is associated with more mucositis than is doxorubicin-->paclitaxel when paclitaxel is administered over 24 hours . High doses of cyclophosphamide can be combined with 24- or 72-hour infusional paclitaxel, and phase II studies of this combination are warranted . Early data suggest that administering biweekly paclitaxel and cisplatin to previously untreated metastatic breast cancer patients is associated with high response rates, and confirmatory studies of this combination and schedule are planned . Preclinical data suggest that cell cycle considerations may be important in combining doxorubicin and possibly other agents with paclitaxel . Paclitaxel is an excellent substrate for P-glycoprotein, the protein product of the multidrug resistance-1 (mdr-1) gene, and phase I trials are under way combining paclitaxel with several known blockers of Pgp function . Finally, pilot studies are under way to determine whether the radiation sensitizing effects of paclitaxel can be exploited as part of radiation therapy for patients with locally advanced breast cancer.

Urol Res, 1995, 22(6), 353 - 60
Establishment and characterization of a multidrug-resistant human bladder carcinoma cell line RT112/D21; Seemann O et al.; A doxorubicin-resistant human bladder carcinoma cell line RT112/D21 was established by continuous exposure of the parental line RT112 to increasing concentrations of doxorubicin over a period of 9 months . RT112/D21 cells expressed significantly more P-170 glycoprotein than the parental line, and rhodamine 123 efflux, as a functional parameter of P-170 glycoprotein activity, was increased . RT112/D21 cells were 96 times more resistant to doxorubicin than RT112 cells, and cross-resistance to epirubicin and vinblastine was present . Sensitivity to methotrexate and mitomycin C remained unchanged . R-verapamil reversed resistance to doxorubicin, epirubicin and vinblastine in RT112/D21 cells but did not affect sensitivity to methotrexate and mitomycin C . In RT112 cells, R-verapamil had no effect on drug sensitivity . Thus, it may be assumed that primary or induced MDR1 gene-encoded P-170 glycoprotein expression is a relevant mechanism of chemoresistance in transitional cell carcinoma, and that chemotherapeutic strategies in combination with chemosensitizers improve response rates.

Jpn J Cancer Res, 1995 Jan, 86(1), 124 - 9
Intracellular carboxyl esterase activity is a determinant of cellular sensitivity to the antineoplastic agent KW-2189 in cell lines resistant to cisplatin and CPT-11; Ogasawara H et al.; KW-2189, a novel antitumor antibiotic belonging to the duocarmycins, possesses marked DNA-binding activity upon activation by carboxyl esterase to its active form, DU-86 . Three duocarmycins, KW-2189, DU-86 and duocarmycin SA, were active against the cisplatin (CDDP)-resistant human non-small cell lung cancer cell lines PC-9/CDDP and PC-14/CDDP, and the multidrug-resistant human small cell lung cancer cell line H69/VP . However, HAC2/0.1, a CDDP-resistant human ovarian cancer cell line which is also resistant to CPT-11 because of decreased intracellular activation of CPT-11, was about 12.8-fold more resistant to KW-2189 . HAC2/0.1 was not resistant to other duocarmycins as compared to its parental cell line, HAC2 . There was no difference between HAC2 and HAC2/0.1 with regard to the intracellular accumulation of KW-2189 . Addition of 130 mU/ml of carboxyl esterase to the culture medium did not influence the sensitivity of HAC2 cells to KW-2189 . However, the sensitivity of HAC2/0.1 cells to KW-2189 was enhanced to the level of HAC2 . These results suggest that HAC2/0.1 is less potent than HAC2 in activating KW-2189 . The carboxyl esterase activity of whole-cell and microsomal extracts from HAC2/0.1 was approximately 60% of that from HAC2 . The cell-free experiment revealed that KW-2189 bound to DNA more efficiently in the presence of HAC2 than HAC2/0.1 cell extract . It was concluded that decreased intracellular carboxyl esterase activity in HAC2/0.1 cells caused decreased intracellular conversion of KW-2189 to its active form, thus producing resistance to KW-2189 . The decreased conversion of CPT-11 to SN-38 in HAC2/0.1 cells might be explained by decreased carboxyl esterase activity.

Anticancer Res, 1995 Jan-Feb, 15(1), 121 - 6
Rhodamine 123: is it an appropriate dye to study P-glycoprotein activity in adriamycin-resistant K562 cells?
Denis-Gay M, Petit JM, Ratinaud MH.
The ability of the P-glycoprotein to efflux rhodamine 123 and adriamycin was evaluated using adriamycin-sensitive and -resistant human leukemia K562 cells . We observed that low temperature or verapamil (a P-glycoprotein blocker) inhibited adriamycin efflux in multidrug resistant cells . In the same conditions, resistant K562 cells did not significantly retain rhodamine 123 . This dye was located in the cytoplasm of resistant cells and did not display spectral properties characteristic of stacked rhodamine 123 molecules in mitochondria of sensitive K562 cells . Thus, in adriamycin-resistant K562 cells, the rhodamine efflux may be due to P-glycoprotein activity and also to a nonspecific targeting of dye in resistant K562 cells.

J Physiol, 1995 Jan, 482, 31S - 36S
Volume-activated chloride currents associated with the multidrug resistance P-glycoprotein; Higgins CF; The ability to regulate volume is an important property of most, if not all cells . In epithelial cells, amongst others, cell volume-activated chloride channels are central to this response . The molecular identities of these channels are not yet known . Expression of the human multidrug resistance P-glycoprotein (P-gp) has been associated with cell volume-regulated chloride currents, although the nature of this association is the subject of debate . Recent data indicate that P-gp acts by regulating the activation of an endogenous channel protein . In this review, evidence associating P-gp with cell volume-activated chloride currents, and the possible mechanisms by which this might be achieved, are discussed.

Clin Infect Dis, 1995 Jan, 20(1), 136 - 42
Bacille Calmette-Guérin vaccination for the prevention of tuberculosis in health care workers; Brewer TF et al.; For 60 years vaccination with bacille Calmette-Guerin (BCG) has been used for the prevention of tuberculosis in health care workers . In 1988 the U.S . Advisory Committee on Immunization Practices removed the category of health care worker from the list of persons for whom vaccination with BCG should be considered . Nosocomial epidemics of tuberculosis, especially those caused by multidrug-resistant strains, have led to the reconsideration of vaccination with BCG for this population . We review the available studies of the efficacy of BCG vaccine in health care workers . Although the studies had too many methodological flaws to be combined in a quantitative meta-analysis, they suggest that vaccination with BCG is effective in reducing the incidence of tuberculosis among health care workers.

Acta Haematol, 1995, 93(1), 13 - 9
Haemostatic abnormalities in multidrug-resistant enteric fever; Koul PA et al.; Twenty-five consecutive patients with multidrug-resistant enteric fever were evaluated and followed for haemostatic abnormalities . Twenty-one (84%) of the patients had evidence of disseminated intravascular coagulation (DIC) and 12 (48%) also had evidence of associated fibrinolysis . Clinical bleeding was observed in 3 (12%) cases, and did not bear any correlation with clotting abnormalities . Protein C activity was found to be decreased in 11 of the 15 cases with DIC, and a block in its activation, as previously postulated, could not be substantiated . DIC was reversed in most cases within 8 days of the institution of specific antibiotic therapy.

Cancer Chemother Pharmacol, 1995, 36(1), 87 - 90
Increased cation transport in mdr1-gene-expressing K562 cells; Brismar T et al.; Cation-transport properties were compared in a human leukemic cell line (K562) and its vincristine-selected, mdr1-gene-expressing sublines (K562/Vcr30 and K562/Vcr150) by the capacity of the cells to accumulate the potassium analogue thallium (201Tl) . Determination of the time course of thallium accumulation in the absence and presence of ouabain, an inhibitor of sodium-potassium adenosine triphosphatase (ATPase), showed that the initial (at 20 min) rate of ouabain-resistant uptake was about 70% higher in the K562/Vcr30 cells than in the parental line . The maximal rate (Vmax) of ouabain-resistant uptake was 78 mmol/h for K562 cells and 115 mmol/h for K562/Vcr30 cells, and the Michaelis constant (Km) was 0.37 and 0.18 mmol, respectively . Bumetanide (50 microM), a specific inhibitor of ouabain-resistant Na-K-Cl cotransport, inhibited the elevated 201Tl uptake in K562/Vcr150 cells but had no effect on cellular vincristine accumulation . Incubation with different multidrug resistance (MDR)-reversing agents (verapamil as well as cyclosporin A and its analogue PSC833) had no significant effect on 201Tl uptake . Membrane depolarization by an elevation of the potassium concentration in the incubation medium did not affect vincristine accumulation in any cell line, which indicated that the changed drug-transport properties in mdr1-gene-expressing cells were not due to membrane hyperpolarization . It was concluded that P-glycoprotein-positive cells have a more efficient ouabain-resistant cation-transport mechanism than to cells without P-glycoprotein . A functional relationship between this phenomenon and MDR was not identified.

Cancer Chemother Pharmacol, 1995, 36(1), 65 - 8
A phase I trial of high-dose oral tamoxifen and CHOPE; Smith DC et al.; Drug resistance is a common phenomenon in clinical oncology . In vitro, tamoxifen has been shown to be an effective inhibitor of P-glycoprotein and a modulator of the multidrug resistance phenotype . We have previously shown that vinblastine can be given safely in combination with tamoxifen at doses that may modulate P-glycoprotein activity . In this phase I trial, tamoxifen (150 mg/m2 twice a day) was given with CHOPE (cyclophosphamide/doxorubicin/vincristine/prednisone/etoposide) in order to assess the toxicities of the combination . Resistance to three of these cytotoxic agents (doxorubicin, vincristine, and etoposide) may be mediated by P-glycoprotein . A total of 13 patients were evaluable on this trial, which showed that the maximum tolerated doses of cyclophosphamide and etoposide were 750 and 80 mg/m2, respectively . The dose-limiting toxicity was myelosuppression with 50% of the patients (3/6) treated at this dose level developing febrile neutropenia and 85% (6/7) developing grade 4 neutropenia . Tamoxifen at a dose of 150 mg/m2 twice a day can be given safely with the lymphoma regimen CHOPE at standard doses, but this combination may result in increased myelosuppression.

Eur J Cancer, 1995, 31A(2), 230 - 7
Resistance mechanisms determining the in vitro sensitivity to paclitaxel of tumour cells cultured from patients with ovarian cancer; Baguley BC et al.; Paclitaxel, a drug which stabilises microtubules, demonstrates marked activity against ovarian cancer . We investigated the sensitivity to paclitaxel of tumour cells from disaggregated solid tumours or tumour-bearing ascites from 7 ovarian cancer patients, and 21 established tumour cell lines (ovarian, melanoma and lung) . Response was quantitated by {3H}-thymidine incorporation in 96-well plates or by colony growth . Dose-response curves to paclitaxel were biphasic with a dose-dependent phase providing an IC50 value (50% reduction in incorporation) and dose-dependent "plateau" phase where the effect was independent of paclitaxel concentration . IC50 values ranged from 2.5 to 110 nM with evidence of multidrug resistance in the two most resistant cell lines . The "plateau" killing values varied from 0.1 log10 to > 3.4 log10 units reduction, and were found to be significantly correlated (r = 0.86; P < 0.0001) with logarithmic culture doubling times of the cell lines . Cellular glutathione levels were measured and found not to be significantly associated with response to paclitaxel . The results suggest that the ratio of paclitaxel exposure time to the culture doubling time is a major factor in paclitaxel cytotoxicity . The relationship between tumour cell cytokinetics and paclitaxel pharmacokinetics in vivo may therefore be crucial in determining clinical paclitaxel response.

Acta Oncol, 1995, 34(2), 235 - 41
Comparative study on reversal efficacy of SDZ PSC 833, cyclosporin A and verapamil on multidrug resistance in vitro and in vivo; Watanabe T et al.; A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein . The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp) . PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition . The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o . 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice . PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50% . This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice . These results demonstrated that PSC833 has significant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.

J Pharm Sci, 1995 Jan, 84(1), 21 - 7
A biophysical model of passive and polarized active transport processes in Caco-2 cells: approaches to uncoupling apical and basolateral membrane events in the intact cell; Ho NF et al.; This report is aimed at the biophysical modeling of transmembrane events involving a passive diffusion and directional pumplike mechanism at the apical (AP) and basolateral (BL) membranes of cultured cell monolayers . The essence of the model is based on experimental evidences for the existence of a saturable, apically polarized transport system in Caco-2 cells for peptides which hindered apical to basolateral flux, enhanced basolateral to apical flux, and showed substrate specificity . This system was further inhibited by verapamil, suggesting some homology with P-glycoprotein, the principal mediator of drug resistance in multidrug resistant cancer cells . Preliminary evidence was also obtained suggesting an additional polarized uptake system for the same peptides in the basolateral membrane . Upon saturation and/or inhibition of the active transport mechanisms with verapamil, the peptide fluxes in apical-to-basolateral direction and the basolateral-to-apical direction converged and became controlled by the passive mechanism . Since the intent of the modeling was to provide useful templates for the design of probing experiments and to delineate and quantify mass transfer mechanisms at the AP and BL membranes and their interrelationships, theoretical equations were developed for a host of kinetic boundary conditions: (a) AP-->BL and BL-->AP transfluxes, (b) bidirectional effluxes from substrate-preloaded cells, (c) undirectional efflux across the AP or BL membrane from preloaded cells, and (d) uptake kinetics via the AP or BL membrane leading to equilibrium . Furthermore, flux expressions were reduced to membrane permeability coefficients to accommodate passive diffusion, saturation, inhibition, and directionality . The diffusional mass transport resistances of the aqueous boundary layers and microporous filter support of the cell monolayer were necessarily included.

J Cancer Res Clin Oncol, 1995, 121(3), 155 - 63
"Atypical" multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3); Cimoli G et al.; Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are cross-resistant to various other topoisomerase-II-targeted drugs but not to vinblastine . The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide(VP16)-induced DNA single-strand breaks, under deproteinizing conditions . In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity . No significant alterations in cellular drug uptake were observed in DX3 cells . Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantrone- or VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance . Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal . However, an assay that specifically measures generation of 5'-protein-linked breaks in 32P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16 . These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drug-cytotoxicity rather than loss of normal topoisomerase catalytic function . These data support the hypothesis that A2780-DX3 cells display an "atypical" multidrug resistance.

Curr Opin Oncol, 1995 Jan, 7(1), 28 - 35
Developments in the treatment of acute leukemia in adults; Volm MD et al.; Since 1977, when it was first demonstrated that a small number of patients with refractory leukemia could be cured by bone marrow transplantation, there has been an international effort to increase the proportion of patients cured . This article reviews recent developments in the treatment of adult leukemia with particular emphasis on reports published during the past year . Several new preparative regimens for transplantation are discussed . New chemotherapeutic strategies reported during the past year have included modulating the activity of cytarabine, the most active agent in autologous myelogenous leukemia, using other agents such as etoposide, mitoxantrone, and carboplatin, and attempting to overcome the multidrug resistance phenomenon . There has been further experience in the use of autologous marrow for transplantation, with several centers reporting excellent results for patients so treated . Biologic-based strategies are an exciting area of exploration . The implications of the landmark discovery of all-trans retinoic acid as a treatment for acute promyelocytic leukemia continue to be investigated . Reports of the use of targeted monoclonal antibodies, cytokines, and other immunotherapies have generated the hope that these strategies might prove to be non-cross-resistant therapies and thus effective against residual disease following conventional chemotherapy.

Wien Klin Wochenschr, 1995, 107(15), 470 - 1
{The resistance status of Mycobacterium tuberculosis in Austria 1992 and 1993}; Stauffer F et al.; Tuberculosis caused by resistant Mycobacterium tuberculosis strains seems to be of increasing importance in some parts of the world . Aim of this study was to investigate the frequency and nature of the resistance occurring among the strains isolated in Eastern Austria in 1992 and 1993 . 7.4% of all strains isolated from 608 patients in 1992 and 6.2% of those isolated from 497 patients in 1993 were resistant to one or more tuberculostatic drugs, 2.3% in 1992 and 3.0% in 1993 of the strains were resistant to isoniazid, 0.5 and < 0.1 respectively, to rifampicin . Multidrug-resistant strains were found in 2.8% of the isolates in 1992 and 2.2% in 1993.

Vestn Ross Akad Med Nauk, 1995, (7), 3 - 6
{Current problems of pulmonary tuberculosis}; Khomenko AG; There has been recently a rise in tuberculosis morbidity rates in the countries of Western and Eastern Europe, in the USA . At the same time high tuberculosis morbidity and mortality rates have remained in the developing countries . Along with tuberculosis endogenic reactivation in adults, there is exogenic superinfection is observed, which causes tuberculosis, including that by multidrug resistant mycobacteria . In the latter, patients were found to have a prolong disease which is characterized by acute progressive forms . This leads to a downward trend in therapeutical efficiency . The high onset drug resistance requires that a combination of 4 or 5 antituberculous agents should be used early in the disease.

Yao Xue Xue Bao, 1995, 30(4), 258 - 62
{Establishment of adriamycin-resistant human ovarian carcinoma cell line and its mechanism of multidrug resistance}; Li PY et al.; A multidrug-resistant cell line (A2780/ADM) of human ovarian carcinoma which can resist 0.8 microgram.ml-1 of adriamycin (ADM) was obtained by step-wise selection exposure to increasing doses of ADM . A2780/ADM cells showed 17-fold higher resistance to ADM than A2780 cells . The doubling times were 43.8 h in A2780/ADM and 26.3 h in A2780 cells . Colony formation rates were 15%-20% in A2780/ADM and 65%-75% in A2780 cells . A2780/ADM cell line was also shown to significantly cross-resistant to vincristine (VCR) and VP-16, but no cross-resistance was found to 5-Fu, PDD or Mel . A further investigation showed that intracellular accumulation of ADM in A2780/ADM was significantly decreased . Expressions of P-glycoprotein and GST-pi were increased in A2780/ADM by means of immunohistochemical method . Verapamil (Ver) combined with ADM was found to increase the sensitivity and reverse the resistance to ADM in A2780/ADM . This study indicates that A2780 ADM has the peculiarity of multidrug resistance and there may be other mechanism of drug-resistance besides MDR related to P-170.

Neoplasma, 1995, 42(4), 195 - 201
Decreased sensitivity of multidrug-resistant tumor cells to cisplatin is correlated with sorcin gene co-amplification; Demidova NS et al.; A set of multidrug resistant (MDR) murine leukemia P388 sublines processing 30-50-fold mdr1 gene amplification was obtained as a result of experimental chemotherapy with rubomycin, ruboxyl, vinblastine, vincristine, or combination of rubomycin and vincristine . Significant differences of developed MDR sublines in response to treatment with cisplatin, tiophosphamide, sarcolysin, and dopad were found . Strong correlation between drug sensitivity and a copy number of gene coding for 19-22 kDa calcium-binding sorcin gene co-amplification were hypersensitive to cisplatin and alkylating agents, the cell sublines showing amplification of sorcin DNA sequences did not possess such collateral sensitivity and even acquired cross-resistance . The dependence of sensitivity to cisplatin on sorcin gene co-amplification was confirmed by analysis of Djungarian hamster DM15 cell sublines that selected for MDR in vitro by colchicine.

Pol J Pathol, 1995, 46(2), 77 - 82
The expression of multidrug resistance (MDR) molecule in acute leukemia and lymphoma; Pituch-Noworolska A et al.; Multidrug resistance (MDR) is associated with expression of P-glycoprotein in the malignant cells as the one of known mechanisms for this phenomenon . The isolated blast cells of 60 patients with acute leukemia and non-Hodgkin's lymphoma (NHL) were assayed for the expression of P-glycoprotein (P-170) with MRK16 antibody . The frequency of P-170 expression was studied in the different subtypes of leukemia and NHL based on blasts phenotype . In acute leukemia and lymphoma with B cell lineage of blast cells the percentage of P-170 positive samples was 41.3%, in the non-lymphoblastic leukemia--35.3% and the T cell lineage--75% of P-170 positive samples . The expression of P-170 molecule was associated with: 1 . T cell origin of blasts, 2 . lymphoma form of proliferation . The P-170 assay selects the group of patients with higher risk of drug resistance for modified therapy.

J Cancer Res Clin Oncol, 1995, 121(7), 407 - 12
Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro; Schwabe K et al.; It was shown that cyclopeptolide SDZ 214-103 (10 microM) is more active in rhodamine-123 accumulation in actinomycin-D-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 microM), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR . In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A . For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated . At equipotent modulating doses of verapamil (10 microM) and cyclosporin A (10 microM) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.

Cancer Chemother Pharmacol, 1995, 36(5), 368 - 72
Effect of tamoxifen on mitoxantrone cytotoxicity in drug-sensitive and multidrug-resistant MCF-7 cells; Desai PB et al.; The influence of the antiestrogen tamoxifen (TAM) on the activity of mitoxantrone (MXN), was evaluated against wild-type MCF-7/WT and their multidrug-resistant variant MCF-7/ADR cells . Multidrug resistance (MDR) in this cell line which was selected for resistance to Adriamycin (ADR), is associated with increased expression of P-glycoprotein (P-gp) . In a clonogenic assay it was observed that TAM (1-10 microM) significantly enhanced the activity of MXN in the MCF-7/ADR but not in the drug-sensitive cell line . Isobologram analysis indicated that the effect of the combination was additive in the parental MCF-7/WT cells and strongly synergistic in the MDR MCF-7/ADR cells . Also, TAM (10 microM) caused a three-fold increase in the steady-state levels (Css) of MXN in MCF-7/ADR cells but did not modulate MXN levels in MCF-7/WT cells . The observed synergism in MCF-7/ADR cells was perhaps due to the increase in Css of MXN that may involve interaction of TAM with P-gp . The combination of MXN and TAM may be useful in the treatment of drug-sensitive and drug-resistant breast cancer.

Cancer Chemother Pharmacol, 1995, 36(5), 361 - 7
Relationship between multidrug resistant gene expression and multidrug resistant-reversing effect of MS-209 in various tumor cells; Baba M et al.; MS-209 is a novel quinoline compound which can overcome multidrug resistance (MDR) both in vitro and in vivo, while having a low level of side effects, and is now being evaluated in a clinical phase II study . Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitate the expression levels of MDR genes in various mouse and human tumor cell lines . The MDR gene and the beta actin gene, as the internal reference standard, were coamplified separately, and the relative expression of the MDR gene was represented by the MDR/beta actin ratio . The in vitro MDR-reversing effect of MS-209 was then compared with the MDR gene expression (MDR/beta actin ratio) . We found a significant correlation between these two parameters . Moreover, a significant correlation was also observed between the level of expression of the MDR1 gene and that of P-glycoprotein in human cell lines . Therefore, the efficacy of MS-209 seems to specifically depend on the level of MDR gene expression (P-glycoprotein) . From these observations, it is suggested that RT-PCR assays of MDR1 gene in tumor biopsy specimens might be an effective means to predict the response of tumor cells to combination therapy with MS-209.

Korean J Intern Med, 1995 Jan, 10(1), 38 - 42
The incidence of drug resistant tuberculosis in 1279 Korean patients; Yum H et al.; OBJECTIVES: In the past decade, the incidence of tuberculosis has been decreased in Korea and the nationwide survey of tuberculosis from 1965 through 1990 suggested a declining tendency of resistant organisms . But the prevalence rate of multidrug resistance of Mycobacterium tuberculosis is still a serious problem in Korea, and the aim of this study is to check the drug resistance pattern in the patients visiting University Hospital, the 3rd referral center . METHODS: We reviewed 1279 cases (522 female, 757 male, mean age 39.4 +/- 16.7) of bacteriologically proven tuberculosis seen during the period from 1986 to 1992 retrospectively . Of 1093 patients, who were indentified in previous medical history, 454 (41.5%) had a history of prior antituberculous chemotherapy . RESULTS: Resistance rate (resistant to 1 or more drugs) was 33.9% . Eleven percent of patients had resistance to a single drug (INH: 80.6%) . Twenty two percent of patients had resistance to 2 or more drugs . Resistance rate is higher (47.4%) in the patients with a history of prior treatment than without a history (25.5%) . CONCLUSIONS: These data suggest that the high rate of multidrug resistance in Korea did not show any decreasing tendency . So, mycobacterial culture and sensitivity tests should be recommended at initial treatment of tuberculosis and potent antituberculosis drugs are strongly recommended.

Cell Biol Int, 1995 Jan, 19(1), 35 - 41
Changes in the nature of P-glycoprotein gene transcripts during the development of multidrug resistance in CHO cells; Vickers SE et al.; We have studied the nature of P-glycoprotein gene transcripts in multidrug resistant Chinese hamster ovary cell lines selected in vincristine . Using a novel, 35S based, reverse transcription polymerase chain reaction assay, we have shown that there is a considerable increase in the level of pgp2 gene transcripts, with levels rising to a hundred fold those observed in sensitive control cells . The level of pgp1 gene transcripts was raised five fold . The levels of pgp3 gene transcripts were close to the limits of detection by the assay, but were increased in the more highly resistant cell lines . An increase in the size of the P-glycoprotein gene transcript was also observed in some of the resistant cell lines.

Oncol Res, 1995, 7(2), 103 - 11
DNA topoisomerase II expression, stability, and phosphorylation in two VM-26-resistant human leukemic CEM sublines; Chen M et al.; We have examined features of DNA topoisomerase II (topo II) isoforms in human leukemic CCRF-CEM cells and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5 . They are about 40- to 50-fold and 150- to > 400-fold resistant to teniposide, respectively, and have increased levels of cross-resistance to other complex-stabilizing topo II inhibitors . Topo II activity in these lines is reduced in proportion to their resistance . However, both sublines carry two identical point mutations in topo II alpha cDNA that have recently been found to confer resistance to etoposide and m-AMSA in transfected yeast cells . Although these data provide a strong molecular basis for this type of multidrug resistance, these findings alone cannot explain the increased level of resistance and cross-resistance, and the further decreased cellular topo II activities in the most resistant CEM/VM-1-5 cells compared to CEM/VM-1 cells . In this study we found that (1) topo II beta is not expressed in CEM/VM-1-5 cells; (2) a 160-kDa protein was consistently detected and coimmunoprecipatated only in nuclear extracts of CEM cells; (3) when nuclear extracts from all three cell lines were incubated at 37 degrees C, an immunoreactive band of 140-kDa appeared by 60-90 min only in samples of CEM cells, not in those of CEM/VM-1 and CEM/VM-1-5 cells; and (4) the in vivo phosphorylation level of topo II alpha protein was decreased > or = 2-fold in both resistant cell lines, compared to that of CEM cells . Thus, cell lines selected for the altered topo II-associated multidrug resistance phenotype may contain multiple alterations in both topo II isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

Verh K Acad Geneeskd Belg, 1995, 57(2), 81 - 103; discussion 103-8
{Tuberculosis: current epidemiological-clinical problems}; Demedts M et al.; Tuberculosis (tb) mortality, morbidity and infection prevalence were very high in Belgium and in the other industrialised countries during the previous century, and the first half of this century . Therefore tb was an "export" pathology, especially towards developing countries . At the end of this century tb-epidemiological indices reached very low levels in the Western world, while tb became endemic in several non-Western countries and so it actually has become an "import" pathology . In the USA, as well as in many European countries, the tb-morbidity incidence started to increase again about ten years ago . The risk groups are, however, not identical in the USA and in Belgium . In the USA, it is particularly the AIDS-epidemic that is the cause of half of the increase in tb-incidence; in addition social outcasts, homeless and IV-drug addicts are important risk groups and due to their poor therapy compliance they are responsible for the many multidrug resistant forms emerging in New York and other large cities . In Belgium elderly males are an important risk group with a tb-incidence of 50 per 10(5) in 1993 (versus an overall incidence of 15 per 10(5)) . Besides, in this group the diagnosis is often made late . A second important risk group consists of allochthones, with an incidence of 54 per 10(5), especially non-Western allochthones, with an incidence of 120 per 10(5) . Above all others are the asylum seekers with an estimated incidence of 400 per 10(5) (which undoubtedly is an underestimation) . In Belgium, the AIDS-epidemic does not represent a major problem so far; only 3.5% of the tb-cases have AIDS or are HIV-positive, and 50% of these are immigrants . Finally, also multidrug resistance is no real problem, since resistance against isoniazide and rifampicin has been found in only 0.5% of the tb-cases . Contact persons of tb-cases, however, still remain a very important risk group with an incidence of more than 200 per 10(5) . The danger is especially great for as long as the diagnosis has not been made in the source of infection and no therapeutic measures have been taken . While the overall tb-threat increased in the last decade, the tb-organisations (in Flanders the VRGT, Vereniging voor Respiratoire Gezondheidszorg en Tuberculosebestrijding) have been more or less dismantled, which in consequence may lead to problems in the future!(ABSTRACT TRUNCATED AT 400 WORDS)

Tsitologiia, 1995, 37(1-2), 118 - 25
{The kinetics of rhodamine 123 efflux from cells with multiple drug resistance under the action of energy metabolism inhibitors}; Gamalei IA et al.; A study was made of the effects of inhibitors of ATP synthesis on the process of rhodamine 123 (R-123) release from sensitive sp2/0-Ag14 cells, multidrug-resistant mouse myeloma spEBR-5 cells and hybridoma IF7, derived from spEBR-5 cells . It has been shown that IF7 cells are cross-resistant to ethidium bromide, colchicine, actinomycin D and adriamycin . However, hybridoma IF7 cells, compared to parental spEBR-5 cells, show a lower resistance index . When studying the dependence of the R-123 efflux rate on glycolysis intensity (effect of 2 mM 2-deoxyglucose) and on the level of oxidative phosphorylation activity (effect of 2 mM KCN and 30 microM dinitrophenol), the following distinctive properties of the R-123 transport system of IF7 cells (compared to spEBR-5 cells) were detected: 1) uptake of R-123 into IF7 cells is similar to that observed for the sensitive sp2/0-Ag14 cells; 2) efflux of R-123 from IF7 cells takes place more intensely; 3) R-123 transport is dependent on the rate of glycolysis and may be inhibited by KCN . It is found that 2,4-dinitrophenol inhibits the R-123 efflux from all the cells . Verapamil reverses the multidrug resistance both in spEBR-5 and IF7 cells . The mechanisms of multidrug resistance of cells are discussed.

Rev Med Interne, 1995, 16(7), 547 - 52
{Multidrug-resistant tuberculosis . Epidemiology, treatment, prevention and diagnostic research}; Perronne C et al.; The recent augmentation of the prevalence of multidrug resistant (MDR) tuberculosis is related to the high incidence of tuberculosis in HIV infected people, especially in those with low social status and no medical care; several nosocomial epidemics of MDR tuberculosis were observed in American and European institutions where HIV-infected persons were hospitalized; these MDR tuberculosis were associated with a high mortality-rate and frequent nosocomial transmission to immunocompromised contacts and care workers . The rapid institution of an adequate treatment with ancient antituberculosis agents (cycloserin, capreomycin, aminoglycosides) and/or new drugs (rifabutine, ofloxacin, sparfloxacin, etc) is necessary to avoid mortality and to diminish transmission . Prevention of MDR tuberculosis transmission is very important: patient isolation, adequate and prolonged therapy, better detection of resistance with gene-amplification methods (PCR) which are under investigation.

J Cancer Res Clin Oncol, 1995, 121(9-10), 582 - 6
Reconstitution of purified P-glycoprotein into liposomes; Naito M et al.; To study the molecular function of the multidrug-resistance gene product P-glycoprotein, we purified and reconstituted it into liposomes . Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein . We found that transport activity was effective reconstituted after solubilization with cholate, glycocholate and taurocholate . Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity . The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis . The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine . The reconstituted P-glycoprotein specifically bound {3H}azidopine and had an ATPase activity that was slightly stimulated when vincristine was added . Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.

Hereditas, 1995, 122(2), 125 - 34
Colcemid resistance in murine SEWA cells: non-Pgy gene amplification at low levels of resistance and preferential Pgy2 gene amplification at high levels of resistance; Wettergren Y et al.; Mammalian cell lines often become multidrug-resistant to cytotoxic drugs by amplification and/or overexpression of the P-glycoprotein (Pgy) genes . However, several malignant cell lines seem to acquire low levels of drug resistance by non-P-glycoprotein mediated mechanisms . We report here on cytogenetical signs of non-Pgy gene amplification in murine SEWA cells during the early steps of selection in Colcemid (COL) . In line TC13COL0.01, rare cells exhibited a homogeneously staining region (HSR) distally in chromosome 16 . As the COL-concentration was raised the HSR-chromosome was retained and, in addition, the cells developed numerous double minutes (DMs) . The DMs, but not the HSR, contained amplified Pgy genes . The HSR may correspond to amplified heat shock protein 70 (Hsp70) genes, detected by Southern analysis . A second low-level COL-resistant line, TC13D70.01, contained DMs but showed no amplification of Pgy, Hsp70, Hsp90, alpha- or beta-tubulin genes . In higher COL-concentration, P-glycoprotein mediated drug resistance was induced . In contrast to actinomycin D-resistant SEWA cells, in which higher amplification levels of Pgy1 than of Pgy2 are regularly present, the COL-resistant lines showed a preference for Pgy2 gene amplification . These results are in line with the suggestion that the murine Pgy1 and Pgy2 genes have overlapping but distinct drug specificities.

Cancer Chemother Pharmacol, 1995, 36(6), 499 - 505
The activity of deoxyspergualin in multidrug-resistant cells; Holmes JA et al.; Deoxyspergualin, a synthetic analogue of the immunosuppressive anti-tumour antibiotic spergualin, has been shown to possess potent in vitro and in vivo anti-tumour activity and is currently in the National Cancer Institute (NCI) decision network . Deoxyspergualin shows similarities in properties and mechanisms of action to the natural-product immunosuppressive agents cyclosporin A and FK506, each of which can act as a modifier of multidrug resistance . We therefore decided to examine the comparative activity of deoxyspergualin in parent and multidrug-resistant cells . Deoxyspergualin contains the polyamine spermidine within its structure . Bovine serum copper amine oxidase catalyses the oxidative deamination of spermidine to produce an aminoaldehyde, ammonia and hydrogen peroxide . These aminoaldehydes are believed to be responsible for the toxicity of polyamines in vitro in the presence of bovine serum . For this reason, all experiments were carried out in medium containing bovine serum and in medium containing horse serum (which is low in copper amine oxidase content) . We used the tetrazolium (MTT) colorimetric assay to determine drug sensitivity and tritiated daunorubicin accumulation together with inhibition of azidopine binding to study specific mechanisms of resistance modulation . The murine cell lines EMT6/P and EMT6/AR1.0 and the human cell lines H69/P and H69/LX4 were, respectively, 32-, 32-, 372- and 483-fold more sensitive to spermidine and 175-, 133-, 321- and 444-fold more sensitive to spermine in the presence of calf serum than in the presence of horse serum . However, these large differential effects were not seen for deoxyspergualin . It appears that in the presence of horse serum, deoxyspergualin may exert its effect by a mechanism other than polyamine oxidation . Deoxyspergualin did not enhance the accumulation of {3H}-daunorubicin in EMT6/AR1.0 cells . Furthermore, deoxyspergualin (1-20 microM) did not restore the sensitivity of EMT6/AR1.0 or H69/LX4 cells to that of the parent lines . P-glycoprotein (Pgp) in membranes prepared from H69/LX4 cells was photo-affinity-labeled with {3H}-azidopine . Deoxyspergualin did not inhibit this labeling . Although deoxyspergualin appears to exert its immunosuppressive effect via a mechanism similar to that of cyclosporin A and FK506, it does not share their ability to modify Pgp-mediated multidrug resistance . However, its lack of cross-resistance and potent in vivo anti-tumour activity make deoxyspergualin a promising candidate for development as an anti-cancer agent.

Cancer Invest, 1995, 13(5), 475 - 9
The expression of P-glycoprotein in canine lymphoma and its association with multidrug resistance; Moore AS et al.; Canine lymphoma is a spontaneous, naturally occurring disease that is a model for non-Hodgkin's lymphoma in humans . Chemotherapy with antineoplastics results in a high rate of remission; however, relapse and clinical drug resistance are usually seen within 8-10 months . The P-glycoprotein product of the mdr gene is thought to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance . To assess the role of mdr gene products in drug resistance in canine lymphoma, membrane preparations of lymphoma cells from 31 dogs with high- or intermediate-grade lymphoma were subjected to Western blotting for detection of P-glycoprotein . In this study, one of 30 samples taken from dogs prior to receiving chemotherapy expressed detectable levels of P-glycoprotein . P-glycoprotein was also detected in biopsy samples from 3 of 8 dogs that had become resistant to chemotherapy . This pattern of expression is similar to that in human non-Hodgkin's lymphoma . These studies suggest that canine lymphoma is a useful model for studying multidrug resistance.

World J Urol, 1995, 13(3), 178 - 85
Gene therapy on renal-cell carcinoma: magic bullet or tragic insanity?
Mickisch GH.
Correction of the aberrant genetic code as a means of rational therapy has been a challenge since the first discoveries of an abnormal genetic link to expression of certain disorders . Our growing understanding of the molecular basis of cancer has also led us into a new era in cancer therapy . The possibility of gene therapy represents one of the biggest potential returns on the investment in molecular biology research over the past several years . As a massive gene therapy attack mounts against many forms of malignancy employing various techniques, strategies, and concepts, there appears to be reason to be optimistic, with expectations thus far decidedly outweighing results . Scientists and clinicians have joined together to target directly the molecular basis of tumorigenesis through the restoration of tumor-suppressor gene function or inhibition of oncogene expression . In addition, scientists mapping the human genome have supplied us with a number of genes that can be used to destroy cancer cells selectively {e.g., the herpes simplex-thymidine kinase (HS-tk) gene}, induce a potent antitumor immune response (e.g., interleukin 2), and afford protection to normal tissues from the toxic effects of standard chemotherapy {e.g., multidrug resistance gene type 1 (mdr 1)} . These new anticancer tools provide new opportunities for more specific tumor cell destruction in vivo without the common regional and systemic side effects related to conventional forms of chemotherapy, immunotherapy, radiation, and surgery . Hence, over the next 5-10 years, gene therapy is likely to become a realistic treatment option for certain cancers.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephron, 1995, 69(3), 277 - 80
MDR1 gene expression in lymphocytes of patients with renal transplants; Gotzl M et al.; The MDR1 gene, a multidrug resistance gene, codes for P-glycoprotein which pumps hydrophobic drugs out of the cells . Since cyclosporins also bind to P-glycoprotein and might be pumped by this transmembrane protein, we determined the expression of the MDR1 gene in the lymphocytes of 32 patients with renal transplants . MDR1 RNA expression of lymphocytes was measured by slot blot analysis and compared to the expression of drug-sensitive KB-3-1 cells and multidrug-resistant KB-8-5 cells . MDR1 RNA expression was detected in the lymphocytes of 9 (28%) patients, whereas no expression was seen in the remaining 23 patients . No association between MDR1 RNA expression and transplant function or hematological parameters was observed . However, none of the 6 patients who had transplants for more than 40 months expressed the MDR1 gene in their lymphocytes . In conclusion, expression of the MDR1 gene does occur in lymphocytes of patients with renal transplants and might reduce the immunosuppressive efficacy of cyclosporins through enhanced efflux of cyclosporins.

Blood, 1995 Jan 1, 85(1), 186 - 93
Increased expression of the multidrug resistance-associated protein gene in relapsed acute leukemia; Schneider E et al.; Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine relative levels of transcripts for MDR1 and the recently described multidrug resistance-associated protein (MRP) in normal lymphohematopoietic cells and in 62 bone marrow aspirates of newly diagnosed and recurrent acute leukemia . Levels of MRP expression in newly diagnosed AML samples were similar to those observed in normal bone marrow cells (CD34-negative and CD34-positive) and in unselected HL60 human promyelocytic leukemia cells, which were used as an internal control throughout this study . In contrast, samples of AML obtained at the time of relapse contained approximately twofold higher levels of MRP RNA (P < .01) . Analysis of paired samples, the first obtained at diagnosis and the second at relapse, from 13 acute myelogenous leukemia (AML) and four acute lymphocytic leukemia (ALL) patients showed that MRP expression was increased at the time of relapse in greater than 80% of patients . In contrast, no consistent changes of MDR1 expression at relapse were observed . These results raise the possibility that increased MRP expression might contribute to leukemic relapse.

Invest New Drugs, 1995, 13(1), 37 - 41
Binding of a new multidrug resistance modulator, S9788, to human plasma proteins and erythrocytes; Urien S et al.; The interactions of S9788 with human plasma proteins have been investigated in vitro by an erythrocyte partitioning technique that allows an estimation of the plasma proteins and erythrocytes binding parameters . S9788 was 98% bound to plasma and blood . Lipoproteins bound S9788 with high affinities (binding constants of 0.645, 12.8 and 87.0 x 10(6) M-1 for HDL, LDL and VLDL, respectively) and accounted for more than 55% of the total circulating S9788 . Albumin and alpha 1-acid glycoprotein also bound S9788 with lower binding constants of 0.022 and 0.245 x 10(6) M-1 . S9788 was mainly distributed in the plasma blood compartment (75-80%) with blood-to-plasma concentrations ratio of 0.6 to 0.7 . These results indicate that, in vivo, the fraction of blood S9788 available for tissue diffusion, i.e., the free drug fraction in blood, should depend on lipoprotein concentration in plasma.

Invest New Drugs, 1995, 13(1), 13 - 21
P-glycoprotein mediated resistance to 5'-nor-anhydro-vinblastine (Navelbine); Adams DJ et al.; Navelbine (NVB, vinorelbine tartrate) is a semisynthetic Vinca alkaloid in which the catharanthine moiety contains an eight-membered ring in place of the nine-membered ring that is present in all naturally occurring members of the vinblastine group . This modification selectively reduces interaction with anoxal vs mititotic microtubules and may account for the lower neurotoxicity with improved antitumor activity that has been observed in clinical trials with breast, lung and ovarian cancer . We were interested in whether the structural modification in NVB would also alter the drug resistance profile . Specifically, our aim was to determine whether NVB, like vinblastine (VBL), participates in P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) . NVB-resistant, murine P388 cells (P388/NVB), were derived in vivo and used in conjunction with a battery of drug-resistant P388 cell lines in vivo and murine and human tumor cell lines in vitro to develop a resistance profile for NVB . P388/NVB bells were cross-resistant to drugs involved in MDR (doxorubicin, etoposide, amsacrine, vinblastine, vincristine and actinomycin D), but not to the alkylating agents, cyclophosphamide, carmustine, and cisplatin, or to the antimetabolites, 5-fluorouracil and methotrexate . P388/NVB cellular resistance to NVB was stable without drug pressure during continuous passage in vivo for more than ten weeks and in vitro for at least five weeks . These cells exhibited increased expression of P-gp, and a 30-fold level of resistance of NVB in vitro, which was completely reversable with verapamil . The MDR phenotype was confirmed in other tumor models . P388 tumors resistant to vinblastine, vincristine, doxorubicin, and etoposide were cross-resistant to NVB in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Chemother Pharmacol, 1995, 37(1-2), 168 - 72
Collateral sensitivity to radiation and cis-platinum in a multidrug-resistant human leukemia cell line; Cho J et al.; Although collateral sensitivity to gamma radiation has previously been described in multidrug-resistant tumor cell lines, we describe here a multidrug-resistant human T-cell acute lymphatic leukemia cell line, L100, which displayed increased sensitivity to both gamma radiation and cis-platinum . Cis-platinum cytotoxicity of parental L0 cells and L100 cells was enhanced, whereas radiation sensitivity of L0 and L100 cells was unaltered by glutathione depletion . These results indicate that disparate mechanisms are operative in the collateral sensitivity of L100 cells to gamma radiation and cis-platinum.

Crit Rev Clin Lab Sci, 1995, 32(3), 221 - 64
P-glycoprotein: clinical significance and methods of analysis; van der Heyden S et al.; Multidrug resistance (MDR) is responsible for a decrease in sensitivity of tumor cells tumor cells to unrelated, naturally occurring anticancer drugs . This resistance is correlated with expression and activity of a membrane protein, P-gp 170, functioning as a drug-extruding pump . It has been well described in in vitro situations; however, the clinical detection and implications are not yet clear . Multiple detection assays have been developed based on the discovery of the MDR gene family and the corresponding protein . Southern, Northern, or Western blot analysis, S1 nuclease protection or PCR-based assays, immunohistochemical detection or functionality tests by flow cytometry have been used extensively . However, by use of these techniques on clinical material, both normal and malignant, contradictory results have emerged . The sensitivity and specificity of a certain technique are always limited by unavoidable parameters, for example, skill of the technician . Moreover, the complexity of the development of resistance against anticancer agents (external determinants), such as the diversity of tumor tissues, the simultaneous presence of other resistance mechanisms, and the low expression level, make MDR detection equivocal and can lead to contradictory results . Previous treatment influencing the MDR profile and inappropriate timing of the test make a possible correlation between MDR expression and chemotherapeutic resistance difficult to establish and can lead to discordant results . In this review, the need for proper criteria is stressed . No single detection technique provides the ideal test to detect MDR . Tandem testing could give more certainty, although small sample size limit this application . Formulation of a standard assay with better definition of a positivity is essential before clinical trials are started.

Acta Haematol, 1995, 94(2), 78 - 83
Multidrug resistance in B-cell chronic lymphocytic leukemia; Grulois I et al.; Multidrug resistance (MDR) was investigated in peripheral blood cells isolated from 40 patients with B cell chronic lymphocytic leukemia (B-CLL) and from 7 healthy volunteers, using a flow cytometric assay that detects cellular efflux of the fluorescent dye rhodamine 123 (Rh 123), which has been demonstrated to be transported from the cell by the P-glycoprotein pump . The proportion of B leukemic cells effluxing Rh 123 and thus displaying MDR was low (14 +/- 17%) in B-CLL and in only 4 cases did the contingent of B leukemic cells showing MDR represent more than 30% of the total leukemic cells . In contrast, a higher proportion of cells effluxing Rh 123 (44 +/- 13%) was demonstrated in normal B lymphocytes . No statistical correlation was found between the number of leukemic B cells displaying MDR and clinical parameters or previous treatment . These results clearly suggest that MDR activity is usually low in B-CLL.

Rheumatol Int, 1995, 15(2), 83 - 6
Expression of a multidrug resistance gene in human rheumatoid synovium; Jorgensen C et al.; The objective of this study was to assess the expression of a multidrug resistance (MDR) phenotype, implicated in the cellular resistance of tumor to chemotherapy, in rheumatoid synovial membrane . Synovial membrane from 16 rheumatoid (RA) patients was studied . Six patients with osteoarthritis constituted the control group . The cell membrane expression of the glycoprotein Pgp 170, encoded by the MDR 1 gene, was determined by an immunoperoxidase technique using two different monoclonal antibodies (JSB 1, C 219) . The polymerase chain reaction (PCR) methods were used in parallel to detect the presence of the MDR 1 gene mRNA in the synovial cells . Pgp 170 was expressed on the cell membrane of five RA patients and MDR 1 cellular transcription was detected in one other RA patient . We did not observe any association between synovial glycoprotein expression and age, disease activity, and a specific treatment with a long-acting drug . However, MDR protein expression was associated with the successive treatment with more than three disease-modifying antirheumatic drugs (DMARDs) . We concluded that the synovial membrane expresses a glycoprotein recognized by the antibodies JSB 1 and C 219 . The absence of concomitant MDR 1 transcription suggests the expression of an atypical MDR phenotype in the synovial membrane, distinct from the Pgp 170 encoded by the MDR 1 gene . The implications of the MDR phenotype and the resistance of RA to DMARDs is further discussed.

Proc Natl Acad Sci U S A, 1994 Dec 20, 91(26), 13033 - 7
Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport; Muller M et al.; The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells . MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell . Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells . The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics . Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

FEBS Lett, 1994 Dec 19, 356(2-3), 287 - 99
P-glycoprotein-mediated efflux of hydroxyrubicin, a neutral anthracycline derivative, in resistant K562 cells; Borrel MN et al.; Hydroxyrubin (OH-Dox), a neutral doxorubicin derivative that is only slightly cross-resistant to doxorubicin (Dox), can be actively pumped out of resistant K562 cells by P-glycoprotein (P-gp) . This efflux is saturable and can be inhibited by verapamil . The Michaelis constant is equal to 2 +/- 0.5 microM . However, the efficiency of P-gp in pumping out the drugs is 2.5 times less for OH-Dox than for Dox . This shows that in order to be pumped out by P-gp a molecule does not necessarily have to have a basic center . The mean influx coefficient for the drug is 5 times higher for OH-Dox than for Dox . In conclusion, the degree of resistance of analogs is related not only to their ability to be recognized and transported by P-gp but also, and probably essentially, to their kinetics of uptake . Both parameters have to be taken into account in the rational design of new compounds capable of overcoming multidrug resistance.

Biochem Pharmacol, 1994 Dec 16, 48(12), 2215 - 22
Benzimidazoles, potent anti-mitotic drugs: substrates for the P-glycoprotein transporter in multidrug-resistant cells; Nare B et al.; P-glycoprotein is though to mediate the energy-dependent efflux of many structurally and functionally unrelated lipophilic compounds . Presently, the molecular mechanism underlying the binding and efflux of drugs by P-glycoprotein is not well understood . However, it has been suggested that two planar benzene ring structures and a cationic charge are commonly found in many drugs that interact with P-glycoprotein . The benzimidazoles (BZs) are potent anti-tumour, anti-fungal and anti-parasitic agents, whose mode of action is thought to result from their inhibition of microtubule functions . Although other classes of microtubule inhibitors, such as colchicine and vinblastine, have been studied extensively with respect to their interaction and efflux by P-glycoprotein, the BZ group of drugs has not been characterized . In this study, we have characterized the interaction of BZ with multidrug-resistant cells and found that resistant cells accumulated substantially less BZ compared with drug-sensitive cells . Furthermore, BZ was more toxic to sensitive than to drug-resistant cells, suggesting that BZ is likely to be a substrate for the P-glycoprotein drug efflux pump . In addition, we used a photoactive analogue of BZ ({125I}ASA-BZ) to demonstrate a direct binding between BZ and P-glycoprotein . Results showing that a molar excess of vinblastine, unmodified BZ, verapamil and rhodamine 123, but not colchicine, inhibited the photoaffinity labelling of P-glycoprotein by {125I}ASA-BZ confirmed the binding specificity of BZ to P-glycoprotein . Protease digestion of {125I}ASA-BZ photoaffinity labelled P-glycoprotein yielded two peptides that were similar to those obtained with other P-glycoprotein-associated drugs, e.g . azidopine and iodoaryl azidoprazosin . Taken together, these results demonstrate a direct and specific interaction between P-glycoprotein and BZ in a manner that is probably similar to other previously characterized P-glycoprotein-associated drugs.

Int J Cancer, 1994 Dec 15, 59(6), 789 - 95
P-glycoprotein expression in the Golgi apparatus of multidrug-resistant cells; Molinari A et al.; The surface and intracellular expression of mdrI-P-glycoprotein in parental drug-sensitive human breast cancer cells (MCF-7) and their multidrug-resistant (MDR) variants has been studied by using the monoclonal antibodies (MAbs) MM4.17 and MRK-16, which recognize 2 different epitopes of the drug efflux pump molecule . Fluorescence microscopic observations showed that P-glycoprotein, in addition to being located at the cell surface, can also be found in the Golgi apparatus of resistant cells . To confirm this finding, Golgi apparatus and P-glycoprotein were double-labelled with wheat-germ agglutinin (WGA) and MAb MM4.17 . Laser scanning confocal microscopy indicated that, in MDR cells, Adriamycin mainly accumulated cytoplasmically in a perinuclear region . This accumulation proved to be modulated by pre-treatment with verapamil or ATP depletion . Moreover, the vital staining of Adriamycin-treated MDR cells, performed with the fluorescent lipid N-{7-(4-nitrobenzo-2-oxa-1,3-diazole)}- 6-aminocaproyl sphingosine (C6-NBD-ceramide), revealed that the anthracyclinic antibiotic was located in the Golgi apparatus . All these results indicate that the drug transporter is located in the Golgi apparatus, in which Adriamycin molecules also accumulate.

Eur J Pharmacol, 1994 Dec 15, 288(1), 105 - 14
Dexniguldipine-HCl is a potent allosteric inhibitor of {3H}vinblastine binding to P-glycoprotein of CCRF ADR 5000 cells; Malkhandi J et al.; Cell membranes were prepared from the multidrug resistant, P-glycoprotein expressing human lymphoblastoid cell line CCRF-ADR 5000 . The P-glycoprotein of these membranes possessed high affinity binding sites for {3H}vinblastine, with a Kd of 8 +/- 2 nM and Bmax of 17 +/- 8 pmol/mg of protein . The binding of {3H}vinblastine to P-glycoprotein was not ATP-dependent, and was inhibited by cytotoxic drugs with the following potency order; vincristine > doxorubicin > etoposide . The 1,4-dihydropyridine and multidrug resistance reversing agent, dexniguldipine-HCl, inhibited binding with a Ki value of 37 nM . The multidrug resistance reversing agent cyclosporin A, and the cytotoxics doxorubicin and etoposide did not alter the kinetics of {3H}vinblastine dissociation from P-glycoprotein; however, the 1,4-dihydropyridines dexniguldipine-HCl and nicardipine accelerated dissociation of {3H}vinblastine . These data suggest that P-glycoprotein possesses at least two allosterically coupled drug acceptor sites; receptor site 1 which binds vinblastine, doxorubucin, etoposide and cyclosporin A, and receptor site 2 which binds dexniguldipine-HCl and other 1,4-dihydropyridines.

J Infect Dis, 1994 Dec, 170(6), 1626 - 30
Qualitative and semiquantitative polymerase chain reaction to predict Plasmodium falciparum treatment failure; Kain KC et al.; Multidrug-resistant falciparum malaria is increasing in most malaria-endemic areas . Rapid methods for predicting treatment failure would aid management and control of drug-resistant infections . In this study, Plasmodium falciparum DNA clearance was examined by qualitative and semiquantitative polymerase chain reaction (PCR) . Thai patients with acute falciparum malaria were prospectively followed by light microscopy and by PCR of P . falciparum DNA eluted from filter paper blood samples . A 206-bp P . falciparum sequence was amplified and detected radiometrically and by high-performance liquid chromatography . Clearance of P . falciparum DNA was significantly delayed in treatment failures compared with that in successfully treated patients (P = .02) . Semiquantitative PCR levels did not drop to < 50% of pretreatment levels until day 3 or later in treatment failures compared with day 1 or earlier for successfully treated parasitemia-matched controls (P = .005) . These results suggest that qualitative and semiquantitative PCR may be useful as a method for monitoring response to therapy.

Gan To Kagaku Ryoho, 1994 Dec, 21(16), 2763 - 70
{Clinical significance of P-glycoprotein expression and multidrug resistance assessed by in vitro thymidine incorporation assay in patients with gastric carcinoma}; Fujii H et al.; Drug resistance in chemotherapy is a significant problem in the treatment of gastric carcinomas as well as other malignant tumors . Multidrug resistant cells frequently overexpress the 170 kDa P-glycoprotein (P-gp) . Twenty-four fresh tumor specimens of gastric carcinoma were assessed by flow cytometric detection of P-gp using monoclonal antibody C219 . Eight patients were P-gp positive . Differentiated gastric carcinomas contained significantly higher concentrations of P-gp positive . Incidence of P-gp positive case was high in advanced stage . Sixteen cases received in vitro chemosensitivity test assessed by thymidine incorporation assay (TIA) . Seven of 9 multidrug resistant cases by TIA were P-gp positive, and all of 7 non-multidrug resistance were negative . Expression of P-gp and multidrug resistance were closely correlated (p < 0.01) . Also, in 89 patients with operable gastric carcinoma, the relationship between multidrug resistance by TIA and their clinicopathologic features as well as their survival lengths were examined . Thirty-one of 89 specimens from gastric carcinoma patients were multidrug resistant by TIA . Patients with multidrug resistant group had a significantly poorer cumulative survival rate than non-multidrug resistant cases (p < 0.01) . The multivariated analyses showed that multidrug resistance analyzed is useful indicator for prognosis (p < 0.1) . We suggest that multidrug resistance cases or P-gp-positive cases of gastric carcinoma are highly malignant, and these determinations are clinically useful.

Semin Oncol, 1994 Dec, 21(6 Suppl 15), 24 - 7
The use of photodynamic therapy in bone marrow purging; Mulroney CM et al.; High-dose chemotherapy and autologous bone marrow transplantation are an effective combination for treating a number of malignant disorders . Clinical trials have demonstrated a potential role for this regimen in the management of acute leukemia and non-Hodgkin's lymphoma . Autologous bone marrow transplantation continues to be limited by high relapse rates, as compared with allogeneic bone marrow transplantation . Two factors are thought to account for this observation . First, autologous transplants lack the immunologic "graft-versus-host" advantage of allogeneic transplants . Second, autologous grafts have the possibility of tumor cell contamination . Methods to reduce tumor cell contamination in autografts include exposure to chemical agents or monoclonal antibodies; long-term marrow cultures; and immunologic manipulation, either with immunomagnetic devices or antibody/complement combinations . Photodynamic therapy (PDT) with porfimer sodium (Photofrin; manufactured by Lederle Parenterals, Carolina, Puerto Rico, under license from Quadra Logic Technologies, Inc, Vancouver, British Columbia, Canada) or benzoporphyrin derivative (BPD verteporfin; BPD-MA; BPD-Quadra Logic Technologies, Inc, Vancouver, British Columbia, Canada) may be an effective means of purging bone marrow . The ability of malignant cells to selectively accumulate photosensitizing agents may account for efficacy of PDT in bone marrow purging . The efficacy of porfimer sodium and BPD has been evaluated in cell lines known to express multidrug resistance (MDR), and the results compared with corresponding MDR-negative cell lines . Multidrug resistance-positive cell lines appear relatively resistant to BPD; porfimer sodium remains active . The reason for the differential effect of MDR positivity on the cytotoxicity of porfimer sodium and BPD is unclear, but is believed to be related to the larger size of the porfimer sodium molecule . Clinical trials evaluating PDT in bone marrow transplantation are under way.

Br J Cancer, 1994 Dec, 70(6), 1118 - 25
Characterisation of a navelbine-resistant bladder carcinoma cell line cross-resistant to taxoids; Debal V et al.; A bladder carcinoma cell line (J82) was selected for resistance to the new vinca alkaloid navelbine . The resistance factor of the resistant subline (J82-NVB) to navelbine was 17 . P-glycoprotein was not detected in the membrane of J82-NVB cells . The lack of cross-resistance to multidrug-resistant (MDR) drugs such as doxorubicin, epipodophyllotoxins and colchicine, the absence of increase in navelbine efflux and the fact that a reduced accumulation of the drug cannot account for the resistance level confirmed that the phenotype of resistance of J82-NVB cells is not a classical MDR phenotype . Moreover, verapamil did not reverse the resistance of J82-NVB cells . The cells were cross-resistant to vinca alkaloids and taxoids which share the same target protein: tubulin . Analysis of microtubules using immunofluorescence showed that disassembly of the microtubular network occurred for the same concentration of navelbine in sensitive and resistant cells . However, after treatment with a concentration of navelbine inducing depolymerisation in both sensitive and resistant cells, reassembly of the microtubular network was observed only in resistant cells . This study suggests that the mechanism of resistance of J82-NVB cells involves recovery from the inhibition of microtubule dynamics induced by drug treatment.

J Cell Physiol, 1994 Dec, 161(3), 393 - 406
Effects of P-glycoprotein expression on cyclic AMP and volume-activated ion fluxes and conductances in HT-29 colon adenocarcinoma cells; Kunzelmann K et al.; The tissue distribution of P-glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelial; where one protein is expressed the other is not . To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp+) . Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp-) cells (as shown using the whole cell patch clamp technique) . Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp-Rev) . Concomitantly, CFTR levels and cAMP-stimulated Cl- conductances increased . The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions . We also studied the possible contribution to Pgp to hypotonically activated (HCS) ion conductances . K+ and Cl- effluxes from Pgp- cells were markedly increased by HCS . This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl- channel blocker DIDS (4,4'-disothiocyano-2,2'-disulfonic stilbene) and required extracellular Ca2+ . In Pgp+ cells, the HCS-induced fluxes were not significantly different from those of Pgp- cells . Verapamil (10 microM), which caused 80% reversal of Pgp-associated drug extrusion, failed to inhibit the HCS-evoked Cl- efflux of Pgp+ cells . Similarly, HCS increased Cl- conductance to the same extent in Pgp-, Pgp+ and Pgp-Rev cells . Verapamil (100 microM), but not 1,9-dideoxyforskolin (50 and 100 microM), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines . Since the inhibition by verapamil was not detected in the presence of the K+ channel blocker Ba2+ (3 mM), it is suggested that verapamil affects K+ and not Cl- conductance . We conclude that hypotonically activated Cl- and K+ conductances are similar in HT-29 cells irrespective of Pgp expression . Expression of high levels of Pgp in HT-29 cells confers no physiologically significant capacity for cell volume regulation.

Int J Cancer, 1994 Dec 1, 59(5), 717 - 23
Intrinsic MDR-1 gene and P-glycoprotein expression in human melanoma cell lines; Berger W et al.; Metastatic malignant melanoma is considered a chemotherapy-refractory malignancy . A few previous studies have delivered contradictory results regarding the presence and functionality of P-glycoprotein (P-gp), a transmembranous protein associated with the classical multidrug resistance (cMDR), in malignant melanoma . Therefore we have investigated this issue on 33 cell lines established from primary and metastatic lesions of human malignant melanoma, comparing different cMDR detection methods . Immunocytochemically 33% of the cell lines stained positive for P-gp . The data correlated with those of a P-gp-radioimmunometric (antibody-binding) assay . When RT-PCR was used for MDR-1 mRNA determination, 76% of the melanoma cell lines scored positive . Slot-blot analysis was seen to be less sensitive than RT-PCR . Results from the functional P-gp assays, using daunomycin (DM) as MDR-substrate, showed no influence of P-gp expression on drug accumulation and cytotoxicity . However, the cMDR-modifier verapamil (VP) significantly increased both parameters in those melanoma cells with the highest P-gp levels . We conclude that cMDR is apparently not the decisive but probably a complementary protective mechanism against toxic agents in malignant melanoma.

Cancer Res, 1994 Dec 1, 54(23), 6122 - 8
Transforming growth factor beta 1 promotes spontaneous transformation of cultured rat liver epithelial cells; Zhang X et al.; The neoplastic transformation of cultured rat liver epithelial cells by various means has consistently been associated with the development of resistance to the mito-inhibitory effect of transforming growth factor beta (TGF-beta), suggesting that such phenotype plays a mechanistic role during the transformation of these cells . We have studied the induction of the "TGF-beta-resistant" phenotype in a clonal strain of early passage WB-F344 normal cultured rat liver epithelial cells, the proliferation of which was markedly inhibited by TGF-beta . The control WB cells in continuous culture slowly developed TGF-beta resistance . However, when the same cells were exposed to step-wise increases of TGF-beta concentration in their culture medium, the development of TGF-beta resistance was accelerated . Cells which had been grown in medium containing 1 ng/ml TGF-beta developed colony-forming capacity in soft agar containing epidermal growth factor . Cells which were grown in media containing 5 and 10 ng/ml TGF-beta demonstrated a low level of colony-forming efficiency in soft agar medium without added epidermal growth factor and tumorigenicity in isogeneic rats . These TGF-beta-resistant cells also exhibited progressively increasing levels of expression of the c-fos and and myc mRNA, and increased resistance to the cytotoxicity of Adriamycin and melphalan . The latter phenomenon was accompanied by an increase in the mdr-1 mRNA expression, cellular glutathione level, and glutathione S-transferase activity . The results suggest that chronic exposure to high concentration of TGF-beta promotes the spontaneous neoplastic transformation of cultured rat liver epithelial cells, and that this process may represent one of the mechanisms of cellular adaptation for induction of the multidrug-resistant phenotype during the carcinogenesis of epithelial cells.

Am J Respir Cell Mol Biol, 1994 Dec, 11(6), 639 - 43
Isolation of the gene for the beta subunit of RNA polymerase from rifampicin-resistant Mycobacterium tuberculosis and identification of new mutations; Donnabella V et al.; Tuberculosis (TB) is one of the most important infections worldwide, with an estimated incidence of 10 million active cases per year . Rifampicin is a key component of the first-line therapy used in the treatment of tuberculosis . In Escherichia coli and Mycobacterium leprae, rifampicin has been shown to inhibit the beta subunit of RNA polymerase . The gene (rpoB) encoding this enzyme has been described in both species . We report the isolation of the homologous functional rifampicin resistance gene from M . tuberculosis . A library was constructed with 15 to 25 kb BamHI-digested DNA fragments from a rifampicin-resistant M . tuberculosis clinical isolate that was ligated into an E . coli-mycobacterial shuttle plasmid . Southern analysis of BamHI-digested DNA from 200 recombinant plasmids was performed and filters were hybridized to a 411 bp fragment of the beta subunit of RNA polymerase from M . tuberculosis . Only DNA from one plasmid (#86) hybridized, which suggested that the gene is found as a single copy per genome . This plasmid was able to transfer rifampicin resistance to sensitive M . smegmatis and thus codes for a functional genetic unit . Sequence analysis in the expected "hotspot" region in eight rifampicin-resistant M . tuberculosis strains (including one multidrug-resistant strain) revealed two novel mutations as well as others previously described.

Cytometry, 1994 Dec 1, 17(4), 343 - 8
Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes; Brezden CB et al.; Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo-1AM was hindered by the low level of accumulation of indo-1 in these cells . CHO cells are known to express basal levels of the multidrug resistance efflux pump P-glycoprotein (P-gp) . Rhodamine-123, which is a known substrate of P-gp, was used to confirm the presence of P-gp in CHO cells . Verapamil and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulation of indo-1 in these cells and therefore allowed for improved intracellular calcium measurements . P-gp overexpressing colchicine-resistant CHO cells (CHRC5) also displayed enhanced indo-1AM loading with P-gp inhibitors . Nondetectable levels of P-gp activity were found in wild-type CEM-CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo-1AM loading in the presence or absence of P-gp inhibitors . Loading of a second calcium fluorescent probe fluo-3AM was improved in CHO cells by P-gp inhibition, whereas the structurally related pH probe BCECF-AM was minimally affected . Because low levels of P-gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes.

J Microsc, 1994 Dec, 176 ( Pt 3), 204 - 10
Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study; Meschini S et al.; The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy . In human melanoma cells (M14), adriamycin was localized inside the nuclei . When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed . In recovered cells, a weakly fluorescent signal was observed in the perinuclear region . When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms . In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells . In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative . When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells . Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative . Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.

J Pak Med Assoc, 1994 Dec, 44(12), 280 - 2
Management of tuberculosis by practitioners of Peshawar; Akhtar T et al.; In this report the drug prescribing practices of practitioners are described . The results indicate that a high proportion (80%) of practitioners still prescribe long duration chemotherapy . The most frequently prescribed drugs are rifampicin (87%), isoniazid (89%) and streptomycin (73%) . Despite the use of these highly effective drugs the duration of illness after diagnosis in 31% is over three years . The possible reasons for the poor control of the disease are noncompliance with treatment and multidrug resistance of mycobacterium tuberculosis . The prescribing practices of the practitioners indicate that they are not receiving continuing education and training on the case management of TB.

Monaldi Arch Chest Dis, 1994 Dec, 49(5), 432 - 8
Mycobacterial infections in AIDS: an overview of epidemiology, clinical manifestations, therapy and prophylaxis; Moroni M et al.; One of the most frequent complications of AIDS is Mycobacterial infections . The incidence of tuberculosis has dramatically increased in all countries as a result of the HIV epidemic . Lately, it has been found that the natural history of new Mycobacterium tuberculosis infection is accelerated by HIV disease . In a wide number of cases the emergence of Mycobacterium tuberculosis nosocomial outbreaks of drug-sensitive and drug-resistant strains has been reported in HIV infected patients . The inadequate efforts to provide complete therapy to this kind of patient has caused the emergence of multidrug-resistant tuberculosis, that is responsible for the increased mortality rate in AIDS patients . A renewed interest in mycobacterial infections has also been kindled by the occurrence of Mycobacterium avium infections in patients with acquired immunodeficiency syndrome . The role of Mycobacterium avium as a pathogen is actually confusing and controversial for clinicians who care for AIDS patients . Disseminated Mycobacterium avium infections occur in a high population of HIV infected patients with low CD4+ cell count . Recent studies reported that rifabutin significantly reduced the incidence of Mycobacterium avium bacteremia, although, new macrolides such as clarithromycin and azithromycin are also effective in the treatment of the infection . Therefore, because of the emergence of macrolides resistance, the use of combination therapy is highly recommended in the Mycobacterium avium infection management.

Leukemia, 1994 Dec, 8(12), 2163 - 8
Expression of the multidrug resistance-associated protein (MRP) in acute leukaemia; Hart SM et al.; A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to investigate and compare transcription levels of the human multidrug resistance gene (MDR1) and the recently described multidrug resistance-associated protein (MRP) in 105 samples from patients with acute leukaemia at presentation and relapse . MRP gene expression was significantly greater in samples from patients with acute lymphoblastic leukaemia (ALL) compared with samples from normal peripheral mononuclear cells (PBMC) and patients with de novo acute myeloid leukaemia (AML) . MRP gene expression was found to be higher in patients with relapsed de novo AML compared to those at presentation but prior therapy did not affect MRP gene expression in ALL . MDR1 gene expression was significantly lower in ALL patients compared to normal PBMC and AML samples . Samples from patients with secondary AML had higher levels of MDR1 expression than those of de novo AML . No changes of MDR1 expression were observed in AML or ALL at relapse . No correlation was observed between MDR1 and MRP gene expression in this group of patients . Our results suggest that MRP expression may be of prognostic importance in AML but the significance of the increased levels we have detected remain unclear.

Eur J Immunol, 1994 Dec, 24(12), 2974 - 81
Heterogeneity in P-glycoprotein (multidrug resistance) activity among murine peripheral T cells: correlation with surface phenotype and effector function; Bommhardt U et al.; P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells . Functional P-gly activity can be conveniently assessed microfluorometrically using the fluorescent dye rhodamine 123 (Rh123), which is an artificial substrate for the P-gly transporter . Here we assess P-gly activity in subsets of mouse peripheral T lymphocytes using the Rh123 efflux assay . Our data indicate that virtually all CD8+ cells extrude Rh123 efficiently, whereas only a subset of CD4+ cells exhibit P-gly activity . Correlation of P-gly activity in CD4+ cells with the expression of a panel of surface markers revealed that cells bearing an "activated/memory" phenotype (CD45RB-, CD44hi, CD62L-, CD25+, CD69+) were exclusively found in the fraction that can extrude Rh123 . In contrast "naive" phenotype CD4+ cells (CD45RB+, CD44lo, CD62L+, CD25-, CD69-) could be further subdivided into two major subsets based on P-gly activity . In functional studies of sorted cell populations the Rh123-extruding subset of "naive" CD4+ cells proliferated more strongly and secreted higher levels of interleukin (IL)-2 than its Rh123-retaining counterpart when activated by a variety of polyclonal stimuli . Furthermore, this subset produced detectable levels of interferon (IFN)-gamma upon stimulation but no IL-4 or IL-10 . As expected, the Rh123-retaining "naive" subset produced only IL-2 after stimulation, whereas the "memory" subset produced IFN-gamma, IL-4 and IL-10 in addition to low levels of IL-2 . Collectively, our data indicate that P-gly activity is a novel parameter that can be used to distinguish a subset of "preactivated" CD4+ cells that would be considered as naive on the basis of their surface phenotype.

Am J Clin Pathol, 1994 Dec, 102(6), 842 - 9
Optimization of immunohistochemical detection of P-glycoprotein in chronic lymphoid disorders; Attal M et al.; To optimize the immunohistochemical detection of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp) in chronic lymphoid disorders, the authors compared the sensitivity of three different monoclonal antibodies (MoAb) directed against P-gp (C219, JSB-1, and MRK 16) by using the APAAP technique on four tissue preparations obtained from lymphoid tumors: Cryostat sections, ModAMEX processed sections, frozen cytospin preparations, and fresh cytospin preparations . Tumor samples were obtained from patients with previously treated chronic lymphocytic leukemia (6 cases) or non-Hodgkin's malignant lymphoma (4 cases) . Lymph nodes (n = 9), spleen (n = 3), and blood (n = 5) were analyzed . JSB-1 MoAb detected P-gp in 4 of 12 cases (33.3%) on either frozen sections or ModAMEX processed sections, and in 6 of 17 cases (35.3%) on frozen cytospin preparations . The sensitivity of JSB-1 was significantly improved when fresh cytospin preparations were used with an incidence of P-gp positive samples as high as 70.6% (P < .05) . C219 MoAb was unreactive with lymphoid cells whatever the technique used, whereas this antibody stained stromal cells . MRK 16 MoAb was equally reactive to JSB-1 on fresh cytospin preparations, but unreactive when the other preparations were used . The specificity of JSB1 MoAb was confirmed by both Western blot analysis and Rhodamine 123 efflux assay . The authors used JSB-1 MoAb on fresh cytospin smears prepared from 28 CLL patients . Overall incidence of P-gp positive cases was 39.2% . Univariate analysis showed that P-gp expression was correlated with prior therapy, refractoriness to treatment, Rai stratification, and time of tissue storage after diagnosis . The authors recommend the use of JSB-1 on fresh cytospin preparations for the immunocytochemical detection of P-gp in chronic lymphoid disorders.

J Parasitol, 1994 Dec, 80(6), 1026 - 30
Antitrypanosomal activity of phosphonylmethoxyalkylpurines; Kaminsky R et al.; Phosphonylmethoxyalkylpurines and -pyrimidines exhibit potent activity against a broad spectrum of DNA viruses . We evaluated some of these nucleotide analogues for antitrypanosomal activity in vitro and in mice . The most active compounds were (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine (HPMPDAP), which inhibited growth of Trypanosoma brucei brucei by 50% (EC50 value) when incubated in vitro for 24 hr with 0.23-5.69 micrograms drug/ml . Both compounds completely eliminated multidrug-resistant T . b . brucei in culture at 1 microgram/ml after 4-5 days exposure . Mice infected with drug-susceptible T . b . brucei were cured with 2 doses of 10 mg/kg HPMPDAP . Two or 5 doses of 50 mg/kg 9-(2-phosphonylmethoxyethyl) adenine (PMEA) or 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP), respectively, were necessary to eliminate T . b . brucei infections in mice . Mice infected with multidrug-resistant T . b . brucei were not cured with the above dosages . The most active compound against Trypanosoma congolense was PMEDAP with an EC50 value of 3.21-11.63 micrograms/ml . Thus, some of the phosphonylmethoxyalkyl purines showed potential as antitrypanosomal compounds at dosages that are below those toxic for mice.

Bioorg Med Chem, 1994 Dec, 2(12), 1403 - 11
Antiproliferative actions of 7-substituted 1,3-dihydroxyacridones; possible involvement of DNA topoisomerase II and protein kinase C as biochemical targets; Bastow KF et al.; 7-Chloro-1,3-dihydroxyacridone (1) reversibly inhibited growth of KB and vero cell lines with IC50's of 35 and 40 microM, respectively, and a topoisomerase II-mediated multidrug resistant KB sub-clone was found to be about three-fold more susceptible to 1 . In contrast, two cell lines of lymphoid origin were killed following treatments with 60 microM and at higher concentrations of 1 . KB cell growth inhibition correlated with a rapid, reversible suppression of thymidine incorporation . Uridine but not leucine incorporation was also rapidly suppressed . The in vitro activities of DNA topoisomerase II and novel protein kinase C-subtype delta were inhibited at effective concentrations in tissue-culture, but 1 did not stimulate intracellular protein-associated DNA breaks nor interfere initially with topoisomerase II-mediated DNA cleavage in KB cells . In addition to antiproliferative effects against cells, the compound was weakly virustatic for herpes simplex virus type I with an IC50 of 8 microM . Limited studies comparing three 1-congeners and citpressine-I, an acridone alkaloid with reported antiherpes activity, demonstrated that 7-substituted 1,3-dihydroxyacridones are novel antiproliferative agents which share similar biological and biochemical properties.

Singapore Med J, 1994 Dec, 35(6), 599 - 601
Multidrug-resistant typhoid fever in Singapore; Oh HM et al.; A study was conducted to determine the clinical and epidemiological characteristics of multidrug-resistant (MDR) typhoid fever in Singapore . Twenty-one of 121 patients with typhoid fever had MDR typhoid fever after recent travel to the Indian subcontinent . Fifty patients with drug susceptible typhoid fever were also analysed for comparison . Nineteen of the MDR S . typhi isolates had resistance to ampicillin, chloramphenicol and trimethoprim-sulphamethoxazole (TMP-SMX) while the remainder had resistance to ampicillin and TMP-SMX . The predominant presenting symptoms were fever and diarrhoea . Eleven patients with MDR typhoid fever were treated with oral ciprofloxacin and nine with intravenous ceftriaxone . The patients with MDR typhoid fever had a longer duration of fever defervescence (8 +/- 5 days) compared to those with drug-susceptible typhoid fever (5.7 +/- 4.16 days) (p < 0.01) . Eighteen patients were cured and one patient defaulted treatment . Two patients relapsed within two months of treatment . The study showed that 17.4% of patients with typhoid fever had imported MDR S . typhi after recent travel to the Indian subcontinent where MDR typhoid fever is prevalent.

Bull Cancer, 1994 Dec, 81 Suppl 2, 84s - 85s
{Methods for diagnosing MDR: immunohistochemistry versus molecular biology}; Mousseau M; The methods of multidrug resistance (MDR) detection in human malignant tumors are numerous . The process of molecular biology (Slot Blot, Northern Blot, Reverse Transcription Polymerase Chain Reaction, PCR) and the process of in situ hybridization or immunohistochemistry are complementary . The process of molecular biology are quantitative and especially sensitive for PCR, by contrast they fail in specificity . This specificity is obtained by in situ hybridization and immunohistochemistry . However, none of these methods give knowledge of GP170 functional activity . The detection of functional GP170 can be identified by rhodamine or daunorubicin intracellular accumulation with flux cytometry or by scintigraphically imaging GP170 expression in vivo with Tc-Sestamibi . The multiplicity of methods for detecting multidrug resistance show that there is no consensus about the best process to employ commonly . In consequence, the alone MDR biological diagnosis should not be the rationale of choice for second line chemotherapy or for using MDR revertant agents . In other words, the MDR biological diagnosis, keeping in mind that other chemoresistant mechanisms (topoisomerases, glutathione system, etc) could be present simultaneously or successively during tumor spreading or during successive antineoplastic treatments.

Bull Cancer, 1994 Dec, 81 Suppl 2, 62s - 68s
{Pleiotropic resistance associated with topoisomerases}; Riou JF et al.; There are at least two well-characterized mechanism of resistance to Topo I and II inhibitors: modifications of intracellular accumulation and reduced formation of cleavable complexes . Limited drug accumulation is usually due to P-glycoproteinMDR or to Multidrug Resistance associated Protein (MRP) . Reduction of Topo I (or II) cleavable complexes not related to drug transport can either be due to decreased enzyme levels or enzyme mutations . For Topo II inhibitors, differential expression of the Topo II isoforms alpha and beta and changes in Topo II phosphorylation may also contribute to resistance . For dual Topo I and II inhibitors, resistance mechanisms are more complex to analyze but may also involve dual Topo I and II alterations . Finally, in a given cell line, several mechanisms are commonly associated in pleiotropic resistance to Topo inhibitors.

Cancer Metastasis Rev, 1994 Dec, 13(3-4), 411 - 31
Regulation of protein kinase C and role in cancer biology; Blobe GC et al.; Protein kinase C (PKC) is a family of closely related lipid-dependent and diacyglycerol-activated isoenzymes known to play an important role in the signal transduction pathways involved in hormone release, mitogenesis and tumor promotion . Reversible activation of PKC by the second messengers diacylglycerol and calcium is an established model for the short term regulation of PKC in the immediate events of signal transduction . PKC can also be modulated long term by changes in the levels of activators or inhibitors for a prolonged period or by changes in the levels of functional PKC isoenzymes in the cell during development or in response to hormones and/or differentiation factors . Indeed, studies have indicated that the sustained activation or inhibition of PKC activity in vivo may play a critical role in regulation of long term cellular events such as proliferation, differentiation and tumorigenesis . In addition, these regulatory events are important in colon cancer, where a decrease in PKC activators and activity suggests PKC acts as an anti-oncogene, in breast cancer, where an increase in PKC activity suggests an oncogenic role for PKC, and in multidrug resistance (MDR) and metastasis where an increase in PKC activity correlates with increased resistance and metastatic potential . These studies highlight the importance and significance of regulation of PKC activity in vivo.

J Cell Sci, 1994 Dec, 107 ( Pt 12), 3281 - 90
Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling; Sardini A et al.; P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter . A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell . Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels . In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated . The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

J Gen Physiol, 1994 Dec, 104(6), 1129 - 61
Swelling-activated chloride channels in multidrug-sensitive and -resistant cells; Ehring GR et al.; Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug-efflux pump for a broad range of substrates . We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug-sensitive and -resistant cell lines . Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents . Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux . Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123 . During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines . The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively . gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV . Cl- channels slowly closed during strong depolarization beyond +60 mV . Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells . gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM) . None of these blockers inhibited R123 efflux . NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler . In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered . Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution . Moreover, the rate of R123 efflux did not change during cell swelling . We conclude that P-glycoprotein and swelling-activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.

J Nucl Biol Med, 1994 Dec, 38(4 Suppl 1), 18 - 21
Nickel-57-doxorubicin, a potential radiotracer for pharmacokinetic studies using PET: production and radiolabelling; Zweit J et al.; The clinical use of anthracyclines, such as doxorubicin (DXR), is hampered by tumour development of multidrug resistance (MDR) . The drug efflux associated with MDR could be characterised in vivo using Positron Emission Tomography (PET) in conjunction with a suitable radiolabelled drug . We are investigating DXR labelled with the positron emitter 57Ni as a potential analogue of the parent drug . Essential to this work is the production of a high purity radionuclide in a suitable chemical form for the preparation of radiolabelled DXR . To optimise production parameters, excitation functions (reaction cross section as a function of beam energy) for proton induced reactions in cobalt were measured up to 60 MeV . The excitation function for the 59Co(p,3n)57Ni reaction shows a maximum cross section of 13.8 +/- 1.5 mb at 38 MeV . The optimum energy range for production of 57Ni was found to be 41-->26 MeV resulting in an experimental thick target yield of 17.8 MBq/muAh . The level of the 56Ni impurity is only 0.21% at the end of bombardment . A radiochemical procedure, based on cation-exchange chromatography, has been developed for the separation of radionickel from the cobalt target and other radiochemical and chemical impurities . The 57Ni activity was eluted, using 2M HCl, from a Dowex-50Wx8(H+) column, in a 95% radiochemical yield . Optimum labelling of DXR has been investigated in terms of pH, reaction time and temperature, achieving radiochemical yields > 94% . DXR labelled with 57Ni therefore shows promise as a radiotracer for pharmacokinetic studies using PET.

Anticancer Drugs, 1994 Dec, 5(6), 655 - 65
Modulation of human P-glycoprotein epitope expression by temperature and/or resistance-modulating agents; Jachez B et al.; Three monoclonal antibodies (mAb), MRK16, MM4.17 and MC57, directed against distinct epitopes on the external domain of human P-glycoprotein (Pgp), were used to follow its expression on multidrug resistant (MDR)-cells . The linear MM4.17 epitope and conformational MRK16 epitope showed a 4-fold higher expression at 37 degrees C than at 4 degrees C, while the detection of the conformational MC57 epitope did not change . Inhibition of Pgp function, by a short pretreatment of the MDR-cells with resistance-modulating agents (RMA), such as SDZ PSC 833 and SDZ 280-446, could not be related to depletion of Pgp from the cell surface, since their expression of the MM4.17 and MRK16 epitopes was found unchanged . However, a substantially higher expression of MC57 epitopes was found on RMA-treated cells than on untreated ones . Since this effect correlated to the strength of different RMA in reversing the MDR phenotype, MC57 epitopes might be more efficiently expressed on inactivate(d) forms of the Pgp molecules, suggesting that RMA might inhibit Pgp function by disturbing the conformation of individual Pgp molecules, their topographical distribution or polymerization status in the membrane.

FEBS Lett, 1994 Nov 28, 355(2), 187 - 91
The antiprogestatin drug RU 486 potentiates doxorubicin cytotoxicity in multidrug resistant cells through inhibition of P-glycoprotein function; Lecureur V et al.; The antiprogestatin drug RU 486 was examined for its effect on doxorubicin cellular retention and cytotoxicity in multidrug resistant cells overexpressing P-glycoprotein (P-gp) . RU 486 was shown to strongly enhance intracellular accumulation of doxorubicin in both rat hepatoma RHC1 and human leukemia K562 R7 drug-resistant cells but had no action in SDVI drug-sensitive liver cells . The antiprogestatin drug when used at 10 microM, a concentration close to plasma concentrations achievable in humans, was able to hugely increase the sensitivity of RHC1 cells to doxorubicin . RU 486 appeared to prevent the P-gp-mediated doxorubicin efflux out of RHC1 cells and was demonstrated to interfere directly with P-gp drug binding sites since it blocked P-gp labelling by the photoactivable P-gp ligand azidopine . These results thus demonstrate that RU 486 can downmodulate anticancer drug resistance through inhibition of P-gp function.

FEBS Lett, 1994 Nov 28, 355(2), 173 - 7
Tamoxifen stimulates phospholipase D activity by an estrogen receptor-independent mechanism; Kiss Z; The effects of tamoxifen (TAM), a widely used agent in the treatment of breast cancer, were examined on phospholipase D (PLD)-mediated phospholipid hydrolysis . In drug-sensitive MCF-7 human breast carcinoma cells TAM, similar to several well-established activators of PLD, had no effect on phospholipid hydrolysis . In an estrogen receptor-deficient multidrug-resistant subline of MCF-7 cells, TAM preferentially stimulated the hydrolysis of phosphatidylethanolamine; two-fold stimulation required 2.5 or 5 microM TAM in the absence or presence of serum, respectively . In NIH 3T3 fibroblasts significant (4- to 4.8-fold) stimulation of phosphatidylethanolamine and phosphatidylcholine hydrolysis in the presence of serum required 10 microM TAM . These data establish that TAM can stimulate PLD activity by an estrogen receptor-independent mechanism.

J Biol Chem, 1994 Nov 25, 269(47), 29715 - 9
NF-IL6, a member of the C/EBP family of transcription factors, binds and trans-activates the human MDR1 gene promoter; Combates NJ et al.; Revealing the regulatory mechanisms involved in P-glycoprotein expression is important to our understanding of multidrug resistance (MDR) in tumor cells . The MDR1 gene encoding P-glycoprotein contained a promoter sequence (-157 to -125) that was found to be homologous with other mdr gene promoters and that specifically interacted with a nuclear protein . The nuclear protein was identified, using a HeLa lambda gt11 cDNA expression library, to be the transcriptional regulator nuclear factor for interleukin-6 (NF-IL6), a member of the C/EBP family of transcription factors that bound an NF-IL-6-like consensus element 5'-TTTCGCAGT-3' . Furthermore, a glutathione S-transferase fusion protein (10.1-glutathione S-transferase) containing the partial NF-IL6 cDNA was also found to specifically interact with the MDR1 promoter sequence . Co-transfection of an NF-IL6 expression vector with a chloramphenicol acetyltransferase reporter gene driven by 1018 base pairs of MDR1 5'-flanking sequences demonstrated that NF-IL6 trans-activated the MDR1 promoter . This trans-activation was significantly reduced when the NF-IL6 element in the reporter gene construct was deleted or mutated . Identification of NF-IL6 as an important transcriptional regulator and the implications of its potential role in MDR1 gene induction in response to a variety of stimuli are discussed.

Cancer Lett, 1994 Nov 25, 87(1), 17 - 23
Presence of antitumor activities in the milk collected from gossypol-treated dairy cows; Hu YF et al.; Two human breast carcinoma cell lines (MCF-7, MCF-7 Adr) and a rat esophageal cancer cell line (RE-B2T) were used to evaluate the antiproliferative potential of gossypol (GP)-containing milk (GP-Milk), which was collected from Brown Swiss dairy cows treated daily with federally allowable 450 ppm of GP for 6 days . Treatment of the cultured cancer cells with GP-Milk for 24 h significantly inhibited the rates of 3H-thymidine incorporation during the ensuing 3-h period in all three tumorigenic cell lines . The inhibitory effects of GP-Milk occurred in a dose-dependent manner in all cases, but the calculated ED50 varied with cell lines . ED50 for GP-Milk was estimated at 10% for wild-type MCF-7 human breast cancer cells, 15% for multidrug-resistant MCF-7 Adr human breast cancer cells and 50% for RE-B2T rat esophageal carcinoma cells . The potential of GP-Milk as a dietary supplement for the prevention and/or treatment of human breast cancer is discussed in this paper.

Biochemistry, 1994 Nov 22, 33(46), 13761 - 8
Transport studies of doxorubicin in model membranes indicate a difference in passive diffusion across and binding at the outer and inner leaflets of the plasma membrane; Speelmans G et al.; The kinetics of passive transport of the anticancer drug doxorubicin were analyzed in relation to membrane composition in large unilamellar vesicles in which DNA was enclosed . Special attention was paid to lipids that are typical for the inner and outer leaflet of the plasma membrane of mammalian cells: Phosphatidylethanolamine and anionic phosphatidylserine versus phosphatidylcholine, sphingomyelin, and cholesterol, respectively . The presence of anionic phospholipids results in a highly efficient incorporation of the drug into biological and model membranes {de Wolf, F . A., et al . (1993) Biochemistry 32, 6688-6695} . Therefore, the effect of drug binding on the amount of free, transportable drug was explicitly taken into account . However, even after correction for binding the permeability coefficient was about 35% lower in membranes containing 50 mol % of the anionic phosphatidylserine than in membranes consisting only of zwitterionic phospholipids (0.71-0.79 versus 1.18-1.25 microns s-1) . This shows that drug binding and insertion also affect the intrinsic transport characteristics of the membranes . As compared to pure phosphatidylcholine, binding was not influenced by the incorporation of sphingomyelin or cholesterol, but equimolar amounts of sphingomyelin and cholesterol in phosphatidylcholine membranes decreased the rate of doxorubicin transport by 60% and 80%, respectively . The inhibitory effect of these two lipids is probably due to a closer packing of the membranes . In accordance, after the acyl chain order was decreased by adding the anaesthetic-like phenethyl alcohol (0.5% v/v), transport was stimulated more than 4-fold . The implications of our findings for the functioning and rate of drug pumping by the multidrug resistance-conferring P-glycoprotein in cancer cells are discussed.

Biochem Pharmacol, 1994 Nov 16, 48(10), 1989 - 92
In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclosporin A at the blood-brain barrier; Sakata A et al.; To evaluate the significance of P-glycoprotein (P-gp)-mediated active efflux on the blood-brain barrier (BBB) permeability of cyclosporin A (CsA) in vivo, we investigated the effects of ATP depletion in the brain and of a multidrug-resistant (MDR) reversing agent on the transport of CsA across the BBB . Using transient brain ischemia obtained by 4-vessel occlusion of vertebral and common carotid arteries in rats to deplete ATP content in the brain, the estimated permeability surface area product (PS) value of {3H}CsA was increased 2.7-fold compared with that in normal rats, whereas the PS value of {14C}sucrose was not altered . Additionally, when quinidine hydrochloride (QND) was infused into the brain through a microdialysis probe implanted in the rat hippocampus, the extravascular extraction of CsA was increased to approximately 2.5-fold of the control, whereas no difference in the extravascular extraction between control and normal rats having no implanted dialysis probe was observed . Furthermore, the efflux rate from brain to blood of CsA was decreased remarkably to 5% of control at steady-state by co-administration of CsA with QND directly into the brain through the dialysis probe . The ATP-dependent and QND-sensitive efflux of CsA from the brain strongly indicates that P-gp in the brain capillary endothelial cells functions as an efflux pump under the physiological state, and that P-gp-mediated efflux of CsA is a major mechanism of the restricted transfer from blood into the brain.

Biochem Pharmacol, 1994 Nov 16, 48(10), 1871 - 81
Comparison of mechanisms responsible for resistance to idarubicin and daunorubicin in multidrug resistant LoVo cell lines; Toffoli G et al.; Two human colon carcinoma drug resistant clones (LoVo-IDA-1 and LoVo-IDA-2) were selected by continuous pressure of LoVo parent cell lines to idarubicin (IDA) . Both cell sublines exhibited a typical multidrug resistance (MDR) phenotype but, despite IDA selection, the resistance index (RIext) was higher for daunorubicin (DAU) (RIext = 101-112) than for IDA (RIext = 20-23) . A similar pattern of cross-resistance was also observed in two (DOX) doxorubicin-selected LoVo cell lines (LoVo-DOX-1 and LoVo-DOX-2) . All the MDR cell lines exhibited decreased drug accumulation and increased intracellular drug tolerance as evidenced by the greater intracellular amount of drug required to cause a 50% growth inhibition (IC50int) compared to their parent cell line . The differences between DAU and IDA RIext exhibited by MDR cells were a function of intracellular resistance . DAU IC50int was 13.9 and 14.9 times higher in LoVo-IDA-1,2 and 6.4 and 6.2 in LoVo-DOX-1,2 cell lines, respectively, than in LoVo-sensitive cells, whereas IDA IC50int was only 3.6 and 3.2 times higher in LoVo-IDA-1,2 and 2.2 and 2.3 in LoVo-DOX-1,2 cell lines, respectively . Conversely, variations in IDA accumulation between resistant and sensitive cells were similar to those observed for DAU {the ratios between DAU uptake in sensitive and resistant cells were almost identical (P = NS) to those observed for IDA} . Differences between IDA and DAU intracellular distribution accounted for the relatively higher DAU intracellular resistance . In fact nuclear/cytoplasmic (N/C) DAU fluorescence ratio was higher (P < 0.01) in sensitive (N/C = 3.4 +/- 2.7) than in MDR cells (N/C ranging from 0.31 +/- 0.2 to 0.41 +/- 0.1) . In contrast, no significant (P = NS) differences were observed in IDA N/C ratios between sensitive and MDR cells (N/C ranging from 0.16 +/- 0.1 to 0.20 +/- 0.1) . In MDR cells, 1-hr VER (10 microM) treatment partially reverted both DAU N/C ratios and intracellular DAU resistance but neither changes in IDA N/C ratios nor variation in intracellular IDA resistance were observed following VER exposure . In conclusion, the greater intracellular drug tolerance that MDR cells show for DAU compared to IDA makes IDA more effective than DAU in MDR cells overexpressing P-glycoprotein (P-gp).

Biochem J, 1994 Nov 15, 304 ( Pt 1), 271 - 9
DNA-binding characterization of a novel anti-tumour benzo{a}phenazine derivative NC-182: spectroscopic and viscometric studies; Tarui M et al.; NC-182 is a novel anti-tumour compound having a benzo{a}phenazine ring . Fluorescence, absorption and c.d . spectroscopy, as well as viscometric titrations, were systematically performed to investigate the interaction mode of this drug with DNA and its effect on DNA conformation, based on comparative measurements with distamycin (DNA minor-groove binder) and daunomycin (DNA-base intercalator) . NC-182 was found to be a potent intercalator of DNA, especially the B-form DNA, although no specificity was observed against the base-pair . The binding of NC-182 to B-DNA behaves biphasically, depending on the molar ratio (r) of drug to DNA: NC-182 acts to render the B-form structure rigid at relatively low r value and to promote the transformation of B- to non-B forms at high r values . It was also shown that NC-182 promotes the unwinding of Z-form DNA to B-form . Viscometric, u.v . 'melting' and c.d . experiments further showed that (1) the DNA duplex structure is thermally stabilized by intercalation with NC-182 and (2) the intercalation of NC-182 into a poly(dA).2poly(dT) DNA structure thermally stabilizes the triplex structure, resulting in a melting point close to that of the duplex structure; the melting curves of triplex and duplex structures coincide at r > 0.06 . These observations make a significant contribution to our understanding of the biological properties of this novel benzo{a}phenazine derivative, a new anti-tumour tumour agent against multidrug-resistant and sensitive tumours.

Arch Biochem Biophys, 1994 Nov 15, 315(1), 41 - 7
Accumulation of fatty alcohol in MCF-7 breast cancer cells; Welsh CJ et al.; The MCF-7 cell line (human breast epithelial cells) accumulates fatty alcohols . The fatty alcohols were identified as C16:0, C18:0, and C18:1 alcohols by thin-layer chromatography and gas chromatography/mass spectrometry . This accumulation of alcohols in MCF-7 was found in cultures of MCF-7 cells obtained from other laboratories but not in a variety of unrelated cell lines . The presence of the alcohols suggested an aberrant ether lipid metabolism in the MCF-7 cells . Therefore, the capacity for either lipid biosynthesis was evaluated using cells incubated with either {14C}stearyl alcohol or {14C}stearic acid . MCF-7 cells incorporated less than 0.4% of the {14C}alcohol into ether-linked phospholipids, whereas the AB589 breast epithelial cells, used as a "normal control" for comparisons, did not accumulate fatty alcohol and incorporated approximately 20% of the radiolabeled alcohol into phospholipids containing ether linkages . Although the MCF-7 cells were unable to effectively incorporate the fatty alcohol into ether linkages, the cells were able to oxidize the alcohol to fatty acid . When incubated with {14C}stearic acid, the conversion to radiolabeled fatty alcohol in MCF-7 cells was approximately four times higher than the alcohol levels found in AB589 cells . While deficient in the ability to synthesize ether linkages, the MCF-7 cells did incorporate radiolabeled hexadecylglycerol, a precursor containing an ether linkage, into phospholipids . Collectively, the data indicate that the MCF-7 cells possess a deficiency in the alkyl DHAP synthase activity . A near absence of ether-linked lipids in the MCF-7 cells was indicated by the radiolabeling studies, and this finding was corroborated by results from HPLC analysis . Analyses of the partial glycerides, obtained from the enzymatic hydrolysis of cellular phospholipids, found only trace levels of ether lipids in the MCF-7 cells . The aberration in ether lipid biosynthesis did not correlate with the expression of the multidrug resistance phenotype in a series multidrug resistant MCF-7 variants . The results are discussed relative to the use of the MCF-7 cells as a model for investigations of ether lipid biosynthesis and the cellular physiology of ether lipids.

Cancer Res, 1994 Nov 15, 54(22), 5917 - 24
Collateral sensitivity of human melanoma multidrug-resistant variants to the polyamine analogue, N1,N11-diethylnorspermine; Porter CW et al.; Certain N-alkylated analogues of the natural polyamine spermine, such as N1,N11-diethylnorspermine (DENSPM), rapidly deplete intracellular polyamine pools by down-regulating the biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and by potently up-regulating the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase . On the basis of previously reported antitumor activity in human tumor xenograft model systems, DENSPM is currently undergoing Phase I clinical trials against human melanoma and other solid tumors . The antiproliferative activity of this analogue against the multidrug resistance (MDR) phenotype was examined in three MDR sublines of human melanoma RPMI-7932 cells, which were shown to be 2-to 10-fold resistant to classical MDR agents . These MDR lines had been separately derived using different selecting agents (Lemontt et al., Cancer Res., 48: 6344-6353, 1988) . Subline functional resistance due to P-glycoprotein was confirmed by decreased retention of rhodamine 123 relative to parent cells as detected by flow cytometry . Although the three sublines were 2- to 10-fold less sensitive than the parent line to classical MDR-type agents, they were found in dose-response studies to be significantly more sensitive to DENSPM than the parent line . In addition, they showed a distinct cytotoxic response after a 48-h treatment with 10 microM DENSPM, which was not apparent in the parent line . Growth sensitivity of the sublines to the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, or the S-adenosylmethionine decarboxylase inhibitor, CGP-48664, was found to be similar to parent cells . The ratio of the key biosynthetic enzyme activities for ornithine decarboxylase and S-adenosylmethionine decarboxylase was found to be 3.5- to 5-fold higher in all three sublines, due mainly to increases in the former enzyme . This imbalance produced unusually high putrescine pools . Although DENSPM down-regulation of decarboxylase activities and potent up-regulation of spermidine/spermine N1-acetyltransferase activity occurred similarly in both parent and variant lines, polyamine depletion was greater in the variant lines . Collateral sensitivity of the MDR sublines to DENSPM is partially attributable to the finding that analogue (and spermidine) uptake in the sublines was about 2-fold higher (after 2 h) than in the parent cells . The presence of disturbances in polyamine homeostasis and increased sensitivity to DENSPM in three independently selected cell line variants suggests that they may be generally associated with the MDR phenotype in human melanoma and possibly other tumor cells . The collateral sensitivity of human melanoma MDR variants to DENSPM represents a possible therapeutic indication which should be considered during the ongoing clinical evaluation of this drug.

Cancer Res, 1994 Nov 15, 54(22), 5902 - 10
Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells; Cole SP et al.; We have previously identified and characterized a novel member of the ATP-binding cassette superfamily of transport proteins, multidrug resistance protein (MRP), and subsequently demonstrated that its overexpression is sufficient to confer multidrug resistance on previously sensitive cells (Cole et al., Science (Washington DC), 258: 1650-1654, 1992; Grant et al., Cancer Res . 54: 357-361, 1994) . In the present study, we have transfected two different eukaryotic expression vectors containing MRP complementary DNA into HeLa cells to study the pharmacological phenotype produced exclusively by overexpression of human MRP . The drug resistance patterns of the two MRP-transfected cell populations were similar . They were characterized by a moderate (5- to 15-fold) level of resistance to doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide, and a low (< or = 3-fold) level of resistance to taxol, vinblastine, and colchicine . The transfectants were not resistant to 9-alkyl anthracyclines, mitoxantrone, or cisplatin . The MRP-transfected cells were also resistant to some heavy metal anions including arsenite, arsenate, and trivalent and pentavalent antimonials but were not resistant to cadmium chloride . Accumulation of radiolabeled vincristine was reduced by 45% in the MRP-transfected cells and could be restored to the levels found in sensitive cells by depletion of ATP . Rates of vincristine efflux did not differ greatly in the sensitive and resistant cells . The cytotoxic effects of vincristine and doxorubicin could be enhanced in a dose-dependent fashion by coadministration of verapamil . Cyclosporin A also increased vincristine toxicity but had less effect on doxorubicin toxicity . The degree of chemosensitization by verapamil and cyclosporin A was similar in MRP-transfected cells and in cells transfected with the vector alone, suggesting that sensitization involved mechanisms independent of MRP expression . Verapamil and cyclosporin A caused a modest increase in vincristine accumulation in the resistant cells but did not restore levels to those of the sensitive cells . Taken together, these data indicate that drug-resistant cell lines generated by transfection with MRP complementary DNA display some but not all of the characteristics of MRP-overexpressing cell lines produced by drug selection in vitro . They further demonstrate that the multidrug resistance phenotype conferred by MRP is similar but not identical to that conferred by P-glycoprotein and includes resistance to arsenical and antimonial oxyanions.

Cancer Res, 1994 Nov 15, 54(22), 5788 - 92
Detection of the M(r) 190,000 multidrug resistance protein, MRP, with monoclonal antibodies; Hipfner DR et al.; MRP is a M(r) 190,000 integral membrane phosphoglycoprotein that is overexpressed in some drug-selected resistant cell lines and has been shown to cause multidrug resistance in transfected cells . Five murine hybridoma cell lines (QCRL-1, QCRL-2, QCRL-3, QCRL-4, and QCRL-6) have been generated which secrete monoclonal antibodies (MAbs) that react specifically with membrane proteins of MRP-overexpressing, multidrug-resistant, drug-selected H69AR cells and MRP-transfected HeLa cells (T5) but not the respective parental (H69) and vector-transfected (C1) cells . The ability of three of these MAbs (QCRL-1, QCRL-2, and QCRL-3) to selectively immunoprecipitate a M(r) 190,000 protein from 35S-labeled H69AR and T5 membranes indicates that these MAbs are specific for MRP . MAb QCRL-1 is also capable of detecting the low levels of MRP present in revertant H69PR cells by immunoblot analysis . Indirect immunofluorescence analyses show that MAbs QCRL-1, QCRL-2, and QCRL-3) strongly and differentially react with fixed T5 and H69AR cells but not with unfixed cells, suggesting that these MAbs recognize intracellular MRP epitopes . The availability of reagents for the specific and sensitive immunodetection of MRP should greatly facilitate biological and clinical studies of this novel drug resistance protein.

Cancer, 1994 Nov 15, 74(10), 2757 - 64
A phase I trial of intrahepatic verapamil and doxorubicin . Regional therapy to overcome multidrug resistance; Saltz L et al.; BACKGROUND . Verapamil can modulate multidrug resistance in vitro, but only at levels that are not tolerable when administered systemically . Regional strategies of drug administration may permit the delivery of high concentrations of a drug to specific areas with lower systemic levels . Colorectal cancers typically express the multidrug resistance phenotype . METHODS . A Phase I trial was performed to determine the maximum tolerable dose (MTD) and dose limiting toxicities of verapamil by hepatic artery infusion, together with doxorubicin, to patients with hepatic metastases of colorectal cancer . Fourteen patients with metastatic colorectal cancer received a 14-hour intrahepatic infusion of verapamil . Six hours after the start of the infusion, a fixed dose of doxorubicin (50 mg/m2) was given, also via the hepatic artery, over a 30-minute period . Patients were followed by cardiac telemetry but were not in an intensive care setting, and no invasive monitoring was used . All patients had received prior intrahepatic chemotherapy . RESULTS . The MTD of intrahepatic verapamil on this schedule in this patient population was 1.2 mg/kg/hour . Hypotension was the dose limiting toxicity . No major objective responses were noted in this heavily pretreated patient population . A dose of 1.0 mg/kg/hour is recommended for Phase II trials . CONCLUSIONS . Based on estimations of normal hepatic artery blood flow, the estimated concentration of verapamil delivered to the hepatic tumors at 1.0 mg/kg/hour is 3.6 micrograms/ml (7.3 microM), which is comparable to concentrations at which an in vitro reversal of MDR is seen . This study demonstrates that the systemic toxicities of an MDR reversal agent can be overcome by regional drug delivery, establishing this approach as an important model system for further study of MDR modulation.

J Biol Chem, 1994 Nov 11, 269(45), 27807 - 10
The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates; Leier I et al.; The multidrug resistance-associated protein (MRP) is the product of an ATP-binding cassette transporter gene overexpressed in some tumor cells resistant to antineoplastic agents . We studied the transport function of MRP in membrane vesicles prepared from HeLa cells transfected with an MRP expression vector and overexpressing this 190-kDa membrane glycoprotein . ATP-dependent primary-active transport into the vesicles was demonstrated for leukotriene C4 (LTC4), LTD4, LTE4, and S-(2,4-dinitrophenyl)glutathione with relative rates, at a substrate concentration of 50 nM, of 1.0, 0.27, 0.14, and 0.16, respectively . The endogenous glutathione conjugate LTC4 had the highest affinity for this transporter with a Km of 97 nM . The Km for ATP was 19 microM . Direct photoaffinity labeling with {3H}LTC4 labeled a 190-kDa membrane protein predominantly in the MRP-transfected HeLa cells . ATP-dependent LTC4 transport was effectively inhibited by the LTD4 receptor antagonist MK 571, whereas cyclosporin A and, particularly, its analog PSC 833 were much less potent . The respective Ki values were 0.6, 5, and 27 microM, respectively . In addition, MK 571 preferentially inhibited photoaffinity labeling of the 190-kDa protein in the MRP transfectants . Our results provide direct evidence that the MRP gene encodes a primary-active ATP-dependent export pump for conjugates of lipophilic compounds with glutathione and several other anionic residues . We conclude that the biosynthetic release of LTC4 from cells is mediated by the 190-kDa product of the MRP gene.

Proc Natl Acad Sci U S A, 1994 Nov 8, 91(23), 11123 - 7
Ribozyme-mediated reversal of the multidrug-resistant phenotype; Scanlon KJ et al.; This study examined the effects of suppressing c-fos oncogene expression on multidrug resistance (MDR) . A2780S human ovarian carcinoma cells with resistance to actinomycin D were isolated and the resultant A2780AD cells exhibited the MDR phenotype . A hammerhead ribozyme designed to cleave fos RNA cloned into the pMAMneo plasmid was transfected into A2780AD cells . Induction of the ribozyme resulted in decreased expression of c-fos, as well as that of the MDR gene (mdr-1), c-jun, and mutant p53 . The transformants displayed altered morphology and restored sensitivity to chemotherapeutic agents comprising the MDR phenotype . An anti-mdr ribozyme separately expressed in A2780AD cells efficiently degraded mdr-1 mRNA . However, reversal of the MDR phenotype by the anti-mdr ribozyme occurred one-fourth as rapidly as that induced by the anti-fos ribozyme . These results reinforce the central role played by c-fos in drug resistance through its participation in signal transduction pathways.

J Clin Oncol, 1994 Nov, 12(11), 2453 - 9
Multidrug resistance in lymphomas; Yuen AR et al.; PURPOSE: To discuss the significance of multidrug resistance (MDR) in human lymphomas and to review recent and ongoing clinical trials using MDR modulators . DESIGN: A medical literature search was used to identify articles that reported results on the expression or modulation of MDR in human lymphomas . This review summarizes the various methods for detecting expression of the mdr1 gene in tumor specimens, the patterns of expression in lymphomas, and recent and upcoming clinical trials using modulating agents to reverse MDR . RESULTS: There is considerable variation in the assays used to evaluate the expression of mdr1 in lymphomas . Current methodology includes reverse transcriptase polymerase chain reaction (rt-PCR) for assay of mdr1 mRNA, and immunohistochemistry or flow cytometry for detection of the multidrug transporter, P-glycoprotein (P-gp) . The preponderance of evidence suggests that mdr1 expression is relatively low in untreated patients (10% to 20% of lymphomas positive), but increases in patients with recurrent disease (50% to 70% positive) . Some evidence suggests that mdr1 expression is a prognostic factor for response to chemotherapy, as well as for subsequent survival . Verapamil and cyclosporine (CsA) have been used as competitive inhibitors of the multidrug transporter P-gp in early clinical trials . Although these studies show some activity in modulating clinical MDR, both verapamil and CsA manifest considerable toxicities at doses below those required for complete inhibition of P-gp function . CONCLUSION: MDR due to the expression of the mdr1 gene is an important factor in the course of patients with lymphomas . Continued clinical trials with more potent and less toxic modulators are needed to define the ultimate benefit of modulating MDR in lymphomas.

Carcinogenesis, 1994 Nov, 15(11), 2541 - 6
Metabolic activation of 2-acetylaminofluorene is required for induction of multidrug resistance gene expression in rat liver cells; Schrenk D et al.; P-Glycoprotein the multidrug resistance (mdr) efflux transporter is encoded by class 1 mdr genes (mdr1) in humans and rodent species . In rat liver and in rat hepatocytes in primary culture, expression of mdr1 genes can be induced with the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF) . As a consequence, increased P-glycoprotein levels led to an accelerated efflux of vinblastine from the hepatocytes and to resistance towards vinblastine-mediated cytotoxicity . N-Hydroxylation, an obligatory initial step in the activation of 2-AAF into electrophilic DNA-binding metabolites is catalyzed predominantly by cytochrome P450 (CYP)1A2, an isozyme present in normal rat liver . In rat hepatocytes in primary culture, mdr1 induction with 2-AAF could be inhibited by addition of the CYP1A-inhibitor alpha-naphthoflavone, indicating the requirement for metabolic conversion of 2-AAF to act as an inducer of mdr1 gene expression . Both N-hydroxy-2-AAF and the mutagenic 2-AAF derivative N-acetoxy-2-AAF (AAAF) were more potent than 2-AAF as mdr1 inducers . mdr1 induction also decreased when deacetylation of AAAF, which strongly accelerates its conversion into a mutagen, was inhibited with paraoxon . Furthermore, rat liver epithelial cells stably transfected with mouse CYP1A2 showed inducibility of mdr1 gene expression with 2-AAF, whereas the parental cell line, which is devoid of CYP1A2 activity, did not . These findings indicate that electrophilic metabolites formed during 2-AAF or AAAF metabolism are responsible for mdr1 induction in rat hepatocytes . The increased mdr1 gene expression may reflect an adaptive cellular response to electrophiles which includes enhanced synthesis of P-glycoprotein aimed to protect the cell from further damage.

Blood, 1994 Nov 1, 84(9), 3113 - 21
Expression of the multidrug resistance associated protein and P-glycoprotein in doxorubicin-selected human myeloid leukemia cells; Slapak CA et al.; Drug-resistant sublines of the human U-937 myeloid leukemia cell line were selected in doxorubicin concentrations of 10, 40, and 200 ng/mL (designated U-A10, U-A40, and U-A200, respectively) . Northern blot analysis showed overexpression of the multidrug resistance-associated protein (MRP) gene, but not MDR1, in U-A10 cells as compared with parental U-937 cells . Prolonged passage of U-A10 cells in 10 ng/mL of doxorubicin had little effect on MRP RNA levels, but increased MDR1 expression . The U-A40 and U-A200 cells, derived by selection of U-A10 cells, showed high levels of both MRP and MDR1 expression . None of the drug-resistant cell lines showed MRP or MDR1 gene amplification as judged by Southern blot analysis . U-A10 cells exhibited minimal decreased net accumulation of anthracycline, whereas U-A40 and U-A200 cells showed more significantly decreased drug accumulation as compared with U-937 cells . Subcellular anthracycline accumulation in U-937 cells as determined by fluorescence microscopy showed daunorubicin fluorescence predominately in the nucleus . However, the drug-resistant cell lines showed minimal nuclear drug accumulation with marked redistribution of drug into a vesicular compartment . Treatment with sodium azide/2-deoxyglucose, 2,4-dinitrophenol, or monensin, but not verapamil, abolished the vesicular accumulation . These studies in doxorubicin-selected U-937 cells indicate that induction of MRP overexpression occurs before that for the MDR1 gene . In addition, the drug-resistant cells possess an energy-dependent redistribution of anthracyclines into a nonnuclear vesicular compartment.

Br J Cancer, 1994 Nov, 70(5), 795 - 8
The use of genetic marking to assess the interaction of sensitive and multidrug-resistant cells in mixed culture; Bradley C et al.; The interaction of normal (CHO-K1) and multidrug-resistant (Adrr) Chinese hamster ovary cells was examined in mixed monolayer and spheroid culture . In order to assess the individual response of the two cell types in mixed culture, CHO-K1 was genetically marked by transfection with a bacterial beta-galactosidase gene . The enzyme product can be detected histochemically and allows identification of the marked cell line, designated CHO-K1-BG . Following administration of doxorubicin or mitozantrone, there was a large difference in the clonogenic survival of CHO-K1-BG and Adrr, whereas the overall survival of a 50:50 mixture of the two cell lines had intermediate values . When the survival of marked and unmarked colonies from mixed culture was assessed separately, there was no detectable alteration in chemosensitivity . We have found no evidence for interaction of sensitive and multidrug-resistant cells in this system.

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