Multidrug resistant

Cancer Res, 1994 Nov 1, 54(21), 5607 - 13
Overexpression of the multidrug resistance-associated protein (MRP) gene in vincristine but not doxorubicin-selected multidrug-resistant murine erythroleukemia cells; Slapak CA et al.; Multidrug-resistant sublines of the murine erythroleukemia cell line PC4 were sequentially selected in increasing vincristine concentrations (5-160 ng/ml) . The low- and intermediate-level resistant cell lines, selected in < or = 40 ng/ml of vincristine, demonstrated resistance to Vinca alkaloids and to an epipodophyllotoxin but little or none to an anthracycline . The expression of murine mdr genes, as analyzed by Northern blotting, revealed a baseline expression of murine mdr2 in parental cells that was unchanged in the drug-resistant cell lines . Overexpression of mdr3 was observed only in the highest-level resistant cell line, PC-V160, whereas mdr1 mRNA was not detected in any of the cell lines . The polymerase chain reaction, using mdr3-specific primers, excluded the possibility that low levels of P-glycoprotein expression contributed to the resistance phenotype in the low and intermediate-level resistant cell lines . Northern blot analysis using a human complementary DNA probe for the multidrug resistance-associated protein (MRP) demonstrated overexpression of murine mrp in each of the vincristine-selected sublines . Genomic amplification of the mrp gene was coincident with mrp overexpression . The expression of mrp was also examined in two series of previously characterized doxorubicin-selected cell lines derived from parental PC4 and C7D murine erythroleukemia cells . In contrast to the vincristine-selected cell lines, overexpression of mrp was not detected . These studies demonstrate that, in murine erythroleukemia cells selected for vincristine resistance, overexpression of murine mrp occurred prior to that for murine mdr . In contrast to human MRP, selection for vincristine, but not doxorubicin resistance, resulted in the overexpression of murine mrp.

Haematologica, 1994 Nov-Dec, 79(6), 500 - 7
Restoring uptake and retention of daunorubicin and idarubicin in P170-related multidrug resistance cells by low concentration D-verapamil, cyclosporin-A and SDZ PSC 833; Michieli M et al.; BACKGROUND . Overexpression of the mdr-1 gene that codes for a 170 Kd transmembrane glycoprotein (P170) is a factor responsible for decreased cell sensitivity to anthracyclines and other drugs, and is related to treatment failure in acute leukemia and other tumors . Several agents, including verapamil and cyclosporine derivatives, can modify P170-related resistance in vitro and can be proposed as adjuvant treatment for leukemia and cancer . MATERIALS AND METHODS . To investigate the optimal conditions for adjuvant treatment, D-verapamil (D-VRP), cyclosporin-A (CyA) and the cyclosporine derivative SDZ PSC 833 (PSC) were used alone and in combination at drug concentrations that can be achieved and maintained in vivo . The drugs were tested for their capacity to restore intracellular daunorubicin (DNR) and idarubicin (IDA) accumulation in high-resistance (CEM VLB 300) and intermediate-resistance (CEM VLB 100) cells to levels found in the non-resistant parental cell line (CCRF CEM) . RESULTS . In intermediate-resistance cells, IDA alone was more easily restored than DNR plus modifiers, and intracellular IDA concentration was returned to the level of non-resistant cells with low-dose D-VRP (1 microM), CyA (0.8 microM) and PSC (0.4 microM) . In high-resistance cells no modifier or modifier combination was able to restore intracellular DNR concentration to the value of non-resistant cells . Intracellular IDA concentration was almost completely restored only with D-VRP (2-3 microM) and CyA (0.8-1.6 microM) in combination or with PSC alone (0.4 microM) . CONCLUSIONS . These data suggest that as far as P170-related resistance is concerned, IDA alone is as efficient as or even more efficient than DNR plus modifiers, and that residual resistance to IDA can be overcome with a combination of D-VRP and Cy-A at a clinically achievable concentration, or with a more powerful modifier like SDZ PSC 833.

Clin Infect Dis, 1994 Nov, 19(5), 916 - 21
Cellular transport of drugs; Steinberg TH; The ability of drugs to enter cells and to reach an adequate concentration within the appropriate intracellular compartment may be an important determinant of the efficacy of therapy for infections due to intracellular pathogens . Antibiotics vary considerably in their ability to accumulate within cells . All solutes can be taken up by endocytosis, and some compounds reach high intracellular concentrations after crossing the plasma membrane by diffusion or by specific transport processes . Cellular organelles have properties that affect the intracellular distribution of drugs and that may account for the net accumulation of drugs within cells . An important example is the ability of acidic organelles to trap lysosomotropic weak bases . Cells also express transport proteins that may limit the intracellular accumulation of drugs by secreting them across the plasma membrane into the extracellular medium . Thus, cellular organic anion transporters can decrease the intracellular accumulation of certain antibiotics, and the multidrug-resistance transporter, or p-glycoprotein, confers resistance to antineoplastic agents . Inhibition of organic anion transport may have therapeutic value . A better appreciation of the mechanisms by which drugs accumulate within cells and within specific intracellular compartments may lead to the design of agents that reach higher concentrations at clinically relevant intracellular sites.

Somat Cell Mol Genet, 1994 Nov, 20(6), 463 - 80
Subclonal heterogeneity of the multidrug resistance phenotype in a cell line expressing antisense MDR1 RNA; Hanchett LA et al.; A multidrug resistant (MDR) cell line was transfected with an antisense MDR1 expression vector and transfectant clones were analyzed for reversion of the MDR phenotype . Only one of 10 antisense-expressing transfectants showed a reduction in drug resistance, MDR1 mRNA and P-glycoprotein . Observations made using rhodamine-123, a fluorescent substrate for P-glycoprotein, revealed that dye retention in individual cells was highly variable within this antisense-expressing clone . Subpopulations were established from the original clone based on differences in rhodamine-123 retention . Rhodamine-123 retention varied inversely with levels of P-glycoprotein and MDR1 mRNA . All subpopulations expressed similar levels of antisense MDR1 RNA yet had dramatic differences in MDR1 mRNA levels . Analysis of vector integration site restriction fragment length polymorphisms confirmed that all populations originated from the same transfectant clone . Nuclear run-on analysis indicated that the mdr1 gene is transcribed at the same rate in all populations, suggesting that the reduction in MDR1 mRNA is mediated posttranscriptionally . Cells with the greatest reduction in MDR1 mRNA accumulate distinct antisense RNA transcripts in the nuclear RNA fraction, suggesting that antisense effectiveness in this system is associated with a nuclear event or process . These results reveal that antisense RNA activity is not necessarily distributed equally within a clonal population.

Mol Biochem Parasitol, 1994 Nov, 68(1), 81 - 91
The P-glycoprotein-related gene family in Leishmania; Legare D et al.; P-glycoprotein gene amplification has been described in several drug-resistant parasitic protozoa . The first P-glycoprotein related gene described in Leishmania was ltpgpA, a gene frequently amplified in arsenite resistant Leishmania . Hybridization experiments indicated that ltpgpA was part of a gene family . In addition to ltpgpA, four novel genes were cloned that are present in two loci: ltpgpB and ltpgpC tandemly linked to ltpgpA on a 800-kb chromosome; and ltpgpD and ltpgpE closely linked on a chromosome ranging from 950 kb to 1400 kb, depending on the Leishmania species . Another P-glycoprotein gene, homologous to the more recently described ldmdr1, was linked to ltpgpD and ltpgpE . Nucleotide sequencing of ltpgpB and ltpgpE revealed that the Leishmania P-glycoprotein-related genes have diverged considerably from the main branch of P-glycoproteins and are more homologous to the recently described multidrug resistance-associated protein found in multidrug-resistant human lung cancer cell lines . Cross-resistance studies and gene transfection experiments indicated that under the conditions tested only ltpgpA and ldmdr1 are involved in resistance to arsenite and antimonials or hydrophobic drugs such as vinblastine respectively.

J Hepatol, 1994 Nov, 21(5), 754 - 63
Effect of colchicine and heat shock on multidrug resistance gene and P-glycoprotein expression in rat liver; Vollrath V et al.; The multidrug resistance genes encode plasma membrane glycoproteins named P-glycoproteins, that act as an ATP-dependent drug efflux pump and decrease the cytosolic concentration of chemotherapeutic agents . It has been hypothesized that in rat liver, this protein may have a physiological role as a biliary transporter of xenobiotics and endobiotics . Some human tumor cell lines turn on the human multidrug resistance gene in response to high temperature and after exposure to toxic chemicals . Accordingly, it has been proposed that the human multidrug resistance gene is a heat shock gene . We have assessed whether two environmental stresses, heat shock or acute exposure to cytotoxic drugs (colchicine, vincristine, vinblastine and daunomycin), induce changes in the expression of multidrug resistance genes in the rat . Total cellular RNA extracted from rat liver was hybridized to a labeled human multidrug resistance gene cDNA probe . Temperature upshift did not increase the steady-state of mdr mRNA levels in the tissues studied, suggesting that the mdr genes are not activated as part of a heat shock response . The mdr mRNA levels increased in rat liver as early as 3 h after a single injection of colchicine, reached a peak (500%; p < 0.05) after around 24 h and returned to constitutive levels after 48 h . Changes in the relative content of mdr mRNA were not detected in kidney, adrenal gland and small bowel, suggesting that the in vivo induction of the mdr gene in the liver is a tissue-specific response . The other cytotoxic drugs that were tested did not increase the steady-state of mdr mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Res Hum Retroviruses, 1994 Nov, 10(11), 1471 - 8
Zidovudine induces the expression of cellular resistance affecting its antiviral activity; Dianzani F et al.; We have previously shown that multidrug-resistant cells expressing the multidrug transporter P-glycoprotein are less sensitive to the antiviral activity of AZT . Subsequently, we addressed the question whether AZT itself is able to induce cellular resistance to the drug . Indeed, CEM cells propagated in the presence of increasing concentrations of AZT become resistant to the antigrowth and antiviral activity of AZT but do not express detectable level of P-glycoprotein . Sensitivity of these cells to other compounds, such as vinblastine, vincristine, ddI, and ddC remained unchanged, indicating that, in contrast to P-glycoprotein-positive cells, AZT-induced resistance is specific for AZT . Interestingly, in AZT-induced resistant cells the intracellular accumulation of AZT and exogenous deoxythymidine, as well as thymidine kinase activity, are significantly reduced when compared with the parental cell line . Our findings show that AZT itself may directly induce the expression of cellular mechanisms leading to the acquisition of specific cellular resistance that can affect its antiviral activity.

Res Commun Mol Pathol Pharmacol, 1994 Nov, 86(2), 195 - 203
Reduced drug resistance in a multidrug resistant cell line by 5,8,11,14-eicosatetraynoic acid; Anderson KM et al.; We examined whether the arachidonic acid competitive antagonist, ETYA (5,8,11,14-eicosatetraynoic acid), modulated drug sensitivity in a cell line that over-expresses the multiple drug resistance protein, MDR1 . ETYA was nontoxic to drug-sensitive parental KB3-1 cells or drug-resistant MDR KB8-5-11 cells, with an IC50 of 190 microM for both lines . ETYA (20 microM) increased rhodamine 123 accumulation in KB8-5-11 MDR cells but not in KB3-1 sensitive cells . Arachidonic acid at 20 microM did not alter rhodamine accumulation in either cell line . Increasing the concentration of ETYA from 40 to 160 microM or incubation beyond 30 min did not increase KB8-5-11 dye retention . Forty microM or more ETYA increased KB3-11 dye retention . In a 6 day proliferation assay of KB8-5-11 cells, a nontoxic (40 microM) concentration of ETYA reduced the IC50 for doxorubicin 4-fold, the IC50 for colchicine 2-fold, but had no effect on the IC50 for vinblastine . ETYA at 40 microM did not alter the IC50 for any drug tested with KB3-1 cells . Therefore: (a) ETYA (20 or 40 microM) modulated resistance of KB8-5-11 cells to several drugs to a limited extent, without potentiating toxicity in the parental line, while arachidonic acid did not . (b) Since cationic rhodamine 123 is concentrated in mitochondria, the extent is dependent upon the transmembrane potential, and increased dye retention due to ETYA may in part be related to altered ETYA-induced cell membrane potential.

Leuk Lymphoma, 1994 Nov, 15(5-6), 453 - 68
Evaluation of the clinical relevance of the anionic glutathione-s-transferase (GST pi) and multidrug resistance (mdr-1) gene coexpression in leukemias and lymphomas; Russo D et al.; By using RNA slot-blot technique, the frequency and the degree of GST pi and mdr-1 gene coexpression were investigated in 23 AML patients, 9 ALL, 9 CLL and 11 cases of NHL in an attempt to study their clinical and prognostic relevance . GST pi and mdr-1 levels were expressed as arbitrary units (U) with respect to the negative controls (U = 0), MCF7 and HL60 sensitive cell lines, and the positive controls (U = 10), MCF7/DOXO and HL60/DNR resistant cell lines . The concomitant GST pi/mdr-1 gene overexpression showed a negative prognostic value in the set of newly diagnosed AML pts (10 cases), furthermore higher GST pi and mdr-1 mRNA levels were averagely detected in the relapsed/resistant ALL pts (4 cases), and in CLL (7 cases) and NHL (8 cases) heavily pretreated patients who were unresponsive to chemotherapy and with a disease progression . These preliminary data show that two different mechanisms of drug resistance can be coexpressed at the same time in those leukemias and lymphomas with a clinically unfavourable course.

Anticancer Res, 1994 Nov-Dec, 14(6B), 2685 - 9
Lacidipine and josamycin: two new multidrug resistance modulators; Crosta L et al.; In this paper we report the results obtained treating a multidrug resistant (MDR) murine erythroleukemia cell line with daunomycin (DNM) in association with two new modulators characterized by a favourable therapeutic index, lacidipine (LCD), a dihydropyridine calcium antagonist, and josamycin (JSM), a macrolide antibiotic . LCD and JSM exhibited a greater MDR reversal activity than verapamil (VRP) and erythromycin (ERY) respectively . The accumulation of DNM in the DRTL cells exposed to modulators was similar to that of the parental cell line FLC . In the case of LCD, it was possible to ascertain that at a very low concentration this molecule can circumvent MDR without modifying DNM accumulation, suggesting that multiple different determinants may be responsible for MDR other than P-170 in this cell line.

Anticancer Res, 1994 Nov-Dec, 14(6B), 2643 - 8
Combined activity of interleukin-1 alpha or TNF-alpha and doxorubicin on multidrug resistant cell lines: evidence that TNF and DXR have synergistic antitumor and differentiation-inducing effects; Borsellino N et al.; We report on the antiproliferative effects that interleukin-1 alpha (IL-1) or TNF-alpha (TNF) in combination with doxorubicin (DXR) exert on DXR-sensitive (B16 melanoma, Friend, K562 and CCRF/CEM leukemias) and -resistant (B16-DXR, FLC-DXR, K562-DXR) cell lines in vitro . Multidrug resistance (MDR) of the latter lines entails cross-resistance to vincristine and overexpression of P-glycoprotein . Il-1 showed only a very marginal growth inhibitory activity and the effects of its combination with DXR were essentially additive in all the cell lines, except in chemosensitive B16, where a slight synergism occurred . TNF demonstrated greater antiproliferative activity in the MDR B16 and Friend tumors than in their parent variants . The combination of TNF and DXR produced synergistic growth inhibition in B16, K562 and, particularly, also in the MDR sublines of these two tumors . In addition, TNF and DXR induced synergistically erythroid differentiation in K562 and multidirectional differentiation in K562-DXR . The synergism was critically schedule-dependent in that it was achieved only when DXR application preceded or was simultaneous with that of TNF . Finally, TNF did not modify drug accumulation and retention in the cells . Our present findings stress especially the fact that DXR and TNF may exert useful antitumor synergism even in MDR lines; however, it is not likely that their interaction will occur at the specific MDR process level.

Anticancer Res, 1994 Nov-Dec, 14(6B), 2605 - 9
Multidrug resistance phenotype evaluation by immunofluorescence and functional tests: comparison of two monoclonal antibodies and three fluorescent dyes in three cells lines; Maynadie M et al.; Various agents have been shown to enhance drug sensitivity of multidrug resistant (MDR) cells and are thus of interest when the MDR phenotype is identified . Detection of MDR cells is of importance and can be carried out either by immunofluorescence with monoclonal antibodies or by functional tests using fluorescent dyes uptake . MDR has been analysed by flow cytometry on three sensitive and resistant cell lines, with MRK16 and C219 monoclonal antibodies directed against P-glycoprotein (P-gp) and with rhodamine 123, Hoechst 33342 and daunorubicin . Resistant cells were revealed by MRK16 and C219 but the results obtained with MRK16 gave higher both percentages of fluorescent cells and mean fluorescence . Fluorescence intensity observed with daunorubicin was lower than with rhodamine 123 . With Hoechst 33342, mean fluorescence was quite identical on sensitive and on resistant cells . It was concluded that MRK16 and rhodamine 123 were well adapted to detect P-gp and evaluate its functional ability.

Anticancer Res, 1994 Nov-Dec, 14(6B), 2589 - 95
Effects of vinblastine, colchicine, and verapamil on rhodamine 123 accumulation in human P-glycoprotein-positive leukemia cells; Lautier D et al.; Multidrug-resistant (MDR) cells have been characterized by reduced accumulation of rhodamine 123 (R123) . We addressed the question of whether R123 could compete with substrates or inhibitors (vinblastine, colchicine, verapamil) of P-glycoprotein (Pgp) overexpressed in MDR cells, using fluorescence image cytometry . Verapamil caused a dose-dependent increase in R123 accumulation . R123 accumulation was increased by vinblastine only at high levels and colchicine had no effect on R123 accumulation . Treatments with two drugs altered R123 accumulation depending on drug concentration ratio . The results indicate that vinblastine, R123 and verapamil can compete for outward transport by Pgp . A dual effect of vinblastine suggests that vinblastine can activate Pgp at low concentrations and inhibit R123 transport at higher concentrations.

J Nat Prod, 1994 Nov, 57(11), 1517 - 22
Indole alkaloids from Peschiera laeta that enhance vinblastine-mediated cytotoxicity with multidrug-resistant cells; You M et al.; Coronaridine {1}, conoduramine {2}, and voacamine {3}, three indole alkaloids isolated from Peschiera laeta, have been found to enhance the cytotoxic response mediated by vinblastine {4} with multidrug-resistant KB cells . Inhibition of vinblastine binding with membrane vesicles isolated from this cell line was also assessed, and the bisindole alkaloids conoduramine {2} and voacamine {3} were found to be more potent inhibitory agents than the monomeric alkaloid, coronaridine {1} . Thus, these compounds appear to function by binding with P-glycoprotein.

Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 957 - 64
Ornithine decarboxylase--a predictor for tumor chemosensitivity; Bachrach U et al.; The activity of ornithine decarboxylase (ODC) was determined in P388 murine leukemia cells treated with adriamycin (ADR) and methotrexate (MTX) . Some of the cell lines were resistant to ADR, MTX or their combinations . A similar pattern was found between the cytotoxicity and the suppression of ODC activity in these cell lines in terms of drug concentrations . In a cell line resistant to one drug, a relatively high concentration of that drug was required to inhibit ODC activity . This effect was independent of the sensitivity of the cells to the other drug . A similar correlation between arrest of growth and the inhibition in the induction of ODC was also observed in human epithelial carcinoma cells . In this case too, the growth of multidrug resistant cells was not affected by vinblastine, neither was the induction of ODC . On the other hand, both the growth and the induction of ODC were inhibited by vinblastine in drug-sensitive cells . These findings suggest that ODC measurements might be used for predicting the chemosensitivity of tumor cells.

Pediatr Infect Dis J, 1994 Nov, 13(11), 990 - 4
Therapy of multidrug-resistant typhoid fever with oral cefixime vs . intravenous ceftriaxone; Bhutta ZA et al.; We randomly allocated 80 children with suspected multidrug-resistant tyhpoid fever to therapy with either cefixime or ceftriaxone . Of these, an alternative diagnosis was subsequently made in 10 children and another 10 were excluded because cultures were negative . In 9 cases the typhoidal organisms isolated were susceptible to first-line drugs . In all, 50 children were randomly allocated to receive therapy with either intravenous ceftriaxone (65 mg/kg/day once daily, Group A, n = 25) or oral cefixime (10 mg/kg/day divided every 12 hours, Group B, n = 25) for 14 days . The two groups were comparable in their clinical characteristics, duration and severity of illness at the time of admission . The time to defervescence was comparable in both groups (8.3 +/- 3.7 vs . 8.0 +/- 4.1 days, P = not significant) . An equal number (3 in each group) failed to respond and underwent a change in therapy . Three children in Group A and one in Group B relapsed . No adverse effects were seen in either group during the course of therapy . Our data suggest that oral cefixime can be used as effectively as parenterally administered ceftriaxone for management of typhoid fever in children.

Kekkaku, 1994 Nov, 69(11), 711 - 7
{New drugs against tuberculosis and nontuberculous mycobacterial infections: a review}; Amitani R et al.; The number of cases with tuberculosis is again increasing in many countries, and recently several nosocomial outbreaks of multidrug-resistant tuberculosis have occurred in the United States . The number of patients with disseminated Mycobacterium avium complex (MAC) infections in AIDS population, and patients with MAC pulmonary disease unassociated with HIV seem to be also increasing . It takes at least 6 to 9 months for an initial treatment of active tuberculosis due to drug-sensitive strains with the standard regimen which includes isoniazid (INH) and rifampicin (RFP) . Treatment for the diseases caused by drug-resistant M . tuberculosis and MAC is much more time-consuming and more toxic than for the diseases caused by drug-sensitive strains, and often unsuccessful . For the reasons described above, the developments of new agents with potent antimycobacterial activities are highly desired . The new agents should also be useful for treating patients who have acquired resistance to many of the currently available drugs . In this review the new antimycobacterial drugs are summarized . Some of them have already been used clinically, but many are still in experimental evaluations . 1) Rifamycin derivatives: rifabutin (RBT), KRM-1648 (KRM), rifapentin (RPT), FCE-22250, FCE-22807, CGP-7040, SPA-S-565 and other rifamycin derivatives . New rifamycin derivatives including RBT, KRM have increased in vitro antimycobacterial activities . RBT and KRM are much more active in vitro and in vivo than RFP against both M . tuberculosis and MAC . KRM seems to be more potent than RBT against MAC in experimental studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticancer Res, 1994 Nov-Dec, 14(6A), 2389 - 93
Effect of S9788, cyclosporin A and verapamil on intracellular distribution of THP-doxorubicin in multidrug-resistant K562 tumor cells, as studied by laser confocal microspectrofluorometry; Sebille S et al.; S9788 modulating resistance effect has been investigated on the activity of THP-DOX against multidrug-resistant K562R cells and compared to that of cyclosporin A and verapamil . Intracellular THP-DOX distribution and particulary its intranuclear concentration, with or without modulators, has been measured using confocal laser microspectrofluorometry . The kinetics of intranuclear accumulation of THP-DOX (1 microM in the medium), as a function of time, were rapid in K562S and K562R cells . Maximum accumulation of THP-DOX is reached in a few minutes (K562S, 400 microM; K562R, 40 microM) . The addition of S9788, cyclosporin A and verapamil (5 microM) after one hour THP-DOX incubation, led to respectively 290, 250 and 114 microM . Uptake of THP-DOX was increased in K562R cells by a factor of 7 when S9788 was added . Results obtained on THP-DOX efflux from nuclei of K562S and K562R cells, after 3 hours of incubation without drug, showed a very short T1/2 (time corresponding to 50% decrease of intranuclear concentration of THP-DOX) in K562R cells (12 min) compared to that in K562S cells (150 min) . The addition of S9788, cyclosporin A and verapamil (5 microM) led to a T1/2 of 90, 30 and 20 min respectively . The T1/2 of THP-DOX was increased in K562R cells by a factor of 7.5 when S9788 was added . We tried to correlate these results with those obtained in growth inhibition study . The IC50 (concentration which induces 50% growth inhibition) of THP-DOX, corresponding to one hour THP-DOX treatment and 3 days culture of K562S and K562R cells were respectively 230 and 7000 nM, and the RI (resistance index) value is 30 . The addition of S9788, cyclosporin A and verapamil (5 microM), during the one hour treatment, led to an IC50 value of 350, 2000 and 5000 nM respectively . S9788 induced an IC50 value 20 times lower than without the modulator . Our study suggests that the higher activity of THP-DOX against K562R cells in the presence of S9788, compared to cyclosporin A and verapamil, is due to a higher intranuclear THP-DOX accumulation and to a strong decrease of drug efflux from K562R nuclei . This could be explained by a higher affinity of S9788 for membrane P-glycoprotein of K562R cells and/or an additional mechanism of action.

Anticancer Res, 1994 Nov-Dec, 14(6A), 2383 - 7
Study of sodium orthovanadate as a reverser of multidrug resistance on lymphoblastic leukemic CEM/VLB100 cells; Colin M et al.; The occurrence of multidrug resistance (MDR) is the major cause of failure of cancer chemotherapy . Finding a way to circumvent this problem is now a major challenge in oncology . Multidrug resistant CEM/VLB100 cells accumulate 10 times less vinblastine (VLB) after 30 min than their sensitive counterparts (CEM cells) . At a non-cytotoxic concentration (1 mM) of sodium orthovanadate (OVN), uptake by CEM/VLB100 cells was increased 4 times while no effect was observed on CEM cells . The action on VLB uptake of OVN and verapamil (VPL), an usual MDR modulator, was additive . In CEM/VLB100 cells, OVN did not alter efflux . Its cellular mechanism of action could involve a transitory stimulation of VLB influx (x3) . OVN uptake in CEM and CEM/VLB100 cells was not significantly different and reached saturation after 30 s (180 pmol/10(6) CEM cells and 150 pmol/10(6) CEM/VLB100 cells) . This OVN uptake was concentration dependent . IC50 of VLB and doxorubicin were decreased by approximately 43 and 62% after 1 hour's exposure to OVN and 48 hours of culture . Under these conditions, OVN was more efficient than OVN.

Anticancer Res, 1994 Nov-Dec, 14(6A), 2371 - 3
Proposals for concomitant use of several modulators of multidrug resistance in clinics; Robert J; Numerous modulators of multidrug resistance present a good in vitro activity which has not always been reproduced in vivo or in clinics . One reason is the fact that the plasma concentration required might exceed the maximum tolerated dose of modulator, due to its own toxicity . In addition, it has been suggested that the different modulators thus far identified might act on different sites of P-glycoprotein and eventually on different targets . It has been shown that the combination of verapamil and quinine, and of verapamil and cyclosporine A, behave synergistically in the circumvention of multidrug resistance . There is therefore a good rationale for the evaluation of protocols combining two different modulators to cytotoxic chemotherapy.

Br J Haematol, 1994 Nov, 88(3), 566 - 74
Development of extended multidrug resistance in HL60 promyelocytic leukaemia cells; Su GM et al.; In an attempt to mimic clinical conditions for the treatment of leukaemia, the HL60 promyelocytic cell line was treated for 18 h with low, clinically relevant, levels of the anthracycline epirubicin and the Vinca alkaloid vinblastine . The resulting drug-resistant sublines not only expressed P-glycoprotein and the MDR phenotype but were also cross-resistant to chlorambucil, methotrexate and cisplatinum, and had increased resistance to radiation . Development of resistance was associated with an aberrant differentiation phenotype with decreased expression of myeloid antigens and expression of glycophorin A, an antigen normally associated with erythroid differentiation . The ability of HL60 cells to terminally differentiate in response to all-trans-retinoic acid (vitamin A acid) was lost in the sublines . These results suggest that either a single novel mechanism is responsible for multiple drug resistance or the initial response to drug treatment is the co-induction of multiple mechanisms . These cells and the method by which they were generated therefore provide a clinically relevant model for the study of the initial events in the development of not only multidrug resistance but also the extended multiple drug resistance usually encountered in the treatment of leukaemia.

In Vivo, 1994 Nov-Dec, 8(5), 835 - 41
Regulation of the multidrug resistance (MDR1) gene expression; Chin KV et al.; The emergence of drug resistance poses a major obstacle to the success of chemotherapy for a large number of human cancers . Development of the multidrug resistance phenotype in human malignancies is an especially pressing problem because the tumors become cross-resistant to multiple chemotherapeutic agents that are both chemically and physically unrelated . The increased resistance to multiple cytotoxic natural product chemotherapeutic drugs is due to overexpression of the mdr gene, which encodes a plasma membrane ATP-dependent efflux pump . Expression of P-glycoprotein is tissue specific and found in a number of normal tissues, including colon, small intestine, kidney, liver and adrenal gland, as well as in the capillaries of brain and testis . The precise physiological functions in these tissue localizations is unclear at present . Intense efforts in many laboratories currently are invested on elucidating the functions of P-glycoprotein and investigating mechanisms that regulate the mdr gene expression.

In Vivo, 1994 Nov-Dec, 8(5), 829 - 34
Modes of resistance to antitumor agents; Kessel D; Drug resistance has complicated chemotherapy of neoplastic disease since the early beginnings of the field . Successful use of drug therapy requires that tumor cells be more drug-responsive than the tissue of origin . This can occur with rapidly-dividing tumors which will be highly-responsive to drugs toxic in the S-phase of the cell cycle . Another drug sensitive population will consist of neoplastic cells unable to repair drug-induced damage which can be repaired by normal host cells . Even if one of these conditions is initially found, mutational events leading to drug resistance often occur . The most thoroughly explored mode of drug resistance involves MDR (multidrug resistance), an outward transport system which limits penetration of a variety of cytotoxic agents into the cytoplasm and nucleus . MDR is expressed by many normal cell types, and attempts at MDR circumvention appear to lead to new modes of host toxicity . It appears that new directions in cancer therapy will be needed to yield responses in the common slow-growing tumors which tend to be inherently drug-resistant.

Eur J Clin Microbiol Infect Dis, 1994 Nov, 13(11), 902 - 7
Tuberculosis at the end of the twentieth century; Sepkowitz KA et al.; Tuberculosis has once again emerged as a significant public health problem in Western countries . Much of the rise has been fueled by the growing numbers of persons infected with HIV . Co-infection with Mycobacterium tuberculosis and HIV has been shown to result in high rates of active tuberculosis, and possibly in acceleration of progression to AIDS . Primary tuberculosis occurs at high rates among dually infected persons, further emphasizing the need for effective isolation of infectious cases . Recent preliminary studies have demonstrated that the survival of persons with multidrug-resistant tuberculosis can be six months and longer, far in excess of the 4 to 12 weeks reported previously . At least seven health care workers have died of occupationally-acquired multidrug-resistant tuberculosis, making control of the spread of tuberculosis in health care settings an urgent public health priority.

Am J Physiol, 1994 Nov, 267(5 Pt 1), C1351 - 8
Expression and function of P-glycoprotein in human mesangial cells; Bello-Reuss E et al.; P-glycoprotein (PGP), responsible for multidrug resistance (MDR) in cancer cells, is normally expressed in kidney proximal tubules and mesangium . PGP expression and function were studied in human mesangial cell cultures . MDR1 gene expression was demonstrated by reverse transcription-polymerase chain reaction . PGP expression was determined using MRK16 monoclonal antibody and its function was assessed by the efflux of rhodamine-123 (R123) . R123 efflux had a half time of 25 +/- 5 s . Efflux was inhibited by cyclosporin A (10 microM), verapamil (10 microM), and vinblastine (100 microM) with half times of 380, 535, and 312 s, respectively . Incubation with MDR1-antisense oligonucleotide decreased R123 efflux (half time = 304 s) . Verapamil, cyclosporin A, and PSC-833 augmented the cytotoxicity of Adriamycin by reducing the 50% maximal growth-inhibitory dose from 730 nM to 130, 110, and 90 nM, respectively . We conclude that human mesangial cells express MDR1 and demonstrate xenobiotic transport inhibitable by several known PGP substrates . Concomitant exposure of mesangial cells to PGP-transported drugs causes intracellular accumulation of toxic PGP substrates and ultimately damages the mesangial cells.

Cancer Lett, 1994 Oct 28, 86(1), 135 - 42
High mdr1- and mrp-, but low topoisomerase II alpha-gene expression in B-cell chronic lymphocytic leukaemias; Beck J et al.; Fifteen samples from 11 patients suffering from chronic lymphocytic leukaemia (CLL; 5 untreated, 6 chemotherapeutically treated) were analysed for their individual gene expression of the multidrug resistance (MDR) associated genes encoding mdr1/P-glycoprotein, mrp, and topoisomerase II alpha/beta-isoenzymes by a complementary DNA polymerase chain reaction (cDNA-PCR) approach . The expression of glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as standard . Thereby, we generally found high mdr1- and mrp-, but low topoisomerase II alpha-mRNA levels . While mdr1 levels of the CLL samples were mostly found to be in the range of values measured in the T-lymphoblastoid, P-glycoprotein MDR cell line CCRF VCR 100, mrp levels were usually found to be 2-4-fold higher compared therewith . This might represent a multifactorial MDR in CLL . In contrast to the low or even absent topoisomerase II alpha gene expression, however, the expression of the topoisomerase II beta gene was generally high in the CLL lymphocytes exceeding the value observed in the cell line CCRF VCR 100 up to 5-fold . mdr1 gene expression correlated significantly with mrp gene expression in samples from patients having received chemotherapy (rs = 0.5833, P < 0.05, n = 10) . In two patients the follow-up analysis revealed combined increases in mdr1- and mrp-gene expression levels in the course of the disease.

Biochemistry, 1994 Oct 25, 33(42), 12665 - 75
Analysis of drug transport kinetics in multidrug-resistant cells using a novel coumarin-vinblastine compound; Bornmann WG et al.; We have synthesized an analogue of vinblastine wherein a coumarin molecule is attached to the C17 of the vindoline moiety via a succinimide bridge (cou-VBL) . cou-VBL exhibits fluorescence similar to that exhibited by coumarin . Chinese hamster ovary fibroblasts (LR73 cells) that exhibit an IC50 for vinblastine (VBL) of about 100 nM in growth inhibition assays are similarly sensitive to the cou-VBL compound . LR73 cells transfected with the mu MDR 3 gene that were subsequently selected on vinblastine (MDR35 cells) exhibit resistance to cou-VBL that is similar to their VBL resistance . A large change in the quantum efficiency of cou-VBL fluorescence accompanies efflux from intact cells, and comparison between cou-VBL and {3H}VBL efflux from the MDR35 cells reveals that the transport kinetics of the fluorescent analogue is very similar (if not identical) to that exhibited by {3H}VBL . Thus, similar to continuous monitoring of fluorescence (CMF) studies performed with the naturally fluorescent chemotherapeutic doxorubicin (Roepe, 1992), cou-VBL may be used in CMF studies aimed at rigorously defining the kinetics of VBL efflux from multidrug-resistant (MDR) cells . Initial data suggest the following: (1) A single exponential term approximates efflux from sensitive cells, whereas two exponentials are required to fit efflux from MDR35 cells . (2) The faster MDR35 term is virtually identical to the single term for the sensitive cells, whereas the other defines a process that is 5-20 times slower than passive diffusion, depending on the drug concentration . (3) The slower component has a much steeper and nearly linear dependence on drug concentration, whereas the fast passive diffusion component becomes asymptotic near 0.3 pM exchangeable drug/microgram of cell protein.

J Biol Chem, 1994 Oct 21, 269(42), 26058 - 65
Transport of polypeptide ionophores into proteoliposomes reconstituted with rat liver P-glycoprotein; Eytan GD et al.; The aim of this study was to examine the peptide transport activity of a naturally occurring P-glycoprotein such as that present in rat liver canalicular membrane vesicles . The peptide ionophores valinomycin and gramicidin D, which are known substrates of P-glycoprotein, served to monitor the P-glycoprotein activity indirectly as the ATP-dependent uptake of 86Rb+ mediated by these ionophores . Canalicular membrane vesicles proved inherently permeable to K+ ions, which prevented assay of transport ionophore activity . Therefore, P-glycoprotein was extracted from canalicular membrane vesicles and reconstituted into proteoliposomes that are relatively impermeable to cations . P-glycoprotein activity in the proteoliposomes was dependent on ATP hydrolysis since it was not observed with non-hydrolyzable analogs of ATP . Maximal ATP-dependent 86Rb+ uptake occurred at 50 nM gramicidin D and at 500 nM valinomycin thus possibly reflecting higher affinity of P-glycoprotein for gramicidin D . Nigericin, which does not participate in the multidrug resistance phenomenon, did not support an ATP-dependent uptake of 86Rb+ . ATP hydrolysis increased the amount of 86RB+ transported into the proteoliposomes . Furthermore, preincubation of the proteoliposomes in the presence of gramicidin D and 86Rb+, allowing for maximal ATP-independent 86Rb+ uptake to occur, did not interfere with subsequent ATP-dependent uptake, indicating the latter to constitute an active transport mechanism . The ATP-dependent component of 86Rb+ uptake occurred neither with liposomes nor with proteoliposomes reconstituted with proteins extracted from sinusoidal vesicles that lack P-glycoprotein . The ATP-dependent uptake was blocked by the known inhibitors of the ATPase activity associated with P-glycoprotein, oligomycin and vanadate, as well as by its established substrates, daunorubicin, doxorubicin, vinblastine, and the tripeptide N-acetyl-leucyl-leucyl-norleucinal . Thus, the reconstituted P-glycoprotein catalyzes the ATP-dependent 86Rb+ uptake that appears to occur by an energy-dependent translocation of the 86Rb(+)-ionophore complex . In this case, the actual substrate of P-glycoprotein is the ionophore-cation complex, which is both hydrophobic and positively charged as are most of the substrates of P-glycoprotein . This is the first demonstration of transport of a naturally occurring polypeptide by proteoliposomes reconstituted with physiologically expressed P-glycoprotein.

J Natl Cancer Inst, 1994 Oct 19, 86(20), 1539 - 45
Quantitative immunocytochemical assays of P-glycoprotein in breast carcinomas: correlation to messenger RNA expression and to immunohistochemical prognostic indicators; Charpin C et al.; BACKGROUND: Chemotherapy failure that is due to cellular drug resistance remains a major problem in most cancer patients . One type of drug resistance that has been characterized is the multidrug resistance phenomenon, which demonstrates a reduced ability of cancer cells to accumulate drugs as a result of the effects of an energy-dependent unidirectional drug efflux pump with a broad substrate specificity . This drug pump is composed of a 170-kd transmembrane glycoprotein referred to as the P-glycoprotein (P-gp) that uses energy in the form of adenosine triphosphate to transport drugs through a channel formed by transmembrane segments . PURPOSE: Our purpose was to detect the levels of P-gp expression in frozen untreated breast carcinomas by immunocytochemical assays and to correlate these levels to current prognostic indicators and, in a few cases, to MDR1 (also known as PGY1) mRNA expression by polymerase chain reaction (PCR) . METHODS: The immunocytochemical expression of the multidrug resistance gene, P-gp, was investigated using a specific monoclonal antibody (JSB1) against P-gp in 5-microns frozen sequential sections of breast carcinomas obtained from 213 patients . Microscopic images of immunostained preparations were evaluated by image analysis and were compared with MDR1 transcription (mRNA) assessed by PCR in 16 patients . Quantitative P-gp immunocytochemical assays were correlated to histoprognostic factors and immunocytochemical indicators . RESULTS: Among the 213 breast carcinomas tested, 113 (53%) were P-gp positive, but in 28% of the tumors, the immunostained surface accounted for less than 5% of the total area stained . Quantitative immunocytochemistry reflecting the amount of intracellular P-gp antigen strongly correlated (r = 0.865; two-sided, P < .0001; Pearson's test) with the quantitative evaluation of the scanner analysis of mRNA transcripts . The P-gp expression was significantly (two-sided, P < .001) correlated with p53 expression in tumors, to cathepsin D and Ki67 (two-sided, P < .01) immunoreactivity, and to a lesser extent, the detection of estrogen receptor antigenic sites (two-sided, P = .019) . P-gp expression was found to be independent of expression of progesterone receptor and pS2, pHER-2/neu, and CD31 in tumors and from patient age, tumor size, histologic types, grades and Nottingham prognostic index, and nodal status . CONCLUSIONS: The quantitative immunocytochemical assays of P-gp are correlated to PCR analysis of MDR1 expression, and such correlations can be useful in evaluating potential multidrug resistance in breast cancer . However, the clinical significance of P-gp immunodetections remains to be further determined.

Biochem Pharmacol, 1994 Oct 18, 48(8), 1641 - 6
Expression and function of multidrug resistance P-glycoprotein in a cultured natural killer cell-rich population revealed by MRK16 monoclonal antibody and AHC-52; Kobayashi Y et al.; Natural killer (NK) cells have been reported recently to be the highest in expressing multidrug resistance (MDR) P-glycoprotein among normal mature lymphoid cells . Using a cultured NK cell-rich population, we have examined the expression and function of P-glycoprotein, in particular its role in NK cell-mediated cytotoxicity, by employing two MDR-reversing agents (nicardipine and AHC-52, a nicardipine analog almost devoid of calcium channel blocking activity) and monoclonal antibody against P-glycoprotein (MRK-16) . The expression of P-glycoprotein was detected by flow cytometry and polymerase chain reaction of reverse transcribed mRNA . P-glycoprotein was functional in terms of rhodamine dye excretion and its susceptibility to the MDR-reversing agents . Since the concentration of nicardipine required for 50% inhibition (IC50) of rhodamine dye excretion (2 microM) was close to that of AHC-52 (5 microM), it was suggested that their inhibitory effects were not due to calcium channel blocking activity, and that ACH-52 is a selective inhibitor for P-glycoprotein . The IC50 of nicardipine for NK cell-mediated cytotoxicity (33 microM) was also close to that of AHC-52 (26 microM), indicating that P-glycoprotein is involved in NK cell-mediated cytotoxicity . In support of this, MRK16 inhibited NK cell-mediated cytotoxicity in a concentration-dependent manner . Both binding of target cells to NK cells and post-binding events were affected by AHC-52, suggesting that P-glycoprotein is involved in several steps in NK cell-mediated cytotoxicity.

Int J Cancer, 1994 Oct 15, 59(2), 282 - 6
Resistance to verapamil sensitization of multidrug-resistant cells grown as multicellular spheroids; Sakata K et al.; The ability of verapamil to overcome resistance to adriamycin in a multidrug-resistant derivative of the V79 cell line (LZ), grown as multicellular spheroids or as monolayers, was examined . Verapamil was much less effective in overcoming resistance to adriamycin in spheroids than in monolayers . Verapamil increased the adriamycin content of cells grown as monolayers, but had no significant effect on the drug content of spheroids . This occurred in spite of the same mdr-I mRNA and protein levels in monolayers and spheroids . When the surviving fraction of cells was normalized to the cellular adriamycin content, cells both in monolayers and spheroids treated with verapamil were still more sensitive to adriamycin than their counterparts not treated with verapamil . The observed resistance of spheroids to adriamycin and verapamil sensitization may be caused by a drug-resistance mechanism that is functional only in spheroids, in addition to the activity of P-glycoprotein . Multicellular tissue architecture and cell-cell contact may play significant roles in this type of multidrug-resistance mechanism.

Int J Cancer, 1994 Oct 15, 59(2), 275 - 81
Daunorubicin efflux against a concentration gradient in non-P-glycoprotein multidrug-resistant lung-cancer cells; Mulder HS et al.; Multidrug-resistant, human non-small-cell lung carcinoma SW-1573/2R120 (2R120) cells, not containing the drug efflux pump P-glycoprotein (Pgp), have reduced initial daunorubicin (DN) accumulation rates and decreased cellular steady-state drug concentrations . Previously we found indications of the presence of a plasma membrane "vacuum cleaner", pumping DN directly from the membrane, and reported evidence of active DN pumping using digitonin . Further evidence of active DN pumping is now provided via a different methodology and the active drug pump flux is estimated . Cells were exposed to a flowing medium containing the cytotoxic agent DN . After reaching a steady state, in which net DN uptake equals net DN efflux, high concentration pulses of vincristine (VCR) were injected into the flowing medium . A rapid increase in cellular DN content was observed, while only a minimal effect was seen in SW-1573 wild-type cells . After passage of the VCR pulse, the extra accumulated DN was effluxed against a concentration gradient . Upon increasing the VCR concentration, a maximum pump inhibition was reached which was similar to the effect of cellular energy depletion . Similar effects were observed for Pgp-containing SW-1573/2R160 (2R160) cells as well as non-Pgp MDR human small-cell lung carcinoma GLC4/ADR cells . With increasing extracellular DN concentrations, saturation of the VCR-induced DN influx was observed (DN medium concentration 2.5 microM at 1/2 Vmax) . At an extracellular DN concentration of 5 microM, higher concentrations of VCR were needed to reach the maximum effect in 2R120 cells than at 0.5 microM DN . This is an indication of competitive interaction between DN and VCR for the putative DN efflux system . In summary, we found indications of inhibition of active DN efflux by VCR and DN efflux against a concentration gradient in non-Pgp MDR 2R120 and GLC4/ADR cells . These features are consistent with the presence of a multidrug transporter, different from Pgp, in the plasma membrane of these cells.

Int J Cancer, 1994 Oct 15, 59(2), 208 - 11
Induction of multidrug resistance (MDR) by transfection of MCF-10A cell line with c-Ha-ras and c-erbB-2 oncogenes; Sabbatini AR et al.; To investigate the relationship between oncogene activation and appearance of multidrug resistance (MDR) we transfected the human breast epithelial cell line MCF-10A, negative for the expression of the P-glycoprotein, with c-Ha-ras and/or c-erbB-2 oncogenes . The appearance of the MDR phenotype was then studied by evaluating mdr-1 mRNA expression, the presence of P-glycoprotein on the cell membrane and the onset of doxorubicin resistance, together with the effect of the reversing agent verapamil . We found that only MCF-10A transfected with both c-Ha-ras and c-erbB-2 oncogenes acquired the MDR phenotype.

Cancer Res, 1994 Oct 15, 54(20), 5368 - 73
Effects of amiodarone, cyclosporin A, and PSC 833 on the cytotoxicity of mitoxantrone, doxorubicin, and vincristine in non-P-glycoprotein human small cell lung cancer cell lines; van der Graaf WT et al.; The multidrug resistance (MDR) modulators amiodarone (AM), cyclosporin A (CsA), and PSC 833 were tested for their potential to modulate cytotoxicity of doxorubicin (DOX), vincristine (VCR), and mitoxantrone (MX) in a sensitive human small cell lung carcinoma cell line GLC4, in its DOX-resistant non-P-glycoprotein subline GLC4-Adr, and in its cisplatin-resistant subline GLC4-CDDP . GLC4-Adr, in which overexpression of the so-called multidrug resistance-associated protein has been demonstrated, is 91-fold resistant for DOX, 22-fold for VCR, and 7.5-fold for MX, compared with its sensitive cell line . AM previously modulated DOX and VCR resistance in the P-glycoprotein-positive human colon cancer cell line COLO 320 . Cytotoxicity was studied in the microtiter well tetrazolium assay . In the small cell lung carcinoma cell lines described above, AM did not increase cytotoxicity of DOX, but increased VCR cytotoxicity; moreover, AM was shown to be a potent modulator of MX cytotoxicity . CsA did not potentiate DOX cytotoxicity, but, at a concentration of 4 microM, it modestly increased VCR cytotoxicity in GLC4 . However, 0.8 and 4.0 microM CsA protected against MX cytotoxicity in GLC4 and GLC4-CDDP, but no effect was observed in GLC4-Adr . At the much higher ID10 concentration CsA modulated MX cytotoxicity 1.6-fold in GLC4-Adr and slightly in GLC4 and GLC4-CDDP . PSC 833, a nonimmunosuppressive CsA analogue, did not alter the cytotoxicity of DOX or MX in these cell lines, but potentiated VCR cytotoxicity in GLC4-Adr at a concentration of 0.4 microM . The modulation of MX cytotoxicity by AM and the protection by CsA was confirmed in a clonogenic assay . In the colony-forming unit granulocyte-monocyte assay, no additional MX toxicity on normal bone marrow by AM was observed . Flow cytometry of cellular MX fluorescence was performed in order to elucidate the mechanism behind the AM-induced increased MX cytotoxicity . This revealed an increase in cellular MX after 1-h incubation of MX combined with AM and an inhibition of efflux from GLC4 and GLC4-Adr; CsA and PSC 833 had no effect on MX efflux . An increase in MX-induced cleavable complexes by AM in GLC4 was observed using the K+/sodium dodecyl sulfate coprecipitation assay, but no effect of CsA was found . In conclusion, AM enhances MX and VCR cytotoxicity in these sensitive, non-P-glycoprotein DOX and cisplatin-resistant small cell lung carcinoma cell lines . It also inhibits efflux of MX and causes more MX-induced cleavable complexes.

Arch Intern Med, 1994 Oct 10, 154(19), 2161 - 7
Tuberculosis susceptibility patterns, predictors of multidrug resistance, and implications for initial therapeutic regimens at a New York City hospital; Weltman AC et al.; BACKGROUND: Multidrug resistance has complicated tuberculosis therapy . We studied antibiotic susceptibilities of Mycobacterium tuberculosis and predictors of multidrug resistance to assist in determining initial drug regimens . METHODS: We conducted a case-control study based on chart review of patients with and without multidrug-resistant tuberculosis, including outpatients and inpatients with culture-proved tuberculosis seen at a large New York, NY, hospital during 1991 and 1992 . Patient characteristics studied included serologic findings for human immunodeficiency virus and the presence of the acquired immunodeficiency syndrome . Descriptive analysis considered potential initial drug regimens . A theoretically effective regimen was assumed to contain at least two drugs to which an isolate was susceptible . RESULTS: For 172 patients, 28.5% of isolates were resistant to isoniazid, at least 20.9% to rifampin, 15.7% to ethambutol, 8.1% to pyrazinamide, 18.6% to streptomycin, 9.9% to ethionamide, 8.1% to kanamycin, and none to capreomycin, cycloserine, and ciprofloxacin; 18.6% were resistant to both isoniazid and rifampin . Chart review of 159 patients showed that acquired immunodeficiency syndrome, human immunodeficiency virus seropositivity, female gender, residence in the Bronx, and race were associated with multidrug resistance . The four-drug regimen of isoniazid, rifampin, ethambutol, and pyrazinamide was theoretically effective for 81% to 85% of patients . No subset of patients would have a markedly better theoretical benefit from that regimen . Only five- or six-drug regimens that used the combinations of capreomycin plus ciprofloxacin, capreomycin plus cycloserine, ciprofloxacin plus cycloserine, or all three drugs together theoretically offered significantly higher effectiveness . CONCLUSIONS: Tuberculosis isolates at our hospital have a high frequency of multidrug resistance . Only five- or six-drug regimens are theoretically adequate as initial therapy for our patients.

Biochem Pharmacol, 1994 Oct 7, 48(7), 1437 - 45
Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells . Activation of P-glycoprotein as a putative mechanism; Critchfield JW et al.; Since P-glycoprotein (P-gp) in normal tissues may serve as a cellular defense mechanism against naturally occurring xenobiotics, we considered whether physiologically active components of commonly ingested plant foods could influence P-gp function . To examine this possibility, a series of flavonoids commonly found in plant foods was tested for their ability to modulate {14C}Adriamycin ({14C}ADR) accumulation and efflux in P-gp-expressing HCT-15 colon cells . Many flavonoids, in the micromolar range, inhibited the accumulation of {14C}ADR . Detailed experiments utilizing flavonoids with the greatest activity in reducing {14C}ADR accumulation, i.e . galangin, kaempferol, and quercetin, revealed that the efflux of {14C}ADR is increased markedly in the presence of these compounds . Flavonoid-induced stimulation of efflux was rapid and was blocked by the multidrug-resistant (MDR) reversal agents verapamil, vinblastine, and quinidine . The magnitude of flavonoid-stimulated efflux in sodium butyrate-treated cells with a 4-fold induction of P-gp protein was similar to that in uninduced cells . {3H}Azidopine photoaffinity labeling of P-gp in crude membrane preparations revealed mild to no competition for binding by flavonoids possessing either activity or inactivity in reducing ADR accumulation . Although flavonoid hydrophobicity was found to be unrelated to flavonoid activity in altering {14C}ADR accumulation, certain structural features were associated with enhancement or diminution of activity . Finally, the significance of flavonoid-related reduction of {14C}ADR accumulation was underscored in cell growth studies, showing partial protection by quercetin against ADR-induced growth inhibition . It is concluded that certain naturally occurring plant flavonoids may acutely upregulate the apparent activity of P-gp.

FEBS Lett, 1994 Oct 3, 352(3), 380 - 4
Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells; Scaggiante B et al.; The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells . A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene . By administering 10 microM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB100 . This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

Hautarzt, 1994 Oct, 45(10), 678 - 84
{Drug resistance of malignant melanoma . Mechanisms and possible modulation}; Schadendorf D et al.; Response rates of metastatic malignant melanoma to cytostatic treatment are disappointingly low . Although immunomodulators such as interferons are more commonly being used in combination with cytostatics, no major breakthrough has been achieved . The mechanisms underlying the high chemoresistance of melanoma cells are so far ill-defined, and investigations are only just being initiated . Several mechanisms of chemoresistance have, however, been studied with other tumours and might be relevant for human melanoma: (1) "Classical" multidrug resistance, determined by the expression of the p-glycoprotein which resembles a membrane pump that eliminates natural and synthetic agents from the cell interior . Different drugs, including calcium antagonists, interfere with its function and can thus modulate chemoresistance . Preliminary data from investigations of these mechanisms indicate that p-glycoprotein is not, however, involved in the multidrug resistance of malignant melanoma . (2) Detoxification, involving glutathione-S-transferases (GST) . GST are a multigene family of enzymes which inactivate alkylating agents by conjugation to glutathione . Their relevance for chemoresistance in melanoma has not yet been clarified . (3) Topoisomerase II, which is involved in DNA recombination and DNA transcription events and represents the target of several inhibitory cytotoxic agents . Low levels of the enzyme render cells resistant to the action of specific drugs . Again nothing is yet known regarding the relevance of this mechanism in human melanoma . Further studies of these potentially important resistance mechanisms are thus urgently needed in order to develop more effective therapies for advanced malignant melanoma.

Mol Pharmacol, 1994 Oct, 46(4), 677 - 84
Mechanisms of resistance to ansamycin antibiotics in human breast cancer cell lines; Benchekroun MN et al.; We recently reported that multidrug-resistant, P-170 glycoprotein-positive, Adriamycin-selected, human breast tumor (MCF7/ADRR) cells were resistant to the benzoquinonoid ansamycin antibiotics geldanamycin (GL) and herbimycin A (HA) and that significantly fewer hydroxyl radicals were formed in resistant cells . We have carried out additional studies to define the mechanisms of cytotoxicity of and resistance to GL and HA, by directly examining the interactions of these drugs with P-170 glycoprotein using photoaffinity labeling . We found that both GL and HA inhibited binding of azidopine to P-170 glycoprotein in a dose-dependent manner . We have developed a 10-fold GL-resistant cell line (MCF7/GLR) by continuous drug exposure . Our studies indicated no significant differences in free radical formation between wild-type MCF7 cells and MCF7/GLR cells . Uptake and efflux studies indicated a small decrease in the GL accumulation but no difference in the efflux of GL in these cells . Verapamil had no effect on cellular accumulation of GL in wild-type MCF7 cells or MCF7/GLR cells . Verapamil significantly increased the accumulation of GL in MCF7/ADRR cells and enhanced GL cytotoxicity 12-fold, suggesting that GL interacted with the P-170 glycoprotein . Using reverse transcription-polymerase chain reaction, we found no expression of the mdr1 gene; however, expression of the multidrug resistance-associated protein was about 2-fold higher in MCF7/GLR cells . Taken together, these studies indicate that the mechanisms of GL resistance are multifactorial . Although decreased free radical formation may not play a significant role in low levels of GL resistance, e.g., in MCF7/GLR cells, both overexpression of mdr1 and decreased free radical formation contribute to GL resistance in highly resistant cells such as MCF7/ADRR cells.

Mol Pharmacol, 1994 Oct, 46(4), 627 - 38
Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen; Lee JS et al.; Fifty-eight cell lines in the National Cancer Institute drug screen were analyzed for their ability to efflux the fluorescent dye rhodamine 123 as a functional assay for P-glycoprotein (Pgp) . Using flow cytometry, the rhodamine fluorescence was measured for each cell line under four incubation conditions, i.e., after accumulation in the presence or absence of the Pgp antagonist cyclosporin A and after efflux in rhodamine-free medium in the presence or absence of cyclosporin A . The results in some cell lines were compatible with Pgp-mediated efflux . There was a significant correlation between mdr-1 expression and rhodamine efflux in the 58 cell lines (r = 0.788, p = 0.0001) . Using the rhodamine efflux data as a seed for COMPARE analysis with the cytotoxicity data on > 30,000 compounds in the National Cancer Institute drug screen database, hundreds of compounds with high correlation coefficients were identified . Selected compounds were tested for reversal of cross-resistance in a multidrug-resistant cell line . A high degree of reversibility, up to 10,000-fold, for some of the compounds was noted in the presence of the Pgp antagonist PSC 833 . This finding suggested that compounds with predominately Pgp-mediated resistance were being identified . Using these compounds as seeds for COMPARE analysis against a more restricted database of 187 standard agents, a series of standard compounds were repeatedly identified as having high correlation coefficients with the newly identified Pgp substrates . These standard agents, including phyllanthoside, bisantrene, and homoharringtonine, constitute an mdr-1 profile . New agents identified as being highly correlated with these compounds may benefit from clinical trials with Pgp antagonists.

Lab Invest, 1994 Oct, 71(4), 595 - 603
A sensitive multilayer immunoalkaline phosphatase method for detection of P-glycoprotein in leukemic and tumor cells in the bone marrow; Haddad G et al.; BACKGROUND: Relatively low levels of the multidrug resistance P-glycoprotein have correlated with poor prognosis in rhabdomyosarcoma, neuroblastoma, acute myelogenous leukemia, lymphoma, myeloma and breast carcinoma . A sensitive, nonradioactive method, less costly and time-consuming than the present molecular biologic techniques, is desirable for direct measurement of P-glycoprotein in tumor cells versus normal cells . EXPERIMENTAL DESIGN: We have devised an immunoalkaline phosphatase method using four antibody layers to amplify the primary signal considerably and refined staining conditions to optimize the 'signal-to-noise' ratio . Immunoalkaline phosphatase is preferred to immunoperoxidase for testing leukemic and tumor cells in bone marrow, because it avoids myeloperoxidase staining in myeloblasts and myeloid progenitors that interferes with P-glycoprotein interpretation . RESULTS: Multilayer immunoalkaline phosphatase detected low levels of P-glycoprotein overexpression, that could not be identified by conventional immunoperoxidase or immunoblot, in a low-resistance (8-fold) cell line with a barely detectable transcript . Our technique also detected increased P-glycoprotein in malignant cells in bone marrow of relapsed acute lymphoblastic leukemia (10/11), acute myelogenous leukemia (2/2), lymphoma (1/1), neuroblastoma (7/7), and rhabdomyosarcoma (2/2) . Increased P-glycoprotein was not identified at diagnosis in 6 patients with acute lymphoblastic leukemia, and two with stage IV and three with stage IVS neuroblastoma that remained relapse-free in the long-term, but was detected in 4 patients with stage IV neuroblastoma and three with rhabdomyosarcoma who ultimately relapsed . CONCLUSIONS: Our new technique is more sensitive than conventional immunoperoxidase and immunoblot for assaying P-glycoprotein in low-resistance cell lines . It may be potentially applicable for detecting low levels of P-glycoprotein overexpression in leukemic and tumor cells in bone marrow . Early identification of low levels of multidrug resistance may be clinically relevant by allowing poor-prognostic patients to receive alternative therapy.

Ann Hematol, 1994 Oct, 69(4), 159 - 71
P-glycoprotein-mediated multidrug resistance in normal and neoplastic hematopoietic cells; Licht T et al.; The multidrug transporter, P-glycoprotein (P-gp), is expressed by CD34-positive bone marrow cells, which include hematopoietic stem cells, and in other cells in the bone marrow and peripheral blood, including some lymphoid cells . Multidrug resistance mediated by P-gp appears to be a major impediment to successful treatment of acute myeloid leukemias and multiple myelomas . However, the impact of P-gp expression on prognosis has to be confirmed in several other hematopoietic neoplasms . The role of P-gp in normal and malignant hematopoiesis and clinical attempts to circumvent multidrug resistance in hematopoietic malignancies are reviewed . The recent transduction of the MDR1 gene into murine hematopoietic cells, which protects them from toxic effects of chemotherapy, suggests that MDR1 gene therapy may help prevent myelosuppression following chemotherapy.

Am J Physiol, 1994 Oct, 267(4 Pt 1), C1095 - 102
ATP-activated chloride channel inhibited by an antibody to P glycoprotein; Zhang JJ et al.; In this report, we present the characteristics of a Cl- channel found in lens fiber cells . The single channel has a conductance of 17 pS, a linear current-voltage curve, is activated by ATP or strong depolarization and is blocked by verapamil, quinidine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 5-nitro-2-(3- phenylpropylamino)benzoate, dideoxyforskolin, and tamoxifen . These properties are similar to those reported for a volume-activated Cl- channel associated with the multidrug resistance (MDR) gene product, P glycoprotein (24) . Confirming this connection, we demonstrate that our lens Cl- channel is inhibited by an antibody to P glycoprotein . The data we present here may, therefore, be the first characterization of the single channel activity of the Cl- channel associated with P glycoprotein.

J Neurosurg, 1994 Oct, 81(4), 587 - 94
Reversal of chemoresistance in malignant gliomas by calcium antagonists: correlation with the expression of multidrug-resistant p-glycoprotein; Kiwit JC et al.; Resistance to multiple drugs is often observed in malignant gliomas . The authors used a microtiter tetrazolium test to analyze primary in vitro chemoresistance and chemosensitivity of 15 early cultures of human malignant glioma exposed to 50 micrograms/ml (1,4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a (ACNU), 50 micrograms/ml cisplatin, 1 microgram/ml vincristine, or combinations of these chemotherapeutic agents . Primary chemoresistance was observed in 87% of tumors for ACNU, in 87% for cisplatin, and in 83% for vincristine . All tumors were examined for expression of multidrug-resistant p-glycoprotein, a transport protein of 170,000 D, by means of immunohistochemical staining with the JSB-1 antibody on paraffinized tumor sections . Eight of 15 specimens (53%) showed positive staining for the monoclonal antibody . Primary chemoresistance was overcome by addition of the calcium antagonists verapamil or nimodipine to the cultures if the original tumor expressed p-glycoprotein (p < 0.01 for verapamil, p < 0.05 for nimodipine) . In tumors not expressing p-glycoprotein, addition of calcium antagonists to the cell cultures did not influence primary chemoresistance . It is concluded from these data that addition of calcium antagonists to the adjuvant chemotherapy of malignant gliomas might overcome primary chemoresistance in tumors expressing the multidrug-resistant phenotype.

J Infect Dis, 1994 Oct, 170(4), 971 - 7
Treatment of multidrug-resistant Plasmodium falciparum malaria with 3-day artesunate-mefloquine combination; Nosten F et al.; Studies of 652 adults and children with acute uncomplicated falciparum malaria were done to determine the optimum treatment of multidrug-resistant Plasmodium falciparum malaria on the Thai-Burmese border . Single-dose artesunate (4 mg/kg) plus mefloquine (25 mg of base/kg) gave more rapid symptomatic and parasitologic responses than high-dose mefloquine alone but did not improve cure rates . Three days of artesunate (total dose, 10 mg/kg) plus mefloquine was 98% effective compared with a 28-day failure rate of 31% with high-dose mefloquine alone (relative risk {RR}, 0.06; 95% confidence interval {CI}, 0.02-0.2; P < .0001) . By day 63, the reinfection adjusted failure rates were 2% and 44%, respectively (P < .0001) . Artesunate also prevented high-grade failures . Both drugs were well tolerated . No adverse effects were attributable to artesunate . Vomiting was reduced significantly by giving mefloquine on day 2 of treatment (RR, 0.40; 95% CI, 0.20-0.79; P = .009 . Artesunate (10 mg/kg over 3 days) plus mefloquine (25 mg/kg) is currently the most effective treatment for falciparum malaria in this area of increasing mefloquine resistance.

Int J Cancer, 1994 Oct 1, 59(1), 83 - 93
Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin; Ujhazy P et al.; Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13) . Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme . Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT . Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines . The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT . All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment . These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active . The parental and MDR cells did not differ, in general, in their sensitivity to adenosine . An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin . The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.

Int J Cancer, 1994 Oct 1, 59(1), 133 - 40
Myeloid and lymphoid cell alterations in normal mice exposed to chemotherapy with doxorubicin and/or the multidrug-resistance reversing agent SDZ PSC 833; Froidevaux S et al.; The cyclosporin SDZ PSC 833 (PSC) is a potent in vivo chemosensitizer for tumor cells with P-glycoprotein(Pgp)-dependent multidrug resistance (MDR) . However, Pgp expression also occurs in CD8+ T cells, NK cells, macrophages and stem cells . In order to find whether PSC might display specific myelotoxicity or potentiate the toxicity of anti-cancer drugs, healthy mice were exposed to single doxorubicin (DOX) and combined (DOX + PSC) chemotherapy protocols known to be near or above the borderline of toxicity for tumor-bearing mice . Mice treated with DOX alone or with (DOX + PSC) showed transient spleen hypoplasia, with a general decrease of all leucocyte lineages and a persistent fall in the numbers of B cells in the bone marrow . In (DOX + PSC)-treated mice, PSC only potentiated the DOX effects without inducing specific depletions of the Pgp-expressing leukocytes (CD8+ and Mac-I+ cells) . Hematopoietic cell grafts from normal mice to (DOX +/- PSC)-treated mice did not correct their B-cell lineage deficiency . When lethally irradiated mice were rehabilitated with hematopoietic cells from (DOX +/- PSC)-treated mice (including those with very reduced survival), all chimeras survived for at least 8 months after the cell graft, at which time their leucocyte population profiles were similar to those of control chimeras.

Cancer Res, 1994 Oct 1, 54(19), 5036 - 40
Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and overexpression of the N-myc oncogene in childhood neuroblastoma; Bordow SB et al.; The MRP gene (Cole et al., Science (Washington DC), 258: 1650-1654, 1992) encodes a membrane-bound glycoprotein the expression of which correlates with non-P-glycoprotein-mediated multidrug resistance in a variety of cultured human cell lines . Using an RNA-polymerase chain reaction assay, expression of this gene was examined in the highly chemoresistant pediatric malignancy, neuroblastoma . MRP expression was observed in 5 human neuroblastoma cell lines and in all 25 primary neuroblastoma tumors of stage I through IVS . Tumors with amplification of the N-myc oncogene were found to have significantly higher MRP expression that those with no amplification (P = 0.0016) . Expression of the MRP gene in the tumor specimens was highly correlated with expression of the N-myc gene (P = 0.0009), while expression of the MDR1 gene, encoding P-glycoprotein, was not related to expression of either the N-myc or MRP genes . Decreased expression of the N-myc oncogene in neuroblastoma cell lines SH-SY5Y and BE(2)-C, following treatment with retinoic acid, was paralleled by down-regulation of MRP gene expression, contrasting with increased expression of the MDR1 gene . Expression of the MRP gene is thus common in both primary neuroblastoma tumors and cultured cell lines, and correlates with amplification and overexpression of the N-myc oncogene, which is central to the malignant phenotype of this disease.

Cancer Res, 1994 Oct 1, 54(19), 5029 - 32
Volume-activated chloride current is not related to P-glycoprotein overexpression; Dong Y et al.; It has been suggested that P-glycoprotein (P-gp), an ATP-dependent transporter responsible for classical multidrug resistance, is also a volume-regulated chloride channel . We reexamined this hypothesis by use of whole-cell patch clamp recordings of three matched pairs of cell lines, which were either drug-sensitive or drug-resistant due to P-gp overexpression . We demonstrate here that volume-regulated chloride-selective currents can be induced in cells with or without P-gp expression . Overexpression of either P-gp or cystic fibrosis transmembrane conductance regulator, the protein product of the CF gene and another member of the ATP-dependent transporters, is associated with a hypotonicity-induced, rapid onset, transient current prior to onset of the volume-sensitive chloride-selective current, an apparent nonspecific effect related to the overexpression of an integral membrane protein . These results suggest that there is no relationship between P-gp and the chloride channel activated by cell swelling.

Curr Genet, 1994 Oct, 26(4), 285 - 94
Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance; Hirata D et al.; A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified . Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs . The Ydr1 protein bears the highest similarity to the S . cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO . The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities . YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance . The two genes had overlapping specificities toward staurosporine and fluphenazine . The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters . The transcription of these genes was also increased by heat shock . The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.

Occup Med, 1994 Oct-Dec, 9(4), 695 - 721
Medical surveillance for workers exposed to tuberculosis; Moline JM et al.; This detailed discussion of medical surveillance techniques addresses such issues as the administration and interpretation of the tuberculin skin test, the importance of BCG vaccine, preventive therapy with isoniazid, the identification of groups at high risk for TB, multidrug-resistant tuberculosis, and regulatory requirements for PPD testing, including CDC guidelines.

Biochem Mol Biol Int, 1994 Oct, 34(4), 773 - 80
Etoposide-resistance in the multidrug-resistant LZ-8 cells; Liengswangwong V et al.; The multidrug-resistant LZ-8 cells were found to exhibit marked resistance to etoposide compared to wild-type, parental V79 cells . The multidrug resistant phenotype did not significantly contribute to this etoposide-resistance . Following exposure of LZ-8 cells and V79 cells to equivalent concentrations of etoposide, there was a dramatic reduction in the number of etoposide-induced stabilized DNA-topoisomerase II complexes in the LZ-8 cells compared to V79 cells, however, this reduction was not found when nuclei isolated from LZ-8 and V79 cells were exposed to equivalent concentrations of etoposide . These results suggest that cytoplasmic factors are involved in the etoposide-resistance of LZ-8 cells.

Acta Med Okayama, 1994 Oct, 48(5), 249 - 55
Immunohistochemical analysis of P-glycoprotein expression in diverse histological types of epithelial ovarian tumors; Kodama J et al.; P-glycoprotein is a transmembrane protein which acts as an energy-dependent drug efflux pump for a variety of anti-cancer drugs . The mdr-1 gene which encodes P-glycoprotein was successfully cloned in 1986 . To investigate P-glycoprotein expression in diverse ovarian tumors, including benign, low malignant potential and malignant, immunohistochemical study was done using a monoclonal antibody (C 219) . Overall, 8 out of the 59 epithelial ovarian tumors (13.6%) expressed P-glycoprotein . It was noted that 5 of the 12 mucinous tumors were found to express P-glycoprotein, while none of the 31 serous tumors were immunohistochemically positive . In 10 malignant ovarian tumors, P-glycoprotein immunostaining was examined both prior to and after chemotherapy . Nine of them did not express any P-glycoprotein before or after chemotherapy . However, one tumor expressed P-glycoprotein after six courses of multidrug resistance-related drug administration . These findings indicate that P-glycoprotein expression is not so common in ovarian tumors, regardless of their malignant potential . Nevertheless, the results suggest a strong association between P-glycoprotein expression and certain histological cell types in epithelial ovarian tumors . It is also possible that P-glycoprotein appears as a result of chemotherapy, but such a phenomenon can not occur unless chemotherapy is administered at high doses for a long period of time.

J Chemother, 1994 Oct, 6(5), 343 - 8
Effect of buthionine sulfoximine on the sensitivity to doxorubicin of parent and MDR tumor cell lines; Crescimanno M et al.; We have studied the interaction of glutathione-depleting concentrations of buthionine sulfoximine (BSO) with the anti-proliferative activity of doxorubicin (DXR) in three tumor lines, the mouse B16 melanoma . Friend erythroleukemia and the human K562 leukemia, both as DXR-sensitive and-resistant (with typical multidrug resistance) variants . BSO significantly enhanced the DXR effects in the wild-type Friend and K562 leukemias, and especially in the drug-resistant subline of Friend leukemia . BSO did not modify DXR accumulation and retention in the latter clone . Moreover, neither BSO nor verapamil used alone completely reversed the resistance to DXR of this cell line; their combination was more efficient and increased its drug sensitivity to a level closer to that of the parental counterpart . These results seem to indicate that the status of glutathione and of the enzymes related to it contributes to the resistance of Friend leukemia to DXR . An interesting additional finding was that BSO significantly synergizes with the antiproliferative effects of vincristine in the drug-sensitive variants of Friend and K562 leukemias.

Anticancer Drugs, 1994 Oct, 5(5), 598 - 600
Effects of lovastatin on a human myeloma cell line: increased sensitivity of a multidrug-resistant subline that expresses the 170 kDa P-glycoprotein; Holmberg M et al.; Using a fluorometric microculture cytotoxicity assay for measuring cell viability and proliferation we examine the cytotoxic effect of lovastatin on a drug sensitive myeloma cell line (RPMI 8226) and a multidrug resistant (MDR) clone (8226/Dox40), that was approximately 100-fold less sensitive to doxorubicin . The RPMI 8226 cells were sensitive to lovastatin with an IC50 of 15.8 micrograms/ml . However, the MDR subline exhibited a collateral sensitivity to lovastatin, with an IC50 of 1.7 microM, thus having a 9.3-fold greater sensitivity to lovastatin than the parental cell line . The combination of doxorubicin and lovastatin did not show any synergistic or antagonistic effects on any of the cell lines . The increased sensitivity to lovastatin of the P-gp 170-expressing MDR cells 8226/Dox40 might be part of a more general phenomenon that merits further investigation.

Trends Microbiol, 1994 Oct, 2(10), 407 - 11
Microbial multidrug-resistance ABC transporters; Ouellette M et al.; Multidrug resistance in tumor cells is often caused by the increased efflux of a wide variety of drugs, mediated by P glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters . The genes encoding members of this superfamily have also been isolated from drug-resistant microorganisms, and the role of microbial ABC transporters in drug resistance is being investigated.

Cell Growth Differ, 1994 Oct, 5(10), 1145 - 52
Enhanced expression by the brain matrix of P-glycoprotein in brain capillary endothelial cells; Tatsuta T et al.; P-glycoprotein (PGP), an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in brain capillary endothelium and could be functionally involved in the blood-brain barrier . To study the regulatory mechanism of PGP expression in brain capillary endothelium, various mouse tissue matrices were tested for their abilities to enhance the expression of PGP in mouse brain capillary endothelial cells (MBEC), which express relatively small amounts of PGP . Of the four tissue matrices we examined, PGP expression in MBEC cultured on the brain matrix increased 2.0-fold . The PGP-inducing activity was similarly detected in bovine brain matrix, and the activity was enriched in the fraction of pl 9.0 by isoelectric focusing . The fraction, named PIC-fraction (PGP-inducing component), increased the PGP expression in MBEC 3.5-fold . By Northern blot analysis, a 3.3-fold enhancement of mdr gene expression was observed in MBEC cultured on the PIC-fraction . The PGP-inducing activity of the PIC-fraction was reduced by the treatment with trypsin but not with collagenase, suggesting that a proteinaceous factor distinct from type I collagen might be responsible for the PGP-inducing activity of PIC-fraction . Although the PIC-fraction increased the PGP expression in other mouse brain capillary endothelial cells, the PIC-fraction did not increase PGP expression in mouse aortic endothelial cells and KB carcinoma cell lines expressing various amounts of PGP . These observations suggest that PGP expression in brain capillary endothelium is specifically regulated by a tissue-specific factor in the brain matrix.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2492 - 4
Loss of function mutation in the yeast multiple drug resistance gene PDR5 causes a reduction in chloramphenicol efflux; Leonard PJ et al.; The yeast (Saccharomyces cerevisiae) PDR5 gene product encodes a 160-kDa protein related to the large ABC family of transporters, including the human MDR1 multidrug resistance p-glycoprotein . Loss of function mutations in PDR5 result in chloramphenicol hypersensitivity . A pdr5::Tn5 loss of function mutant exhibits a markedly impaired efflux of chloramphenicol compared with that of an isogenic PDR5 (wild-type) control.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2380 - 6
Characterization of rifampin-resistance in pathogenic mycobacteria; Williams DL et al.; The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases . Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens . It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species . DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M . tuberculosis from diverse geographical regions throughout the world . In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed . No mutations were observed in rifampin-susceptible strains . No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M . tuberculosis strains . Drug-resistant M . tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene . However, mutations are not correlated with IS6110 profiling outside of epidemics . The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment . Rifampin-resistant strains of M . leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M . tuberculosis . This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M . tuberculosis.

Ann Oncol, 1994 Oct, 5(8), 733 - 9
Biochemical modulation of 'classical' multidrug resistance by BIBW22BS, a potent derivative of dipyridamole; Jansen WJ et al.; BACKGROUND: Modulators of the 'classical' multidrug resistance (mdr) phenotype have low efficacy in patients with solid tumors . We analyzed BIBW22BS, 4-{N-(2-hydroxy-2-met- hyl-propyl)-ethanolamino}-2,7-bis(cis-2,6-dimethyl-morpho- lino)-6-phenylpteridine, a derivative of dipyridamole, for its higher potential to modulate mdr . MATERIALS AND METHODS: Four human malignant cell lines: BRO, A2780, GLC4, SW1573, the Pgp-positive sublines: BRO/mdr1.1, 2780AD and the non-Pgp sublines: GLC4/ADR, SW1573/2R120 were used in vitro to investigate BIBW22BS as a modulator of the antiproliferative effects of vincristine and doxorubicin and to compare the potency of BIBW22BS with that of dipyridamole, verapamil, bepridil and flunarizine . BRO/mdr1.1 s.c . well-established xenografts in nude mice were used to study the modulating properties of BIBW22BS 50 mg/kg i.v . followed after one h by vincristine 1 mg/kg i.p . or doxorubicin 8 mg/kg i.p . weekly x 2 . RESULTS: BIBW22BS was 20- to 100-fold more potent than dipyridamole in the reversal of resistance in the Pgp-positive sublines . Reversal of resistance was obtained in a dose-dependent manner and was complete at concentrations of 0.5-2.5 microM . At non-toxic, equimolar concentrations of 1.0 microM BIBW22BS showed higher modulating potency than the calcium-channel blockers . BIBW22BS did not affect resistance in the non-Pgp sublines . BRO/mdr1.1 s.c . xenografts have stable multidrug-resistance characteristics upon serial transplantation . BIBW22BS, vincristine, or doxorubicin as single agents were not effective in vivo, while the addition of BIBW22BS could significantly reduce the tumor growth expressed as the T/C% of vincristine from 109% to 48% and that of doxorubicin from 55% to 32% . However, reversal of vincristine resistance in BRO/mdr1.1 xenografts was not complete when compared to the efficacy of vincristine in BRO xenografts . CONCLUSION: The results encourage the further preclinical development of BIBW22BS as a modulator of 'classical' multidrug resistance in cancer patients.

Minerva Pediatr, 1994 Oct, 46(10), 463 - 70
{Use of cyclosporin and verapamil in association with chemotherapy in the treatment of pediatric patients with advanced-stage neoplasms . A pilot study}; Miniero R et al.; Multidrug resistance represents one of the most important factors that may lead to a therapeutic failure in some patients affected by malignancies . One of the best known mechanisms is linked to the genic amplification or the overproduction of a membrane glycoprotein, GP170, that is the product of the gene MDR1 . The existence of drugs (calcium blockers, cyclosporine, tamoxifen, reserpine, quinidine) able to bind themselves to gp170 and to paralyze its activity in vitro is well known . We studied 20 pediatric patients (median age 9 years) affected by acute lymphoblastic leukemia (ALL), osteosarcoma, neuroblastoma and medulloblastoma, in advanced stage of disease . We employed in all cases the association of cytostatics with verapamil (50-70 mg/m2 i.v.) and cyclosporine (5-8 mg/kg i.v.) with different infusion schedules . In leukemias we administered vincristine (1.5 mg/m2), and daunomycin (40 mg/m2), in solid tumors VP16 (150 mg/m2) and adriamycin (60 mg/m2) . Seventy-two therapeutic courses were performed: 39 in ALL, 16 in osteosarcoma, 16 in neuroblastoma and 1 in medulloblastoma . On the whole 5 complete remissions were achieved in ALL patients and 1 in an osteosarcoma patient . We did not observe a significant myelosuppression during treatment, therefore few infectious complications occurred; furthermore electrocardiographic changes have been mild and promptly resolved after temporary discontinuation of verapamil infusion . Our data suggest a synergy of verapamil and cyclosporine in the inhibition of multidrug resistance induced by gp170, without the occurrence of heavy toxicity . The results obtained in ALL patients are encouraging., especially in view of a possible subsequent bone marrow transplantation, while in solid tumors they are not as satisfying.

Br J Haematol, 1994 Oct, 88(2), 348 - 56
High expression of the multidrug resistance-associated protein (MRP) in chronic and prolymphocytic leukaemia; Burger H et al.; The expression of the multidrug resistance-associated protein (MRP), a new glycoprotein involved in drug resistance, was investigated in tumour samples from 80 patients with chronic B-cell malignancies by a quantitative RNase protection assay . In B-cell chronic lymphocytic leukaemia (B-CLL) (n = 32), either treated (n = 18) or untreated (n = 14), a high percentage of patients (20/32: 63%) had relatively high expression levels of the MRP gene (25U or more) . In addition, hyperexpression of the MRP gene was demonstrated in 4/10 (40%) untreated patients with B-cell prolymphocytic leukaemia (B-PLL) . In contrast, low MRP mRNA expression levels were detected in hairy cell leukaemia (n = 7), non-Hodgkin's lymphoma (n = 13) and multiple myeloma (n = 18) . Statistical analysis of MRP expression in untreated CLL (mean +/- SD 29.2 +/- 18.5 U) versus treated CLL (mean +/- SD 26.7 +/- 13.7 U) did not show significant differences in MRP expression between the two groups . Southern blot analysis did not reveal amplification of the MRP gene in the leukaemia samples with elevated MRP mRNA levels . We conclude that B-PLL and B-CLL frequently display high MRP expression and that this hyperexpression is probably due to transcriptional activation and/or increased mRNA stability.

Br J Haematol, 1994 Oct, 88(2), 318 - 24
Retrovirus-mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells; Bertolini F et al.; We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer . Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line . Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol . In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells . Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors . (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction . MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls) . (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene . After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells . (4) Cytochemical localization by APAAP staining of P-glycoprotein . No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane . In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Bull Cancer, 1994 Oct, 81(10), 894 - 6
{Modulation of adriamycin cytotoxicity on K 562 and K562adri cells by interferon alpha and/or all-trans-retinoic acid}; Dufour P et al.; Multidrug Resistant (MDR) plays a major role in chemoresistance . Alpha Interferon (IFN) and all trans retinoic acid (ATRA) have antiproliferative effect and IFN regulates several genes, some of them implicated in the regulation of MDR gene expression . We have studied the modulations of adriamycin cytotoxicity by IFN and/or ATRA on K 562 (MDR-) and resistant to adriamycin K 562 adri (MDR+) cell lines . We observed an important increase of adriamycin cytotoxicity on both K 562 and K 562 adri by low dose of IFN and ATRA . Studies of MDR gene expression shows an increase in K 562 adri after exposure to IFN or ATRA . So the observed effect is not due to a down regulation of MDR gene expression but probably to the own antiproliferative effect of IFN and ATRA in combination with the cytotoxicity of adriamycin.

Bull Cancer, 1994 Oct, 81(10), 891 - 3
{Characterization of the mechanism of cross-resistance to vinca alkaloids and taxoids in the human J82 bladder tumor cell line}; Debal V et al.; A phenotype of resistance to the new vinca alkaloid Navelbine was induced in the J82 human bladder carcinoma cells . The resistance factor of the resistant cell line (J82-NVB) to Navelbine was 17 . The resistance phenotype of these cells is not a multidrug-resistance (MDR) phenotype . J82-NVB cells lack overexpression of P-glycoprotein and cross-resistance to MDR drugs like doxorubicin, epipodophyllotoxins or colchicine . Navelbine efflux was similar in sensitive and resistant cells, and resistance could not be explained by a difference of drug accumulation in these two cell lines . The cells were cross-resistant to vinca alkaloids and taxoids whose targets are microtubules . Immunofluorescence study of microtubules showed that depolymerization occured for the same Navelbine concentration in sensitive and resistant cells . This concentration induced growth inhibition in sensitive but not in resistant cells . Moreover, depolymerization induced by Navelbine treatment was reversible, after drug removal, in resistant cells only . This study suggests that J82-NVB cell resistance mechanism involves alterations of microtubule dynamics, allowing recovery of microtubules functions after treatment.

Immunobiology, 1994 Oct, 191(4-5), 337 - 43
Epidemiology of tuberculosis: the impact of HIV and multidrug-resistant strains; Crawford JT; The recent reemergence of tuberculosis in the United States and other developed countries has been attributed to a number of factors including a decreased emphasis on tuberculosis control and immigration . Perhaps the most significant factor is the association of tuberculosis and HIV/AIDS . Infection with HIV greatly increases susceptibility to infection and increases the risk of developing active disease . In addition, progression of tuberculosis can be very rapid in patients with greatly reduced immune function . The increasing incidence of drug resistance is also contributing to the difficulty in controlling tuberculosis . Multidrug-resistance not only adversely affects the patient but contributes to prolonged infectiousness.

Zhonghua Nei Ke Za Zhi, 1994 Oct, 33(10), 666 - 8
{Detection of P-glycoprotein expression in patients with acute leukaemia and clinical significance}; Feng K et al.; Anti-P-glycoprotein monoclonal antibody JSB-1 and alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunocytochemical staining technique were used to study the relation between P-glycoprotein expression and clinical multidrug resistance (MDR) in 42 patients with acute leukaemia (23 ALL and 19 ANLL) . 10 of 17 patients who were diagnosed as refractory or relapsed acute leukaemia were positive with P-glycoprotein expression, while only 3 of 14 newly diagnosed and 1 of 11 who were in complete remission were positive . The preliminary results indicated that there was a close association between the P-glycoprotein expression and the clinical resistance to chemotherapy in some patients.

Cancer Lett, 1994 Sep 30, 85(1), 59 - 63
The riminophenazine agents clofazimine and B669 reverse acquired multidrug resistance in a human lung cancer cell line; Van Rensburg CE et al.; The potential of the riminophenazine agents clofazimine and B669, at therapeutically relevant concentrations, to reverse P-glycoprotein-mediated multidrug-resistance (MDR) in a human lung cancer cell line (H69/LX4) has been investigated in vitro . Cyclosporin A, a well-documented MDR-modifying agent, was included for comparison . Clofazimine, B669 and cyclosporin A at minimally cytotoxic concentrations of 1, 0.5 and 5 micrograms/ml, respectively, were equally effective in restoring sensitivity to vinblastine, doxorubicin, daunorubicin and mitomycin C in the H69/LX4 cell line . All three chemosensitizing agents also increased the accumulation of {14C}vinblastine by H69/LX4 cells . Riminophenazines, which are relatively non-toxic, non-carcinogenic and non-myelosuppressive agents, are promising contenders for evaluation in experimental and clinical oncology as modulators of acquired MDR.

J Biol Chem, 1994 Sep 16, 269(37), 22983 - 9
Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein . Strategic location of a tryptophan residue; Baubichon-Cortay H et al.; The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction . NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage . Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity . TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations . A similar affinity binding was monitored by quenching of intrinsic fluorescence . The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides . TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%) . The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment . A high quenching was observed upon nucleotide interaction with similar affinity . The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.

Cancer Res, 1994 Sep 15, 54(18), 4958 - 66
Differences between drug-sensitive and -resistant human leukemic CEM cells in c-jun expression, AP-1 DNA-binding activity, and formation of Jun/Fos family dimers, and their association with internucleosomal DNA ladders after treatment with VM-26; Kim R et al.; Although the DNA topoisomerases are critical intracellular targets of a number of clinically important anticancer drugs, the mechanism(s) by which inhibition of these enzymes causes cell death are poorly understood . We found that treatment of human leukemic lymphoblasts (CCRF-CEM) with teniposide (VM-26), under conditions that stabilize DNA-topoisomerase II complexes, caused the formation of internucleosomal DNA ladders . However, it appeared unlikely that the VM-26-stabilized DNA-topoisomerase II-cleavable complexes directly produce these internucleosomal DNA ladders, since similar nucleosomal DNA ladders were observed following either continuous or a short (1 h) exposure of cells to VM-26 . Under continuous exposure to VM-26, the internucleosomal DNA ladders were associated with the transient induction of c-jun mRNA in a dose-dependent fashion, reaching maximum expression at 6 h after treatment with VM-26 and being down-regulated to basal levels by 12 h . The induction of c-jun mRNA by VM-26 apparently preceded DNA ladder formation . However, in CEM sublines selected for resistance to VM-26 (CEM/VM-1 and CEM/VM-1-5; approximately 50- and 140-fold resistant, respectively) and which display the phenotype of multidrug resistance associated with altered DNA topoisomerase II (at-MDR), we found that the induction of c-jun mRNA by VM-26 and subsequent DNA ladder formation were progressively attenuated in proportion to the resistance of the cells, apparently due in part to decreased stabilization of DNA-topoisomerase II-cleavable complexes . Further, the attenuated induction of c-jun in the at-MDR cells was found to be associated with a decreased rate of c-jun transcription and an increase in the instability of its mRNA following VM-26 treatment . The attenuation of c-jun mRNA induction was also reflected in decreased production of c-Jun protein in the at-MDR cells . Of interest was the fact that no significant induction of c-fos mRNA by VM-26 was observed in either CEM or at-MDR cells . Furthermore, the induction of c-jun was related to the activation of AP-1 DNA-binding activity in a time- and dose-dependent manner in CEM cells, whereas the activation of AP-1 binding was attenuated in at-MDR cells in proportion to their resistance to VM-26 . Using Jun and Fos family member antibody inhibition experiments in gel-mobility shift assays, we found that AP-1-binding activity appeared to be preferentially mediated by c-Jun/Fra-1 heterodimers in both CEM and at-MDR cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Pharmacol, 1994 Sep 15, 48(6), 1129 - 36
Competitive inhibition by genistein and ATP dependence of daunorubicin transport in intact MRP overexpressing human small cell lung cancer cells; Versantvoort CH et al.; In several multidrug resistant tumor cell lines without overexpression of P-glycoprotein (non-Pgp MDR), a decreased accumulation of drugs has been shown to contribute to resistance . We have recently reported that daunorubicin (DNR) accumulation was decreased in the multidrug resistance-associated protein overexpressing GLC4/ADR non-Pgp MDR small cell lung cancer cell line due to an enhanced energy-dependent efflux which could be inhibited by the isoflavonoid genistein . The purpose of this work was 2-fold: (i) to investigate the mechanism by which genistein inhibits the DNR efflux in the GLC4/ADR cells; and (ii) to characterize the dependence of DNR transport on ATP concentration in intact GLC4/ADR cells . The active transport of DNR in GLC4/ADR cells appeared to be a saturable process with an apparent Km of DNR of 1.4 +/- 0.4 microM . Genistein increased the apparent Km value of DNR, suggesting that this agent is a competitive inhibitor of DNR transport . These data provide additional evidence that energy-dependent DNR transport in GLC4/ADR cells is a protein-mediated process . In addition, genistein decreased cellular ATP concentration in a dose-dependent manner in sensitive as well as in resistant cells . Marked inhibition of DNR transport activity in intact GLC4/ADR cells was found when cellular ATP concentration was decreased below 2 mM by sodium azide or 2-deoxy-D-glucose . Thus, since DNR transport in intact GLC4/ADR is already inhibited at modest cellular ATP depletion, a limitation in ATP supply might open ways to make MDR cells more susceptible to drug toxicity.

Biochem J, 1994 Sep 15, 302 ( Pt 3), 649 - 54
Phorbol ester selectively stimulates the phospholipase D-mediated hydrolysis of phosphatidylethanolamine in multidrug-resistant MCF-7 human breast carcinoma cells; Kiss Z et al.; The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells . In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid . In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho . The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well . However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells . These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.

Int J Cancer, 1994 Sep 15, 58(6), 860 - 4
Possible involvement of multidrug-resistance-associated protein (MRP) gene expression in spontaneous drug resistance to vincristine, etoposide and adriamycin in human glioma cells; Abe T et al.; The multidrug-resistance phenotype in human tumors is partly associated with over-expression of the 170 kDa-P-glycoprotein encoded by the multidrug-resistance-1 (MDR1) gene . Another related, but non-P-glycoprotein, multidrug-resistance-associated protein (MRP) gene encodes a 190 kDa-membrane ATP-binding protein . Glioblastoma multiforme is a highly malignant primary neoplasm of the central nervous system which is refractory to anti-cancer chemotherapy, but the mechanism underlying this drug resistance is unknown . Out of glioma cell lines, 2, namely IN500 and T98G, which had elevated MRP mRNA levels, showed the highest resistance to multiple anti-cancer agents such as etoposide, vincristine and adriamycin, and decreased intracellular accumulation of etoposide . In the remaining 5 cell lines, various degrees of sensitivity to adriamycin and etoposide appeared to correlate with their respective MRP mRNA levels . Our study proposes that MRP may be involved in spontaneous multidrug resistance in human gliomas.

Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1210 - 6
Chinese hamster ovary cells resistant to okadaic acid express a multidrug resistant phenotype; Tohda H et al.; Two Chinese hamster ovary cell clones resistant to okadaic acid (OA) were isolated . The OA-resistance was associated with resistance to colchicine, Vinca alkaloids and inhibitors of DNA topoisomerase (topo) II . Drug accumulation assays showed that the intracellular levels of OA, vinblastine and vincristine, but not the topo II inhibitor etoposide, were significantly lowered in the OA-resistant mutants than in the parental cells . These results, together with the finding of an increased level of P-glycoprotein (P-gp) in the mutant cells, indicate that the resistances to OA, Vinca alkaloids and colchicine are due to a P-gp-mediated mechanism . Resistance to topo II inhibitors, however, was associated with reduced activity of topo II . Thus, at least two events, overexpression of P-gp and reduction of topo II activity, occurred in a single OA-resistant cell line, contributing to expression of the MDR phenotype.

Cancer Lett, 1994 Sep 15, 84(2), 163 - 72
Glutathione S-transferases and P-glycoprotein in normal rat hepatocytes and hepatoma cells: analysis using flow cytometry; Mehta R et al.; Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated antibodies, in combination with flow cytometry (FCM), we have developed a technique to detect the alpha, mu and pi isozymes of GST in cell suspensions from normal rat liver, and in H4IIE cells, a rat hepatoma cell line . Cell suspensions fixed in 1% paraformaldehyde were observed to require cell membrane permeation with lysolecithin to allow access and binding of antibodies to immunoreactive proteins within the cytoplasm . FCM analysis indicated normal rat hepatocytes to be positive for GST alpha and mu, but not GST pi, and the H4IIE cells to be positive for all three GST isozymes . Further analysis by FCM for the expression of P-glycoprotein (mdr), a membrane-associated protein product of the multidrug resistance gene, showed an association between the presence of GST pi and mdr in the two cell types . Thus, mdr was detected in significant amounts in H4IIE cells, but not in rat hepatocytes . The method described here has potential applications in screening, sorting and further characterisation for GST pi-positive hepatocytes for mechanistic studies during sequential rat liver carcinogenesis, as well as for characterisation of human tumors for the expression of different GST isozymes and P-glycoprotein during therapeutic management.

Cancer Res, 1994 Sep 15, 54(18), 4980 - 7
Prevalence of multidrug resistance related to activation of the mdr1 gene in human sarcoma mutants derived by single-step doxorubicin selection; Chen G et al.; Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones . Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells . After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks . Surviving colonies were scored and harvested . Clones were propagated and analyzed for drug resistance phenotype . Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction . Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833 . Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry . Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance . At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation . All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin . Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations . No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110 . Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2 . In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil . Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events . Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.

Cancer Res, 1994 Sep 15, 54(18), 4892 - 8
The inability of the mouse mdr2 gene to confer multidrug resistance is linked to reduced drug binding to the protein; Buschman E et al.; The mouse mdr gene family is composed of three members designated mdr1, mdr2, and mdr3 . A full-length mdr2 complementary DNA clone has been introduced in an amplifiable eukaryotic expression vector (pEMC2b1) which directs amplification and overexpression of a bicistronic mdr2-dihydrofolate reductase mRNA after stepwise methotrexate selection of transfected mutant dihydrofolate reductase Chinese hamster ovary DUK cells . Independent cell clones expressing low to high amounts of mdr2 cellular mRNA and Mdr2 protein in their membrane fraction could be obtained by this selection procedure . Comparison of drug survival characteristics of cell clones expressing similar amounts of either Mdr1 or Mdr2 proteins revealed that Mdr1 but not Mdr2 could confer readily detectable levels of colchicine or vinblastine resistance . Labeling experiments using membrane-enriched fractions and a photoactivatable analogue of ATP showed that the Mdr2 protein was properly inserted in the membrane of transfected cells and could bind this ligand with an apparent affinity similar to that of Mdr1 . However, labeling studies with the photoactivatable drug analogue iodoarylazidoprazosin showed considerably reduced binding of this ligand to Mdr2 as compared to Mdr1 . Our findings demonstrate that Mdr2 cannot confer drug resistance and suggest that this inability is linked to reduced drug binding to the Mdr2 protein.

Cancer Res, 1994 Sep 15, 54(18), 4833 - 6
ATP-dependent transport of glutathione S-conjugates by the multidrug resistance-associated protein; Jedlitschky G et al.; The ATP-dependent transport of the endogenous glutathione conjugate leukotriene C4 (LTC4) was more than 25-fold higher in membrane vesicles prepared from human leukemia cells (HL60/ADR) overexpressing the multidrug resistance-associated protein than from drug-sensitive parental HL60 cells or revertant cells . Similar results were obtained with S-(2,4-dinitrophenyl)glutathione as substrate . Photoaffinity labeling detected preferentially in the HL60/ADR membranes a 190-kilodalton protein binding {3H}LTC4 and 8-azido{alpha-32P}ATP . The {3H}LTC4-labeled 190-kilodalton protein was immunoprecipitated by an antiserum against the COOH-terminal sequence of multidrug resistance-associated protein . Our results indicate that multidrug resistance-associated protein mediates the ATP-dependent transport of LTC4 and structurally related anionic amphiphilic conjugates.

Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8822 - 6
The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump; Zaman GJ et al.; The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs . We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA . MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol . The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant . The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient . Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells . We conclude that MRP is a plasma membrane drug-efflux pump.

Biochemistry, 1994 Sep 13, 33(36), 11008 - 15
Novel Cl(-)-dependent intracellular pH regulation in murine MDR 1 transfectants and potential implications; Roepe PD et al.; Previously {Luz et al . (1994) Biochemistry 33, 7239-7249}, we determined that Cl(-)- and -HCO3-dependent pHi homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991) . Via single-cell photometry experiments performed under various conditions, we are now able to separate Na(+)-dependent and Na(+)-independent components of Cl-/-HCO3 exchange in the MDR transfectants and the parental LR73 cells . Cl(-)-dependent, Na(+)-independent reacidification of pHi, mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein . Analysis of H+ flux at different pHi shows that Cl(-)-dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pHi = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells) . MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl(-)-dependent pHi regulation are more severe . Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pHi homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein . Verapamil reverses the anomalous behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1994 Sep 9, 269(36), 22853 - 7
A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein; Szczypka MS et al.; Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides . The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR) . S . cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells . Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance . Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the delta F508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification . Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional . These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport.

J Mol Biol, 1994 Sep 2, 241(5), 736 - 8
Preliminary crystallographic analysis of a Fab specific for P-glycoprotein with and without bound peptide; Vasudevan S et al.; The antigen-binding fragment (Fab) of anti-peptide monoclonal antibody C219 raised against the multidrug resistance associated P-glycoprotein has been crystallized with and without bound peptide . The crystals of the Fab in the absence and presence of peptide belong to space groups P2(1) and P2(1)2(1)2(1), respectively . The volumes of both crystal forms are consistent with the presence of four Fab molecules per asymmetric unit . Diffraction data to 3.2 A resolution have been collected on a San Diego Multiwire Area Detector system from both crystal forms . Determination of the molecular replacement solutions is underway.

J Clin Oncol, 1994 Sep, 12(9), 1771 - 7
Quinidine as a resistance modulator of epirubicin in advanced breast cancer: mature results of a placebo-controlled randomized trial; Wishart GC et al.; PURPOSE: To evaluate the effect of quinidine, a putative modulator of P-glycoprotein-mediated drug resistance, on the response rate and toxicity profile of epirubicin in patients with advanced breast cancer . PATIENTS AND METHODS: Between 1989 and 1992, 223 eligible patients were randomized in double-blind fashion to receive epirubicin 100 mg/m2 by intravenous (i.v.) bolus and prednisolone 25 mg orally twice daily, along with either placebo or quinidine (250 mg) capsules, taken for 4 days before and 2 days after chemotherapy . Treatment was continued for a maximum of eight courses . RESULTS: Ten eligible patients did not complete the first cycle of treatment . Of the remaining patients, 106 in the placebo arm received 619 courses of treatment, and 107 in the quinidine arm received 612 courses . The median cumulative dose of epirubicin in both arms was 600 mg/m2 . The median quinidine level (measured before epirubicin administration in 288 courses) was 5.5 mumol/L; at this concentration, the drug partially reverses anthracycline resistance in multidrug-resistant (MDR) breast carcinoma cells in vitro . There were no statistically significant differences in hematologic or gastrointestinal toxicity between the two arms . The response rate in the placebo arm was 44% (6% complete remission {CR}, 38% partial remission {PR}), and in the quinidine arm was 43% (4% CR, 39% PR) . Surviving patients have been monitored for a median time of 74 weeks, and there is no significant difference in the overall or progression-free survival between the two arms . The median survival times were 59 weeks for placebo and 47 weeks for quinidine patients . The estimated relative death rate (quinidine/placebo) was 1.2 (P = .247; 95% confidence interval {CI}, 0.88 to 1.63) . CONCLUSION: Quinidine at this dose does not significantly alter the toxicity profile, response rate, or survival after epirubicin chemotherapy in patients with advanced breast cancer . This may be due to ineffective modulation of P-glycoprotein by quinidine or the lack of expression of mdr-1 in a sufficient proportion of cells in these tumors, or alternative mechanisms underlying resistance to epirubicin.

Clin Exp Immunol, 1994 Sep, 97(3), 373 - 9
Reduced NK activity correlates with active disease in HIV- patients with multidrug-resistant pulmonary tuberculosis; Ratcliffe LT et al.; There has been a global increase in the incidence of multidrug-resistant pulmonary tuberculosis (TB) . As there are no previous reports of immune function in HIV- patients with multidrug-resistant pulmonary TB, a comprehensive assessment of cellular immunity in this setting was undertaken . This involved a prospective, case-controlled study which included five patients with active multidrug-resistant pulmonary TB and five matched controls with active non-resistant infection, and documented the changes in immune parameters which occurred upon clinical resolution . Patients with multidrug-resistant TB had significantly lower fresh natural killer (NK) cell activity than matched controls with non-resistant pulmonary TB (P < 0.05) . This was a specific abnormality, as there were no significant differences in antigen-specific cytotoxicity or lymphocyte proliferation in the case-controlled study . Follow-up assessment of the patients with multidrug-resistant infections indicated that clinical improvement correlated with a moderate increase in NK cell activity . Impaired NK cell function may be involved in the pathogenesis of multidrug-resistant TB.

Br J Cancer, 1994 Sep, 70(3), 409 - 14
Effects of a new antioestrogen, idoxifene, on cisplatin- and doxorubicin-sensitive and -resistant human ovarian carcinoma cell lines; Sharp SY et al.; Pyrrolidino-4-iodotamoxifen (idoxifene) is a new non-steroidal antioestrogen currently undergoing phase I clinical evaluation . Using idoxifene and tamoxifen and two additional analogues of tamoxifen (3-hydroxytamoxifen and 4-iodotamoxifen) and the imidazole-based calmodulin inhibitor, calmidazolium, a strong positive correlation (r2 > 0.95) was observed between cytotoxicity and inhibition of calmodulin-dependent cyclic AMP phosphodiesterase (e.g . mean IC50 across four human ovarian carcinoma cell lines of 4.5 microM for idoxifene and 6.3 microM for tamoxifen) . Using two parent human ovarian carcinoma cell lines (41M and CH1; both oestrogen receptor negative) in which acquired resistance to doxorubicin or cisplatin has been generated, we have determined the ability of idoxifene to overcome resistance in these lines . At a non-toxic concentration of 2 microM, idoxifene appeared at least as effective as the clinically used multidrug resistance modifiers verapamil and tamoxifen in overcoming doxorubicin resistance in two acquired resistant cell lines shown to overexpress the P-170 efflux glycoprotein . Non-cross-resistance between cisplatin and idoxifene was observed in two acquired resistant cell lines possessing contrasting mechanisms of resistance to cisplatin (41McisR6 reduced drug transport and CH1cisR6 resistance mediated at the level of DNA) . In one of four cell lines (CH1), synergism between idoxifene and cisplatin was observed by median effect analysis . However, with the 41M and its 6-fold cisplatin-resistant variant, antagonism was observed . These observations made by median effect analysis appeared to be unrelated to platinum uptake or removal of platinum-induced DNA interstrand cross-links . These in vitro data suggest that idoxifene may be usefully combined with doxorubicin in the clinical setting, but caution should be exercised in combining it with cisplatin in the treatment of certain tumours.

Mol Gen Genet, 1994 Sep 1, 244(5), 501 - 11
PDR3, a new yeast regulatory gene, is homologous to PDR1 and controls the multidrug resistance phenomenon; Delaveau T et al.; The Saccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised . Mutations pdr3-1 and pdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene . The sequence of the wild-type PDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein . The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes . Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites . The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus . The salient feature of the PDR3 protein is its similarity to the protein coded by PDR1, a gene responsible for pleiotropic drug resistance . The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved . The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.

Med Clin North Am, 1994 Sep, 78(5), 1067 - 79
Pneumonia in patients with HIV infection; Rosen MJ; The epidemiologic shift in HIV-infected populations from homosexual men to intravenous drug users and their sexual partners, together with the wide application of antipneumocystis prophylaxis and a better understanding of the broad range of HIV-associated illnesses, has changed our concept of the spectrum of lung infections that occur in patients with HIV infection . Bacterial pneumonia, not PCP, is the most common lower respiratory infection . Newer therapies of mild-to-moderate PCP increase the treatment options . The worldwide increase in tuberculosis cases is attributable to coinfection with HIV, and multidrug-resistant tuberculosis is now a serious threat, especially in the inner cities . Fungal pneumonias occur with increased frequency in patients with HIV infection, depending on the geographic factors and the severity of immunodeficiency.

EMBO J, 1994 Sep 1, 13(17), 4036 - 41
Yap1p, a yeast transcriptional activator that mediates multidrug resistance, regulates the metabolic stress response; Gounalaki N et al.; Overexpression of the YAP1 transcriptional activator renders yeast cells resistant to multiple metabolic inhibitors . In an effort to identify other gene products required for this phenotype we have isolated genomic mutations which neutralize this effect . One such mutation was further characterized and the affected gene was shown to be identical to TPS2 which encodes trehalose phosphate phosphatase, an enzyme catalysing the second step in trehalose biosynthesis . We have analysed the transcriptional regulation of the TPS2 gene and have shown that its transcription is induced by a variety of stressful conditions caused by metabolic inhibitors, osmotic shock and heat shock . This transcriptional activation is mediated by multiple stress promoter elements (C4T) and requires the function of Yap1p as well as reduced activity of the cAMP-regulated protein kinase . Using an appropriate reporter gene we have shown that Yap1p is generally required for transcriptional regulation through the C4T stress element . These results show that the YAP1 protein has a pivotal role in the metabolic stress response and the acquisition of stress tolerance.

Cancer Res, 1994 Sep 1, 54(17), 4676 - 9
Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells; Abbaszadegan MR et al.; The multidrug resistance-associated protein (MRP) gene is a member of the ATP-binding cassette transporter gene superfamily and may be partially responsible for clinical drug resistance . Reverse transcriptase-polymerase chain reaction was used to measure MRP mRNA in normal hematopoietic cells from bone marrow and peripheral blood as well as patients with high risk acute myelocytic leukemia and multiple myeloma . All normal peripheral blood cells, regardless of cell lineage (CD4, CD8, CD14, CD15, CD19, CD56), expressed a similar basal level of MRP mRNA . Specimens from bone marrow containing mixed lineages also expressed a similar basal level of MRP expression . In patients with acute myelocytic leukemia, 10 of 12 (83%) of the specimens had detectable MRP mRNA, but the level of expression was similar to that of normal blood cells and low compared to a cell line known to overexpress MRP (H69/AR) . All myeloma patients (12 of 12) had detectable MRP mRNA expression at levels comparable to normal peripheral blood and bone marrow cells . We conclude that MRP is commonly expressed in normal hematopoietic cells as well as certain hematopoietic malignancies . The therapeutic relevance of MRP expression is unknown, but these studies emphasize the importance of measuring MRP expression in normal cells as a point of reference and comparison for detection in malignant cells . We also recommend obtaining sequential specimens from patients, which may reveal an increased expression of MRP from baseline as the disease progresses and becomes resistant.

Oncology, 1994 Sep-Oct, 51(5), 440 - 5
Enhanced expression of the multidrug resistance gene in vindesine-resistant human esophageal cancer cells; Saito T et al.; We developed and characterized a new series of low- and high-grade multi-drug-resistant (MDR) cell lines of human esophageal carcinoma . Eight vindesine-resistant clones, SH-1-V1, SH-1-V2, SH-1-V3, SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7, and SH-1-V8 were isolated from the human esophageal cancer cell line, SH-1, by stepwise selection on exposure to increasing doses of vindesine . SH-1-V1 to SH-1-V8 acquired resistance to vindesine, in a stepwise manner, from 3- to 115-fold over findings in the parental SH-1 cells . The most resistant clone, SH-1-V8, was cross-resistant to other anticancer agents such as vincristine, actinomycin D, and daunomycin, thereby suggesting acquisition of the MDR phenotype . In SH-1-V8 cells, cellular accumulation of vincristine decreased and an MDR reversal agent, cepharanthine, potentiated the cytocidal action of vindesine . The expression of MDR1 mRNA was enhanced and amplification of the MDR1 gene was observed in clones SH-1-V4, SH-1-V5, SH-1-V6, SH-1-V7 and SH-1-V8; expression of MDR1 mRNA was detectable without gene amplification in the remaining 3 clones . The enhanced expression of the MDR1 gene may be involved in the acquisition of vindesine resistance in human esophageal cancer cells.

Cytometry, 1994 Sep 1, 17(1), 84 - 93
Flow cytometric determination of the multidrug-resistant phenotype in acute leukemia; Maslak P et al.; Expression of the multidrug-resistant (MDR) phenotype was investigated in acute leukemia using a monoclonal antibody (HYB-241) directed against a cell surface epitope of the 180 kd P-glycoprotein (gp180) by flow cytometric analysis of clinical samples . Samples from sixty-four patients were tested (37 with acute myelocytic leukemia, 20 with acute lymphocytic leukemia, and 7 with blastic chronic myelocytic leukemia) . A D value (derived from Kolmogorov-Smirnov test) greater than 0.15 was considered positive (+) . Eight of 32 newly diagnosed patients were positive for gp180 compared with 22 of 32 relapsed/refractory (R/R) patients (P < 0.001) . Of the new patients, vinca/anthracycline-based induction therapy failed in 3/6 gp180(+) and 5/18 gp180(-) patients . In the R/R group, 15/16 gp180(+) and 3/6 gp180(-) patients failed to achieve complete remission (P < 0.05) . In vitro drug accumulation studies performed with verapamil failed to show a correlation with clinical response . However, in a subset of patients, a striking correlation (r = .97, P = .001) was noted between the presence of gp180 as determined by the D value and the functional activity of the P-glycoprotein as expressed by increased daunorubicin accumulation in the presence of verapamil . The results suggest that 1) newly diagnosed patients can express gp180, 2) P-glycoprotein is expressed in 69% of R/R patients, 3) response in R/R patients is effected by the presence of gp180, and 4) expression of gp180 is highly correlated with its function as a drug-efflux pump in a subset of the patients studied . The complexity of clinical drug resistance is underscored by the finding that the MDR model is not applicable to all cases . In such instances, other mechanisms may play a predominant role.

Cytometry, 1994 Sep 1, 17(1), 50 - 8
Scanning microspectrofluorometry of rhodamine 123 in multidrug-resistant cells; Millot JM et al.; Scanning microspectrofluorometry has been developed to perform the mapping of fluorescence spectra from all locations in a living cell . This new method has been applied to study the molecular environment of rhodamine 123 (R123) in sensitive (K562, CEM) and multidrug-resistant (K562-R, CEM/VLB100) tumor cells . All cells exposed to R123 showed a similar distribution of fluorescence in the perinuclear region . A lower cytoplasmic fluorescence intensity corresponding to a reduced drug accumulation was observed in resistant cells, as expected in the multidrug resistance process . Fluorescence emission spectra of R123 are useful to probe the polarity of the R123 environment . Thus, fluorescence spectra of R123-treated cells have been analyzed as a linear combination of model spectra: R123 in water and R123 in tensio-active Triton X-100 . In sensitive cells, emission spectra of R123 underwent a red shift, equivalent to those observed in isolated coupled mitochondria . This suggests the formation of a complex in hydrophobic sites . In contrast, R123 spectra were less shifted in resistant cells, showing two types of both hydrophobic and hydrophilic binding sites . This could be related to an intracellular redistribution of R123 in resistant cells.

Free Radic Biol Med, 1994 Sep, 17(3), 191 - 200
Free radical formation by ansamycin benzoquinone in human breast tumor cells: implications for cytotoxicity and resistance; Benchekroun NM et al.; The benzoquinonoid ansamycin antibiotics, geldanamycin and herbimycin A, are potent cytotoxins against tumor cells in vitro . We have examined the mechanism of their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7) cells and we have found that multidrug-resistant MCF-7/ADRR cells that exhibit the MDR phenotype and the overexpression of P-170-glycoprotein, were cross-resistant to geldanamycin and herbimycin A . Verapamil, which binds competitively with P-170-glycoprotein, enhanced geldanamycin cytotoxicity 12-fold only in resistant cells, suggesting that geldanamycin may interact with the drug efflux protein . Geldanamycin and herbimycin A, like adriamycin, were reductively activated by the NADPH-cytochrome P450-reductase and formed reactive .OH . The formation of .OH was significantly lower in resistant cells . In contrast to adriamycin, the formation of .OH was unaffected by the addition of DNA, indicating that a DNA-complexed drug was redoxactive and may, therefore, may be more effective in killing tumor cells at the DNA level . These observations indicate that both the decreased free radical formation and interactions with P170 glycoprotein may be important in geldanamycin and herbimycin A resistance in multidrug resistant human breast tumor cells.

Todays OR Nurse, 1994 Sep-Oct, 16(5), 13 - 9; quiz 44-5
Tuberculosis prevention and control: the particulate respirator controversy; Jackson MM; 1 . Increasing numbers of new cases of tuberculosis have been reported each year since 1984/1985 . It is estimated that from 1985 through 1992 there were over 50,000 cases in excess of projections . Many of these outbreaks have been caused by strains of multidrug resistant Mycobacterium tuberculosis . 2 . The wearing of a particulate respirator {PR} may provide additional protection because of the tighter facial fit and better filtration capability as compared with a standard surgical mask . 3 . The only type of PR certified by the National Institute for Occupational Safety and Health and approved for use by the Occupational Safety and Health Administration to have filtration efficiency for particles as small as 1 microns are high-efficiency particulate air respirators, which are expensive and can be intimidating to patients.

Am J Physiol, 1994 Sep, 267(3 Pt 1), C688 - 99
ATP is not required for anion current activated by cell swelling in multidrug-resistant lung cancer cells; Jirsch JD et al.; During whole cell recording with 4 mM ATP and 0.1 mM GTP in the pipette, outwardly rectifying Cl- currents (155 +/- 20.5 pA/pF) were repetitively activated on reduction of bath solution osmolarity from 290 mosM (control) to 210 mosM . These currents were sensitive to 0.1-1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid . Omission of ATP from the pipette solution reduced the current magnitude to 42.7 +/- 9.5 pA/pF and prevented repetitive activation . More hyposmotic solutions (160 mosM) usually elicited current repetitively despite an ATP-free pipette solution . In cells depleted of ATP (to < 5% of control) by preincubation with 2-deoxyglucose (10 mM) and rotenone (100 nM), hyposmotic solutions failed to activate significant current . Cell volume increased to 230 +/- 18% of control (19.1 +/- 1.2 microns) in 210 mosM bath (normal cells) but only to 114 +/- 13% of control in ATP-depleted cells exposed to 160 mosM solution . This failure of ATP-depleted cells to swell in hypotonic external solutions was reversed by overnight pretreatment with cytochalasin D (2 micrograms/ml; n = 6) but not by colchicine (250 microM; n = 8) . In outside-out patches of membrane dialyzed with zero ATP and excised from swollen cells, we observed sustained activation of a 53-pS outwardly rectifying channel (chord conductance, +100 mV; open probability approximately 1.0) . In cell-attached patches from normal and ATP-depleted cells, we activated similar channels by suction . ATP does not appear to be an absolute requirement for the activation of this Cl- channel in H69AR cells but may be essential for the normal volume response and channel activation mediated through cytoskeletal elements within cells.

Mol Pharmacol, 1994 Sep, 46(3), 562 - 7
Epoxide metabolite of quinine and inhibition of the multidrug resistance pump in human leukemic lymphoblasts; Wigler PW et al.; Multidrug resistance (MDR) in neoplastic cells is usually due to decreased cellular retention of drugs such as vincristine or doxorubicin . An ATP-dependent drug efflux pump has been detected in MDR-1-phenotypic cells; inhibition of the MDR pump is probably the primary mechanism for reversal of MDR . Although quinine (SQ1) and quinidine are reversal agents and inhibitors of the MDR pump, the results from in vivo experiments and in vitro experiments with these diastereomers are contradictory . These observations suggest that an oxidized metabolite of SQ1 is a more potent inhibitor of the MDR pump than is the parent compound . The chemical synthesis of the epoxides of SQ1 and quinidine is reported . The epoxy compounds have been tested as inhibitors of the ATP-dependent MDR pump in human CEM/VLB100 cells . The procedure is based on preloading the cells with an inhibitor and a low concentration of a substrate, rhodamine 123 (R123) . After several cold rinses, the cell suspension is passed through a filtration-flow apparatus and the R123 in the filtrate (determined by fluorescence measurements) reveals the initial efflux of R123 through the MDR pump . When tested as an inhibitor of the MDR pump, quinine-10,11-epoxide is approximately 8-fold more potent than SQ1.

Leuk Res, 1994 Sep, 18(9), 683 - 91
In vitro drug resistance in acute myeloid and chronic B-lymphocytic leukaemic blasts and in normal blood and marrow populations; Baines P et al.; The sensitivities of AML and BCLL blasts to daunorubicin have been determined, using an in vitro (MTT) assay of resistance, and compared with the sensitivities of normal haemopoietic populations and cells of the multidrug-resistant, T-lymphoid line CEM VLB100; The role of the drug-efflux pump, P-glycoprotein, was determined by adding the 'modifier' cyclosporin and by measuring numbers of P-glycoprotein positive cells by immunofluorescence . ID50s for 17 cases of de novo AML varied from 5 to 300 ng/ml giving a median of 105 ng/ml which was similar to the median of 11 normal marrow mononuclear cell preparations (80 ng/ml) but considerably less than the median ID50 of eight blood lymphocyte samples (3500 ng/ml) . ID50s for five relapsed and two refractory AML samples ranged from 27 to 240 ng/ml, well within the de novo range: we had obtained presentation samples for two of these and, in both cases, ID50s were lower at relapse . ID50s, however, were raised in seven marrow mononuclear cell populations taken soon after remission induction (ID50 for remission MNC and normal MNC = 200 and 80 ng/ml, respectively); this may reflect either a property of regenerating populations, or an activation of cellular resistance mechanisms following chemotherapy . ID50s for 17 cases of BCLL ranged from 7 to 200 ng/ml with a median of 48 ng/ml which was significantly lower than the ID50 of AML blasts or of blood lymphocytes . Cyclosporin induced less than two-fold reductions in ID50s of blood lymphocytes, marrow mononuclear cells and de novo AML and BCLL blasts despite giving log reversals in resistance in the CEM VLB100 line . This reflected numbers of P-glycoprotein positive cells in our samples, which were high in CEM VLB100 but low in fresh normal or leukaemic cell suspensions . For both de novo AML and BCLL groups, however, the change in ID50, on addition of cyclosporin, was significant . These data imply a minor role for P-glycoprotein in drug resistance of leukaemic blasts . Nevertheless, there was a positive correlation between daunorubicin ID50s in de novo AML and time to remission which confirms that in vitro chemosensitivity assays can provide a useful measure of in vivo resistance.

Leukemia, 1994 Sep, 8(9), 1492 - 7
Expression of P-glycoprotein in de novo acute myelogenous leukemia at initial diagnosis: results of molecular and functional assays, and correlation with treatment outcome; Ino T et al.; We investigated the expression of P-glycoprotein (P-gp) in 52 adults with de novo acute myelogenous leukemia (AML) at the initial diagnosis . We tested 52 patients by flow cytometry using the MRK16 monoclonal antibody (MoAb) . To investigate the phenotype for multidrug resistance, 41 of the patients were analyzed using rhodamine 123 (Rh123) . We found that 14 (27%) of the 52 patients were positive for P-gp expression by MRK16 MoAb using a cutoff of 5% positive cells . There was a significant correlation between the results of the two analyses (p < 0.01) . We suggest that flow cytometry using MRK16 MoAb is acceptable for use in detecting P-gp expression in clinical samples . Among the 52 patients, 43 (83%) obtained a complete remission (CR) and 45% of remitters were predicted to be alive and in a CR after 8 years . Although the rate of CR on the MRK16-positive patients was comparable to that of the MRK16-negative patients, the MRK16-positive patients were prone to relapse . We conclude that determination of P-gp expression of de novo AML at initial presentation did not significantly influence the outcome of treatment.

Cancer Res, 1994 Sep 1, 54(17), 4557 - 63
Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies; Flens MJ et al.; We have generated rat and murine monoclonal antibodies against multidrug resistance-associated protein (MRP), a M(r) 180,000-195,000 membrane glycoprotein involved in a non-P-glycoprotein multidrug resistance of human tumor cells . The antibodies were raised against two different segments of MRP and found to be suitable for protein blot analyses, immunohistochemical and cytochemical studies, as well as flow cytometry of permeabilized cells . The antibodies do not cross-react with the human P-glycoproteins . Immunocytochemistry using MRP-overexpressing tumor cells of different histogenetic origins showed that MRP is predominantly located in the plasma membrane . Immunoelectron microscopy confirmed the plasma membrane location of MRP . The MRP antibodies provide a sensitive and specific tool for studies on MRP-mediated multidrug resistance.

Methods Find Exp Clin Pharmacol, 1994 Sep, 16(7), 505 - 12
Selective activation of anticancer prodrugs by monoclonal antibody-enzyme conjugates; Wallace PM et al.; Several recent reports have demonstrated that anticancer drugs can be generated site-selectively at solid tumors by monoclonal antibody-enzyme conjugates targeted to antigens on tumor cell surfaces . The first step in this drug targeting approach involves the delivery of the enzyme conjugate to a tumor cell population . After the conjugate has localized within the tumor and cleared from non-target tissues, a relatively non-cytotoxic drug precursor (prodrug) is administered . Upon contact with the targeted enzyme, the prodrug is converted into a toxic drug . Several examples are presented to illustrate this targeting strategy . Monoclonal antibody-beta-lactamase conjugates have been developed to activate a panel of anticancer prodrugs that are mechanistically dissimilar . The antitumor activities of the monoclonal antibody-beta-lactamase conjugate/prodrug combinations exceed those obtained by systemic drug administration, and are immunologically specific . In another example involving targeted cytosine deaminase for the generation of 5-fluorouracil, it is shown that as much as 17 times more drug can be delivered within a tumor compared to when 5-fluorouracil is administered alone . The method of using targeted enzymes for prodrug activation can be extended to include prodrugs that release very potent drugs, such as palytoxin, a marine natural product, and to treat cells that have the multidrug resistance phenotype . Some of the requirements for successful therapy with this approach for cancer therapy are discussed.

Pept Res, 1994 Sep-Oct, 7(5), 265 - 9
Preliminary experimental anticancer activity of cecropins; Moore AJ et al.; The cecropins are a group of peptides that were first isolated from the hemolymph of the giant silk moth, Hyalophora cecropia . In preliminary studies, these novel peptides were shown to be active against several bacteria and mammalian lymphomas and leukemias in vitro . The mechanism of action of the cecropins is thought to involve pore formation at the cytoplasmic membrane . The potential anticancer activity of cecropin B, cecropin P1 and Shiva-1 was investigated against a panel of mammalian cell lines in vitro . Cell lines showed a range of sensitivities to cecropin B (IC50 3.2 to > 100 microM), and two cell lines with the multidrug-resistant phenotype were sensitive to the peptide . In vitro cecropin B activity was virtually complete within one hour . Preliminary in vivo studies showed that cecropin B increases the survival time of mice bearing murine ascitic colon adenocarcinoma cells . Future studies will address structure/activity relationships of similar peptides in order to optimize antitumor activity.

Anticancer Res, 1994 Sep-Oct, 14(5A), 1943 - 50
In vitro antiproliferative effects of albumin-doxorubicin conjugates against Ewing's sarcoma and peripheral neuroectodermal tumor cells; Gabor F et al.; The 3-5 year survival rates of patients with disseminated Ewing's sarcoma (ES) or the closely related peripheral primitive neuroectodermal tumors (PNET) remain low, even under aggressive treatment involving highly toxic multidrug chemotherapeutic regimens . ES and PNET are sensitive to doxorubicin, but may escape treatment by expression of the multidrug-resistant phenotype and/or other mechanisms . In this study, we have identified albumin as growth supporting factor for ES and PNET cells in IGF-I-supplemented serum-free tissue culture medium . To investigate the specificity and toxicity of albumin-based drug conjugates, doxorubicin was coupled to bovine serum albumin (BSA) by either a two step glutaraldehyde or carbodiimide-C4-spacer technique, yielding monomeric DOX-albumin conjugates with conjugation numbers ranging from 3-20 moles DOX/mole BSA . Cellular uptake of fluorescein-isothiocyanate-(FITC)-labeled albumin and DOX-albumin conjugates could be demonstrated by flow cytometric measurements of cell-associated fluorescence and confocal microscopy . The cytostatic activity of these conjugates against ES/PNET cell lines, a neuroblastoma (LAN-1) and prostate cancer carcinoma cell line (PC-3) and normal lymphoblasts was tested in short-term proliferation assays (48 h) . The results show a high selectivity of the DOX-albumin conjugates for ES/PNET cell lines, with highest growth inhibition by conjugates with low DOX conjugation numbers (n = 3) in serum-supplemented medium (17-32 fold loss of activity compared to free DOX), followed by 20-DOX-C4-albumin in serum-free medium and low activity of the other conjugates . In conclusion, DOX-albumin conjugates inhibit the growth of ES/PNET cell lines selectively, showing low activity against the unrelated carcinoma line PC-3 and sparing normal lymphoblasts . The inverse correlation of activity and conjugation number demonstrates a low cytotoxic activity of DOX in acid-stable binding to monomeric albumin, pointing to a selective cytostatic activity of the modified albumin against ES and PNET cells, even in the presence of a 100 fold excess of unmodified serum albumin.

J Antimicrob Chemother, 1994 Sep, 34(3), 313 - 9
Molecular genetics of drug resistance in Mycobacterium tuberculosis; Zhang Y et al.; Tuberculosis (TB) is the single largest killer among infectious diseases . The recent resurgence of TB together with outbreaks of multidrug resistant tuberculosis has focused attention on understanding the mechanisms of such drug resistance . Because of the relative neglect of TB research in the past and late arrival of mycobacterial genetic tools, the molecular mechanisms of drug resistance in TB remained largely unknown until very recently . In this paper we review recent progress on the mechanisms of resistance to three major anti-TB drugs; isoniazid, rifampicin and streptomycin . While the resistance mechanisms for rifampicin and streptomycin are similar to those found in other bacteria, isoniazid susceptibility and resistance is unique to Mycobacterium tuberculosis . So far, mutations in two chromosomal loci, katG and inhA have been found to be involved in isoniazid resistance in TB . Identification and characterization of mutations responsible for resistance opens up new possibilities for rapid detection of drug resistant strains . Molecular understanding of drug resistance and drug action in M . tuberculosis may eventually lead to rational design of new anti-TB drugs.

Clin Lab Haematol, 1994 Sep, 16(3), 261 - 72
Validation of a single point flow cytometric assay for determining P-glycoprotein activity in multidrug resistant cell lines; Chin-Yee I et al.; P-glycoprotein, a transmembrane protein which acts as an energy dependent efflux pump, has been implicated as one mechanism of multidrug resistance (MDR) in human tumours . Commonly employed assays measure P-glycoprotein immunohistochemically or mdr1 messenger RNA . In this study we compared a single point flow cytometric assay for determining activity of P-glycoprotein with cellular expression of P-glycoprotein determined by Western blot . Five cell lines, with varying levels of multiple drug resistance, were incubated with daunorubicin (DNR) in the presence (treated) and absence (control) of cyclosporine or verapamil, agents known to inhibit the activity of P-glycoprotein . The treated cell lines, along with non-treated controls were examined for intracellular concentrations of DNR measured by fluorescence intensity using a flow cytometer . The ratio of fluorescence intensity expressed in the treated/control was used as an index of functional activity of P-glycoprotein . Functional activity of the P-glycoprotein as determined by flow cytometry correlates highly with cellular content of P-glycoprotein measured by western blot (correlation coefficients of r = 0.90-0.98 for the various cell line combinations) . This method represents a rapid single point flow cytometric assay which may be suitable for screening clinical samples for P-glycoprotein activity.

Somat Cell Mol Genet, 1994 Sep, 20(5), 423 - 35
Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein; Sumizawa T et al.; Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 microgram/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent . One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine . The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells . These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1 . Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells . The MRP gene was amplified in all the MDR cells that overexpressed MRP mRNA . DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells . These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.

Tokai J Exp Clin Med, 1994 Sep, 19(1-2), 39 - 46
Expression of the human multidrug resistance gene (MDR1) and prognostic correlation in human osteogenic sarcoma; Imanishi T et al.; The overexpression of P-glycoprotein (P-Gp), encoded by the human multidrug resistant gene (MDRA), decreases lipophilic drug accumulation in multidrug resistant cells in vitro . It is still not clear whether P-Gp contribute to the problem of multidrug resistance (MDR) in osteogenic sarcoma (OS) . We examined the MDR1 expression of 20 OS specimens (11 primary tumors, 10 xenografts, 1 overlapping), by Northern blotting and by the reverse transcriptase-polymerase chain reaction (RT-PCR), and evaluated by the relationship between MDR1 expression and patient prognosis . RT-PCR revealed MDR1 expression in 9/20 OS; 5/11 primary tumors and 5/10 OS-xenografts; northern blotting revealed MDR1 expression in only 5/20 OS . One primary OS specimen and its corresponding xenograft had similar levels of MDR1 expression . All 20 patients with OS were treated with chemotherapeutic protocols including doxorubicin, cisplatin, methotrexate and ifosfamide . Eight of 9 OS-patients expressing MDR1 were resistant to chemotherapy and had a poor prognosis . The relationship between MDR1 expression and poor prognosis in the 20 OS-patients was significant (p < 0.01) . The results support the assumption that MDR1 expression is related to MDR in human OS.

Anticancer Res, 1994 Sep-Oct, 14(5A), 1735 - 42
Overexpression of P-glycoprotein but not its mRNA in multidrug resistant cells selected with hydroxyrubicin; Zhao JY et al.; Previous studies have revealed that cultured cells treated with lipophilic natural products containing aromatic rings and basic amino group usually yielded multidrug resistant (MDR) variants . These MDR cells overexpress P-glycoprotein (P-gp), most often due to gene amplification or transcriptional activation of mdr/P-gp genes . Doxorubicin (Dox) is an anthracycline that belongs to this group of compounds . To explore the possible resistance mechanism(s) to anthracyclines that do not involve P-gp, we use a Dox analog, hydroxyrubicin (HyR) or WP159, which contains a C3' hydroxy group in replacement of the amino group in the sugar moiety of Dox thereby reducing basicity and eliminating positive charge in the parental compound to establish HyR-resistant cell lines . These resistant cells displayed the MDR phenotype and overexpressed P-gp as analyzed by Western blot analyses and immunohistochemical staining using two different anti-P-gp antibodies . Strikingly, the levels of P-gp mRNA in the majority of these MDR cells remained comparable to those in the drug-sensitive counterparts by slot blot hybridization . These results implicate that the basic center of the selecting agent is a critical determinant for generating diverse MDR variants, and that HyR may have a posttranscriptional effect on P-gp biosynthesis . This is the first report suggesting that cultured cells exposed to a particular selecting agent may give rise to particular subtype of MDR variants.

Cytometry, 1994 Sep 1, 17(1), 33 - 8
Isolation and characterization of a Chinese hamster ovary cell mutant with improved staining for indo-1; Wieder ED et al.; Chinese hamster ovary (CHO) 10B2 cells do not stain well with indo-1 and thus cannot be used for experiments to measure intracellular calcium using this dye . We have isolated a mutant CHO cell line (CHO IS1) that stains quite well with indo-1 and that has virtually identical growth characteristics and heat sensitivity as the parent line . The mutant was isolated by sorting individual mutagenized cells with high indo-1 fluorescence and cloning them . Since it has been reported that cells with multiple drug resistance (MDR+) can pump out various fluorescent dyes, the mutant and parent lines were characterized for Hoechst 33342 staining, Adriamycin toxicity, and P-glycoprotein expression, which are markers of the MDR phenotype . P-Glycoprotein was measured with the C219 antibody using flow cytometry . Multidrug-resistant cells (CHRC5) were used as positive controls . The IS1 cells stained as well with Hoechst 33342 as fixed 10B2 cells, and much better than unfixed 10B2 cells . The IS1 cells were 10- to 30-fold more sensitive to Adriamycin than the 10B2 cells, and both cell lines were much more sensitive than the CHRC5 cells . The amount of P-glycoprotein was similar in both 10B2 and IS1 cell lines, but was about fivefold lower than the CHRC5 cells . Thus, the poor staining for indo-1 in the 10B2 cells may not be caused by the P-glycoprotein MDR pump, but by a different efflux pathway . Alternatively, the P-glycoprotein may be altered and less efficient in the CHO IS1 cells.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 506 - 12
Detection of soluble P-glycoprotein in culture media and extracellular fluids; Chu TM et al.; Multidrug resistance (MDR) is a unique phenomenon in cancer patients and is commonly associated with an overexpression of the human MDR gene mdr1, which encodes an energy-dependent Mr 180 kDa membrane bound protein, known as P-glycoprotein . P-glycoprotein serves as a membrane efflux to pump the drugs out of the cancer cells . Western blot analysis, using a newly generated monoclonal antibody F4 which recognizes specifically an extracellular epitope of human MDR1 P-glycoprotein, reveals that soluble P-glycoprotein is detected in the cultured media of viable adriamycin-resistant human ovarian carcinoma 2780AD cells, whereas those of the drug-sensitive parent A2780 cells contain no detectable level of soluble P-glycoprotein . Soluble P-glycoprotein also is detected in extracellular fluids of cancer patients, such as malignant ascites and serum, and is not detectable in serum samples of normal healthy individuals . The Mr of soluble P-glycoprotein is the same as that of membrane bound P-glycoprotein . The presence of soluble P-glycoprotein in extracellular fluids may provide the basis for its use as a quantitative parameter of MDR and as a means to lessen or reverse MDR.

Biochemistry, 1994 Aug 30, 33(34), 10313 - 8
Modulation of P-glycoprotein by protein kinase C alpha in a baculovirus expression system; Ahmad S et al.; The modulation of P-glycoprotein by protein kinase C alpha (PKC alpha) was examined in a baculovirus expression system . PGP was phosphorylated in membrane vesicle preparations in vitro only when coexpressed with PKC alpha, and phosphorylation was Ca(2+)-dependent and inhibited by the PKC inhibitor Ro 31-8220 . PGP and PKC alpha were tightly associated in membrane vesicles and were coimmunoprecipitated with antibodies against either PGP or PKC alpha . Photoaffinity labeling of membrane vesicles with {3H}azidopine indicated that drug binding to PGP was slightly increased in the presence of PKC alpha . In contrast, PGP ATPase activity was increased by PKC alpha as well as by verapamil, but only PKC-stimulated activity in the presence of verapamil was inhibited by Ro 31-8220 . Mutation of serine-671 to asparagine in the linker region of PGP abolished PKC alpha-stimulated ATPase activity, and also inhibited to a lesser degree verapamil-stimulated ATPase activity . These results indicate that PKC alpha in a positive regulator of PGP ATPase activity and suggest that this mechanism may account for the increased multidrug resistance observed in MDR1-expressing cells when PKC alpha activity is elevated.

Cancer Lett, 1994 Aug 29, 84(1), 75 - 83
Modulation of rhodamine 123 uptake by nigericin in sensitive and multidrug resistant leukemic cells; Canitrot Y et al.; We have investigated the effect of the ionophore nigericin (NIG) in multidrug resistant (MDR) cells, using intracellular accumulation of the fluorescent dye rhodamine 123 (R123) . NIG increased the accumulation of R123 in half of the murine MDR RFLC3 population but not in the human MDR CEM/VLB 100 cells . Co-treatment of RFLC3 with NIG plus verapamil showed additive effect on the accumulation of R123 . The increase in R123 accumulation observed in RFLC3 was not the consequence of a direct effect of NIG on P-glycoprotein and was accompanied by a redistribution of the dye throughout the cell and a high cytotoxicity, which prevents the use of NIG as a resistance modulating agent.

Biochem Pharmacol, 1994 Aug 17, 48(4), 709 - 16
Comparative study of intracellular calcium and adenosine 3',5'-cyclic monophosphate levels in human breast carcinoma cells sensitive or resistant to Adriamycin: contribution to reversion of chemoresistance; Mestdagh N et al.; Multidrug resistance (MDR) corresponds to the cross-over resistance of tumour cells to structurally unrelated cytotoxic chemotherapeutic drugs . One of the mechanisms causing this resistance is the enhanced expression of a transmembrane drug efflux pump P-glycoprotein (P-170) . Reversal of P-glycoprotein-associated MDR has received much attention in recent years . In experimental cell lines, P-170 and the glutathione redox cycle seem to contribute to this phenomenon; P-170 may be inactivated by calcium and calmodulin antagonists and the glutathione redox cycle altered by buthionine sulphoximine (BSO) . Treatment of human MCF-7 breast cancer cells with chemosensitizers (CS), such as verapamil, trifluoperazine or BSO, for 72 hr resulted in an enhanced sensitization of cells to Adriamycin, trifluoperazine being the most potent compound in the reversion of chemoresistance . In these Adriamycin sensitive or resistant cells, treated or not by the CS, the possible role of calcium and cyclic adenosine monophosphate (cAMP) in mediating the reversion of chemoresistance to Adriamycin was investigated . It was found that intracellular calcium was approximately 2-fold higher in resistant than in sensitive cells, the opposite was true for cAMP . Modifications in calcium and cAMP levels were observed in MCF-7 resistant cells after treatment with verapamil and BSO; trifluoperazine had no effect on these two parameters . These results seemed to rule out any implication of calcium and cAMP levels in the contribution of these three chemosensitizers in the mechanisms of reversion of chemoresistance to Adriamycin.

Cancer Res, 1994 Aug 15, 54(16), 4488 - 92
Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin; Zhu Q et al.; Non-P-glycoprotein multidrug resistance of HL60/ADR cells appears to be related to overexpression of the MRP gene . Recent studies suggest that this gene may play an important role in a new form of cell resistance to certain chemotherapeutic agents . To examine mechanisms regulating transcriptional activity of this gene, a 2.2-kilobase 5'-flanking sequence of MRP has been isolated from a genomic library prepared from HL60/ADR cells . The 2.2-kilobase DNA fragment linked to the chloramphenicol acetyltransferase (CAT) gene in a reporter plasmid was found to be capable of driving expression of this gene in transient transfection experiments . This DNA containing promoter activity has been sequenced in its entirety and found to contain multiple putative regulatory sites . A series of deletion mutants linked to the CAT reporter gene was used to examine functional domains of the 2.2-kilobase sequence . The results suggest that promoter activity is contained in nucleotides -91 to +103 in a GC-rich region of the MRP genome . Promoter activity contained within this sequence, however, is modulated by both positive and negative regulatory elements . Certain of the regulatory sites contain consensus sequences for positive and negative regulatory elements which have been found in the promoter regions of other genes . Primer extension analysis indicates the presence of multiple major transcriptional start sites from the MRP promoter . Sequence analysis of MRP genomic and complementary DNAs has defined the exon/intron boundaries and the organization of a portion of the 5'-end region of the MRP genome . The results of these studies thus provide new insight in site-specific domains which may function in the regulation of MRP gene expression.

Cancer Res, 1994 Aug 15, 54(16), 4313 - 20
Glutathione-associated enzymes in anticancer drug resistance; Tew KD; The importance of thiol-mediated detoxification of anticancer drugs that produce toxic electrophiles has been of considerable interest to many investigators . Glutathione and glutathione S-transferases (GST) are the focus of much attention in characterizing drug resistant cells . However, ambiguous and sometimes conflicting data have complicated the field . This article attempts to clarify some of the confusion . The following observations are well established: (a) tumors express high levels of GST, especially GST psi, although the isozyme components vary quite markedly between tissues and the isozymes are inducible; (b) nitrogen mustards are good substrates for the GST alpha family of isozymes which are frequently overexpressed in cells with acquired resistance to these drugs; (c) most drugs of the multidrug-resistant phenotype have not been shown to be GST substrates and although GST psi is frequently overexpressed in multidrug-resistant cells, most indications are that this is an accompaniment to, rather than a cause of, the resistant phenotype; (d) transfection of GST complementary DNAs has produced some lines with increased resistance to alkylating agents . Most studies of the relationships between GST and resistance have overlooked the potential importance of other enzymes involved in the maintenance of cellular glutathione homeostasis, and this has complicated data interpretation . Translational research aimed at applying our knowledge of glutathione pathways has produced preclinical and clinical testing of some glutathione and GST inhibitors, with some encouraging preliminary results . In brief, GSTs are important determinants of drug response for some, not all, anticancer drugs . Caution should be encouraged in assessing cause/effect relationships between GST overexpression and resistance mechanisms.

J Biol Chem, 1994 Aug 12, 269(32), 20503 - 8
Cellular control of human multidrug resistance 1 (mdr-1) gene expression in absence and presence of gene amplification in human cancer cells; Kohno K et al.; The Kst-6 cell line is a human KB carcinoma cell line that has a stably integrated chloramphenicol acetyl-transferase (CAT) reporter gene under the control of the human mdr-1 promoter . Using Kst-6 cells as the parental cell line, five vincristine-resistant sublines, designated Kst-V5, -V25, -V50, -V75, and -V100, were isolated by exposure to increasing concentrations of drug . These sublines showed increased resistance to vincristine when compared with parental Kst-6 cells . Two sublines, V25-1 and V25-2, were further isolated from Kst-V25 after culture in the presence of vincristine, and V100-1 and V100-2 were also isolated from Kst-V100 . Southern analysis demonstrated mdr-1 gene amplification in Kst-50, Kst-V75, Kst-V100, V100-1, and V100-2 cells, respectively, but not in Kst-V5, Kst-V25, V25-1, and V25-2 cells . In contrast, increased mdr-1 expression was documented by Northern analysis in Kst-V25, V25-1, and V25-2 cells and five cell lines with mdr-1 amplification . Southern blot analysis utilizing a CAT probe demonstrated a stable copy number in all vincristine-resistant sublines . However, Northern analysis documented increased CAT expression in Kst-V5, Kst-V25, V25-1, and V25-2 cells but reduced mRNA levels in the cell line with amplification of the endogenous mdr-1 gene . Expression of the CAT gene was increased along with the endogenous mdr-1 gene in the early steps of the selection but decreased with the onset of gene amplification . There appeared almost similar mRNA levels of two trans-acting factor genes, SP-1 and MDR-NF1/YB-1, which are supposed to be involved in mdr-1 gene promoter activation, among all cell lines used in this study . These findings suggest that transcription of both the CAT gene fused to the mdr-1 promoter and the endogenous mdr-1 gene is enhanced through activation by trans-acting factors in the early steps of drug selection . However, the quantity of trans-activating factor is limiting, and with the onset of gene amplification, less is available for activation of the CAT gene, resulting in decreased expression.

J Biol Chem, 1994 Aug 5, 269(31), 19910 - 5
Membrane topology of multidrug resistance protein expressed in Escherichia coli . N-terminal domain; Bibi E et al.; Expression of eukaryotic polytopic membrane proteins in Escherichia coli could provide an invaluable system for structure-function studies . Recently, the functional expression of a mouse multidrug resistance protein (Mdr1) in E . coli was described (Bibi, E., Gros, P., and Kaback, H . R . (1993) Proc . Natl . Acad . Sci . U . S . A . 90, 9209-9213) . In the present study, the phoA gene fusion approach has been utilized to determine the membrane topology of the N-terminal domain of Mdr . The results support the idea that the N-terminal half of Mdr contains six transmembrane helices (TMs) . However, our observations suggest that the previously proposed TM4 (amino acid residues Thr214-Ala232) is located at the periplasmic face of the membrane . Alternatively, we propose that another stretch of amino acid residues (Leu243 (out) to Ile260 (in)) traverses the membrane . This assignment is indicated also by the following observations: 1) deletion of a segment containing the originally predicted TM4 (delta T214-K241) does not reverse the orientation of the Mdr-alkaline phosphatase hybrid that is located in the following cytoplasmic loop; 2) a "sandwich" chimera, in which alkaline phosphatase is inserted in-frame between amino acid residues Leu226 and Ser227, exhibits high alkaline phosphatase activity . The existence of an externally exposed hydrophobic domain in Mdr may have special structural and functional implications, and these may also be relevant to other members of the ABC superfamily.

J Natl Cancer Inst, 1994 Aug 3, 86(15), 1152 - 8
Mutation rates and mechanisms of resistance to etoposide determined from fluctuation analysis; Jaffrezou JP et al.; BACKGROUND: The major known mechanisms of resistance to etoposide include altered expression of its target enzyme, topoisomerase II (Topo II), and the multidrug-resistant phenotypes encoded by the mdr1 and MRP (multidrug resistance-associated protein) genes . There is little information regarding the distribution, frequency, and origin of these mechanisms in cancer cells . PURPOSE: We performed fluctuation analysis experiments with the human sarcoma cell line, MES-SA, to assess 1) if selection or induction mechanisms are involved in resistance to etoposide, 2) mutation rates for cellular resistance to etoposide, and 3) the nature of the single-step selected surviving clones . METHODS: Three groups of 10 flasks were seeded with more than 2000 cells each and allowed to grow to near confluence (approximately 3 x 10(6) cells per flask) . After reseeding, each group received etoposide for 1 week at a final concentration of 0.5 microM (group A), 1.0 microM (group B), and 5.0 microM (group C) . Surviving colonies in each of the 30 populations were scored and individually harvested . RESULTS: Mutation rates were estimated at 2.9 x 10(-6) (group A), 5.7 x 10(-7) (group B), and 1.7 x 10(-7) (group C) per cell generation . Of 61 propagated colonies, four of 26 from group A, five of 19 from group B, and none of 16 from group C were stably resistant . Analysis of variance supported the hypothesis of spontaneous mutations rather than induction, conferring etoposide resistance in groups A and B . Five of the stably resistant clones were cross-resistant to doxorubicin . Analysis by polymerase chain reaction failed to detect the expression of the multidrug-resistant gene mdr1 messenger RNA (mRNA) in any of the clones . No increase in expression of the MRP gene was observed . However, a significant decrease in both Topo II alpha and II beta mRNA (30%-70%) was found in six of seven stably resistant and six of six unstably resistant mutants . CONCLUSIONS: Our study demonstrates that resistance to etoposide arises spontaneously, with most clones surviving either stochastically or through very labile mechanisms of resistance . The experimental design has derived a set of resistant mutants from a single-step selection . In those clones, decreased expression of Topo II is the predominant mechanism selected . IMPLICATIONS: These findings suggest that stable resistance to etoposide chemotherapy may be acquired by selection of spontaneously arising mutants rather than induction by drug exposure . The stably resistant clones may represent descendants from a single mutational event in each population.

Biochem Pharmacol, 1994 Aug 3, 48(3), 609 - 12
Role of P-glycoprotein in dolastatin 10 resistance; Toppmeyer DL et al.; Dolastatin 10, a cytotoxic pentapeptide isolated from the mollusk Dolabella auricularia, exhibits potent antitumor activity . The present studies demonstrated that sublines of murine PC4 and human U-937 leukemia cells expressing a multidrug resistance (MDR) phenotype are cross-resistant to this agent . We also demonstrated that such resistance was reversed by verapamil . While these findings suggested the involvement of the P-glycoprotein (P-gp) in dolastatin 10 resistance, we performed similar studies in a CHO cell line transfected with the human mdr1 cDNA . Expression of P-gp in the transfected cells was associated with resistance to dolastatin 10 by a verapamil-sensitive mechanism . The demonstration that photoaffinity labeling of P-gp was decreased in the presence of dolastatin 10 further supports the interaction of this cytotoxic peptide with P-gp . Taken together, these findings suggest that resistance to dolastatin 10 is conferred, at least in part, by P-gp and that this cytotoxic peptide is a novel member of the MDR phenotype.

Biochemistry, 1994 Aug 2, 33(30), 8921 - 9
GTP-stimulated phosphorylation of P-glycoprotein in transporting vesicles from KB-V1 multidrug resistant cells; Lelong IH et al.; We have previously shown that GTP can replace ATP as an energy source to support vinblastine transport by the multidrug transporter P-glycoprotein (Pgp) in plasma membrane vesicles isolated from the multidrug resistant cell line KB-V1 {Lelong et al . (1992) FEBS Lett . 304, 256-260} . Like {gamma-32P}ATP, {gamma-32P}GTP was also able to phosphorylate Pgp in vitro . Unlabeled GTP enhanced the phosphorylation of the transporter by {gamma-32P}ATP, whereas unlabeled ATP inhibited incorporation of label . While phosphorylation by {gamma-32P}ATP was Mg(2+)-dependent, the enhanced phosphorylation of Pgp by GTP was supported by Mg2+ or Mn2+ and to a lesser extent, Ca2+ . Specific inhibitors of cAMP-dependent protein kinase, protein kinase C and cGMP-dependent protein kinase, did not affect phosphorylation . The phosphoprotein phosphatase inhibitor okadaic acid slightly enhanced phosphorylation, and vanadate more dramatically increased phosphorylation of the transporter . Tryptic maps of Pgp phosphorylated peptides indicate that addition of GTP altered the relative labeling of phosphopeptides . These results suggest that the overall phosphorylation of Pgp in vitro is determined by several different protein kinases and phosphatases, at least one of which may be GTP-regulated.

Cancer, 1994 Aug 1, 74(3), 834 - 41
The effects of cyclosporine on the pharmacokinetics of doxorubicin in patients with small cell lung cancer; Rushing DA et al.; BACKGROUND . The authors compared the pharmacokinetics of doxorubicin when administered with and without concomitant high dose cyclosporine for multidrug resistant (MDR) tumor modulation in small cell lung cancer . METHODS . Eight patients with small cell lung cancer served as their own controls and were studied first during an initial course of doxorubicin without cyclosporine, and then subsequently during a cyclosporine-modulated doxorubicin course . All patients received cyclophosphamide and vincristine in each course . Doxorubicin was administered as a 1-hour infusion after a 2-hour cyclosporine loading infusion, and cyclosporine was infused continuously for the next 48 hours . Serum concentrations of doxorubicin, doxorubicinol, and cyclosporine all were assayed by high-pressure liquid chromatography . Pharmacokinetic analysis of doxorubicin included area under the curve (AUC), clearance, apparent volume of distribution at steady state (Vss), and elimination half-life (T1/2) . The percent of change and surviving fraction of leukocyte count and platelets were determined as pharmacodynamic indices . RESULTS . Cyclosporine modulation increased the AUC0-36 of doxorubicin by 48% (P = 0.042) and the AUC0-36 of doxorubicinol by 443% (P = 0.0001), whereas the doxorubicin clearance declined by 37% (P = 0.0495) . No difference was found in the Vss or T1/2 for doxorubicin when cyclosporine was added to the regimen . The ratio of the doxorubicinol AUC0-36 to the doxorubicin AUC0-36 increased significantly with cyclosporine modulation (8.88 vs . 2.19; P = 0.001) . Drug-related toxicity was also greater with the cyclosporine-modulated course of doxorubicin . A 91% reduction in the leukocyte count followed the modified course, compared with an 84% reduction following the initial course (P = 0.0074) . A more prolonged and greater degree of myelosuppression was observed and a significant relationship was found between the systemic exposure to doxorubicin (defined by AUC) and the surviving fraction of the leukocyte count (r = -0.69; P = 0.006) . Similarly, the reduction in the platelet count was significantly greater after the cyclosporine-modulated course (72.8%) than after the initial course (36.4%) (P = 0.0016) . A significant correlation was found between the AUC of doxorubicinol and the surviving fraction of platelets (r = -0.71; P = 0.004) . In addition, patients showed decreased performance status associated with significant weight loss and severe myalgias . CONCLUSIONS . The addition of high dose cyclosporine for MDR modulation resulted in the significant alteration of doxorubicin disposition and remarkable toxicity in all patients . The mechanisms responsible for the decreased doxorubicin clearance may include cyclosporine's ability both to interfere with P-glycoprotein in normal tissues and to selectively inhibit the cytochrome P-450 enzyme system . Further study of this potentially significant drug-drug interaction is warranted.

Cancer Res, 1994 Aug 1, 54(15), 4138 - 43
Development and molecular characterization of a 2',2'-difluorodeoxycytidine-resistant variant of the human ovarian carcinoma cell line A2780; Ruiz van Haperen VW et al.; 2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity . In order to be active it must be phosphorylated by deoxycytidine kinase (dCK) . We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd . The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000 . Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd . AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine . There was no specific dCK activity in extracts from AG600 cells . Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts . Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs . Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000 . The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd . Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed . This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay . Injection of AG6000 cells s.c . into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors . No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors . These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.

Clin Infect Dis, 1994 Aug, 19(2), 299 - 308
Environmental control of tuberculosis: continuing controversy; Segal-Maurer S et al.; The incidence of tuberculosis--and, more important, that of multidrug-resistant tuberculosis--have risen drastically in the past decade . Nosocomial outbreaks have alerted health-care workers to the hazards of the spread of tuberculosis . The use of environmental control modalities (e.g., ventilation, air filtration, and ultraviolet irradiation) and personal protective devices has been explored in the medical, legislative, and public forums . New regulations and legislation have created controversy over the recommendations and their interpretation . In this review we present the theory behind the rational selection of environmental-control modalities and personal protective devices . We also offer suggestions about the application of specific control techniques and the revision of existing facilities to comply with new standards.

Clin Pharmacokinet, 1994 Aug, 27(2), 104 - 19
Clinical pharmacokinetics of halofantrine; Karbwang J et al.; Halofantrine is a phenanthrenemethanol antimalarial that is effective against asexual forms of multidrug-resistant Plasmodium falciparum malaria . It has no action on gametocytes or hypnozoites in the liver . The drug is administered as a racemic mixture but the (+)- and (-)-enantiomers show no difference in activity in vitro . Three formulations for oral administration are available for human use, i.e . tablets, capsules and suspension . Toxicity studies in animals suggest that halofantrine has very low toxicity both in short term and long term animal studies, and there has been no evidence of mutagenicity in these studies . Phase I, II and III clinical trials of halofantrine conducted in several tropical countries found the drug to be well tolerated and effective against multidrug-resistant P . falciparum malaria when 500mg was administered every 6 hours for 3 doses . The majority of clinical adverse effects reported, including nausea, vomiting, abdominal pain, diarrhoea, orthostatic hypotension, prolongation of QTc interval, pruritus and rash, have been mild and transient . There is wide interindividual variation in halofantrine absorption . The maximal plasma concentration (Cmax) is achieved approximately 6 hours after oral administration . Bioavailability is not dose-proportional for doses over 500mg, but there is a dose-proportional increase in Cmax and area under the plasma concentration-time curve (AUC) for doses between 250 and 500mg . In patients with malaria the bioavailability of halofantrine is decreased . The mean half-life of absorption is 4 hours and Cmax is significantly lower than that obtained in healthy individuals . Furthermore, halofantrine absorption is enhanced when the drug is taken with fatty food . Therefore, halofantrine should be taken with food to ensure optimal absorption in patients with malaria . The terminal elimination half-life is 5 days in patients with malaria . Halofantrine is biotransformed in the liver to its major metabolite N-debutyl-halofantrine . Plasma concentrations of this metabolite are observed soon after administration of halofantrine, but in much lower concentrations . The elimination half-life is similar to that of halofantrine . There have been increasing reports of halofantrine treatment failure, particularly in the eastern part of Thailand . The majority of treatment failures have been associated with incomplete drug absorption . The dose-dependent cardiotoxic effects (e.g . cardiac arrhythmia) are a major concern, particularly when the bioavailability of the drug cannot be predicted . Ongoing and future studies should aim at developing more appropriate drug formulation(s) and/or optimising dosage regimens . This will allow therapeutic concentrations to be achieved with minimum adverse effects, particularly cardiotoxicity.

Am J Physiol, 1994 Aug, 267(2 Pt 1), C650 - 8
Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells; Luckie DB et al.; The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein) . Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line . However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels . Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance . At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages . Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity . These results indicate that response curve to hypotonicity . These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.

Br J Cancer, 1994 Aug, 70(2), 239 - 43
Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme; Holm PS et al.; A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4) . This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA . In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA . We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system . A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype . The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line.

Biochem J, 1994 Aug 1, 301 ( Pt 3), 759 - 64
Ascorbic acid increases drug accumulation and reverses vincristine resistance of human non-small-cell lung-cancer cells; Chiang CD et al.; A human lung-cancer PC-9 subline with acquired resistance to vincristine (VCR), a chemotherapeutic agent, was established with incremental increases of the drug . The resistant PC-9 subline (PC-9/VCR) shows a 12-fold increase in resistance to VCR and a unique cross-resistance pattern: high cross-resistance to the potent VCR analogue colchicine (6.9-fold) and vinblastine (2.5-fold); lower cross-resistance to actinomycin D (1.8-fold), cisplatin (1.2-fold) and adriamycin (1.3-fold) and a sensitivity to melphalan and VP-16 which is similar to that of the parental cell line . A reduced accumulation of VCR in the resistant cells was demonstrated . Interestingly, the VCR resistance of the PC-9/VCR cell line was partially reversed by ascorbic acid, and the drug uptake was enhanced . In contrast, ascorbic acid had no effect on drug tolerance and drug accumulation was not observed in either PC-9 parental cells or known multidrug-resistant (MDR) cells, suggesting that VCR resistance in PC-9/VCR cells results essentially from reduced drug accumulation . It is worth noting that, whereas reduced drug accumulation in the PC-9/VCR cells was susceptible to modulation by ascorbic acid, the increased efflux rate characteristic of the resistant cells was not . Further, there was a higher efflux rate in resistant cells than in parental cells . DNA Southern- and RNA Northern-blot hybridization analyses indicate that PC-9/VCR cells do not contain amplified mdr genes or overexpress P-glycoprotein . In addition, the calcium-channel blocker verapamil, which acts as a competitive inhibitor of drug binding and efflux, did not affect the resistant phenotype of PC-9/VCR cells . These findings suggest an ascorbic acid-sensitive drug uptake mechanism which is important in mediating VCR resistance per se in human lung-cancer cells; this differs from the P-glycoprotein-mediated MDR mechanism.

Int J Cancer, 1994 Aug 1, 58(3), 376 - 9
Endogenous tumor necrosis factor functions as a resistant factor against adriamycin; Maeda M et al.; One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) and heat is the induction of reactive oxygen molecules . Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF and heat by inducing manganous superoxide dismutase (MnSOD) to scavenge the reactive oxygen molecules . Intracellular hydroxyl radical production is also involved in adriamycin-induced cytotoxicity . In this study, we therefore examined the possibility that enTNF may act as a protective protein against adriamycin-induced cytotoxicity in a manner similar to that in which it protects against exogenous TNF and heat . Adriamycin-sensitive L-M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with an expression vector which directs the synthesis of non-secretory-type human TNF (enTNF) . The stable transformants became resistant to adriamycin with increased levels of MnSOD . Conversely, when HeLa (human uterine cervical cancer) cells, which originally produce an appreciable amount of enTNF, were transfected with an anti-sense TNF mRNA expression vector to inhibit enTNF synthesis, their intracellular MnSOD activity was suppressed and adriamycin sensitivity was enhanced . However, no alterations in expression of multidrug-resistant gene products--P-170 glycoprotein, glutathione S-transferase pi (GST-pi) and the intracellular concentrations of glutathione (GSH)--were observed in these transfectants as compared to their parent cells . These results indicate that enTNF exerts its intracellular protective effect against adriamycin-induced cytotoxicity by the same mechanism as that against exogenous TNF and heat, namely scavenging reactive oxygen with induced MnSOD.

J Clin Oncol, 1994 Aug, 12(8), 1621 - 9
Paclitaxel in doxorubicin-refractory or mitoxantrone-refractory breast cancer: a phase I/II trial of 96-hour infusion; Wilson WH et al.; PURPOSE: A phase I study of paclitaxel infused over 96-hours was performed to determine toxicity, maximum-tolerated dose (MTD), and pharmacokinetics in patients with incurable lymphomas and solid tumors . A phase II study was performed at the MTD of paclitaxel in patients with doxorubicin/mitoxantrone-refractory metastatic breast cancer . PATIENTS AND METHODS: In the phase I study, paclitaxel dose levels ranged from 120 to 160 mg/m2, administered on a 21-day cycle . Patients with metastatic breast cancer who had either no response or a partial response (PR) to doxorubicin or mitoxantrone and had measurable disease were eligible for the phase I and II studies . Expression of the multidrug resistance (mdr-1) gene was determined in tumor biopsies by mRNA quantitative polymerase chain reaction . RESULTS: Twelve patients received a total of 73 cycles of paclitaxel on the phase I study . Dose-limiting mucositis and/or grade IV granulocytopenia was reached at 160 mg/m2, and 140 mg/m2 was selected as the phase II dose . Thirty-six consecutive patients with metastatic breast cancer were treated, of whom three were not assessable . The median age was 49 years, with disease in the liver and/or lung in 76% . Patients received a median of two prior regimens for metastatic disease, and 73% had no response to prior doxorubicin or mitoxantrone . Of 33 patients treated with paclitaxel, 16 patients (48%) achieved a PR and five (15%) achieved a minor response (MR) . With a median potential follow-up duration of 60 weeks, the median progression-free and overall survival durations were 27 and 43 weeks, respectively . No correlation was found between extent of prior treatment or prior response to doxorubicin/mitoxantrone, and response to paclitaxel . Paclitaxel pharmacokinetics showed a correlation between both granulocyte and mucosal toxicity, and serum steady-state concentrations (Css) more than 0.07 mumol/L . Patients with liver metastases had significantly decreased paclitaxel clearance and higher paclitaxel Css . Levels of mdr-1 were uniformly low in all tumor biopsies studied . CONCLUSION: The recommended phase II dose of paclitaxel is 140 mg/m2 in patients without liver metastases and 105 mg/m2 in patients with liver metastases . Ninety-six-hour infusions of paclitaxel were effective and well tolerated in patients with doxorubicin/mitoxantrone-refractory breast cancer . Prolonged infusion schedules may be more effective than shorter schedules and deserve further study.

J Cell Physiol, 1994 Aug, 160(2), 213 - 26
Stable differentiation of a human colon adenocarcinoma cell line by sodium butyrate is associated with multidrug resistance; Ho SB et al.; Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers . The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line . LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium . Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned . One subclone was designated LS174T-D . The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate . It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions . This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line . LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase . The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells . Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold) . Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines . Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines . However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells . Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin . Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation . This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

Mol Cell Biol, 1994 Aug, 14(8), 5202 - 11
Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells; Kuo MT et al.; Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes . These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells . However, the mechanism of this breakage is unknown . Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31 . This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments . Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene . These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells . Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents . Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

Gen Physiol Biophys, 1994 Aug, 13(4), 287 - 98
Time dependence of {3H}-vincristine accumulation by L1210 mouse leukemic cells . Effect of P-glycoprotein overexpression; Breier A et al.; Overexpression of P-glycoprotein (P-GP) accompanied by multidrug resistance (MDR) to diverse groups of cytostatics was developed by long-term adaptation of mouse leukemic cell line L1210 to vincristine . Two resistant sublines of cells characterized by ID50 values for vincristine 1.05 mg/l (L1210/VCR-1) and 2.3 mg/l (L1210/VCR-2), respectively, were used . The sensitive parental cell line L1210 had the ID50 value for vincristine around 0.01 mg/l . Overexpression of P-GP induced by the adaptation procedure was found to be accompanied by an increase in the mean cell diameter from 10.28 +/- 1.60 microns (mean +/- SD, n = 122) for sensitive L1210 cells to 17.82 +/- 2.59 microns (n = 120) and 37.26 +/- 5.72 microns (n = 121) for L1210/VCR-1 and L1210/VCR-2 resistant cell sublines, respectively . Significant decrease in ability to accumulate {3H}-vincristine from cultivation medium was observed for both resistant cell sublines in comparison to sensitive cells . Accumulation of {3H}-vincristine by sensitive cells is secured only by passive diffusion of the drug across the plasma membrane . Contrary to that, active efflux of drug operating against its diffusion across the plasma membrane should be assumed as a factor influencing the {3H}-vincristine accumulation by resistant cells . Indeed, the time dependence of {3H}-vincristine accumulation by sensitive cells could be fitted using simple monoexponential kinetic dependence in contrast to biexponential kinetic dependences that are necessary for fitting {3H}-vincristine accumulation by both resistant cell sublines . Kinetic analysis of the experimental data indicates that accumulation of {3H}-vincristine by sensitive cells grows to a plateau reflecting probably the equilibrium of drug concentration in the intracellular and extracellular space . On the contrary, accumulation of {3H}-vincristine by both resistant cell sublines was stabilized after an initial growth on a considerably lower level than it was observed for the sensitive cells in the equilibrium.

J Assoc Physicians India, 1994 Aug, 42(8), 599 - 600
A study of culture positive multidrug resistant enteric fever--changing pattern and emerging resistance to ciprofloxacin; Daga MK et al.; The present prospective study was carried out to observe the changing trends in the clinical pattern and multidrug resistance in typhoid fever . Fever was the main presenting feature . Other associated features were headache, vomiting, diarrhoea, altered sensorium and jaundice . Out of 78 patients, one patient died due to enteric encephalopathy and other due to septicaemia with peripheral circulatory failure . 12 patients responded to chloramphenicol and gentamycin . 51 patients responded to ciprofloxacin, while remaining 9 patients responded to combination of cefotaxime and amikacin . Three patients showed in vitro resistance to ciprofloxacin and two out of these also showed no response in vivo . This study re-emphasises the changing pattern, prolonged course and role of quinolones especially ciprofloxacin in the management of drug resistant typhoid fever, but at the same time indicates that ciprofloxacin is not the drug of choice in all cases of typhoid fever and resistance to it may be seen in some cases, where other drugs have to be used.

Chest, 1994 Aug, 106(2), 431 - 4
Preventive therapy for contacts of multidrug-resistant tuberculosis . A Delphi survey; Passannante MR et al.; OBJECTIVE: Several outbreaks of multidrug resistant tuberculosis (MDR-TB) have recently occurred in which healthcare workers and others have become infected . Given the lack of clinical data to guide preventive therapy for such contacts, a Delphi survey of a panel of 31 TB therapy experts was undertaken to identify a consensus regimen . DESIGN: An initial questionnaire presented three scenarios describing persons with significant exposure to MDR-TB and with new tuberculin skin test reactions > 15 mm (except one anergic patient) without evidence of disease . Panelists were asked to suggest possible preventive therapy regimens . METHODS: During a second round survey, the panel members were asked to review the suggested regimens provided for each scenario and to rank them from one to nine as extremely inappropriate to extremely appropriate . Results of this second survey were tabulated and shared with the members of the panel who were then asked to rerank each regimen in light of the previous cumulative panel responses . RESULTS: No specific regimen achieved initial positive consensus by predefined criteria . In two of the three scenarios the no treatment option, however, was deemed clearly inappropriate . The data were also analyzed by what percentage of respondents who ranked a regimen as at all appropriate (ie, six or more on the nine point scale) . For scenarios involving a nurse, an HIV-positive tuberculin reactor, and a patient who was anergic HIV-positive, treatment with pyrazinamide 1,500 mg daily with ciprofloxacin 750 mg twice a day for 4 months received a somewhat appropriate rating from more than 50 percent of respondents . CONCLUSIONS: The management of persons exposed to and infected by patients with MDR-TB has become a serious problem in the context of MDR-TB outbreaks . This panel of experts agreed that some form of preventive therapy was warranted; however, they were not able to reach defined consensus on what regimen should be used, although a regimen of pyrazinamide 1,500 mg daily with ciprofloxacin 750 mg twice a day for 4 months was considered somewhat appropriate . Clinical data on the efficacy of alternative preventive therapy regimens for such contacts are urgently needed.

Curr Opin Obstet Gynecol, 1994 Aug, 6(4), 373 - 6
Re-emergence of tuberculosis; Baker DA; Tuberculosis (TB) has remained a major infectious disease in the world . With the AIDS epidemic, the emergence of TB as a cause of severe and life-threatening infection has become a common-place occurrence in many countries . In the USA, the increase of new cases of TB average at the rate of 20% each year . Of great alarm is the findings of multidrug-resistant TB (MDR-TB) with outbreaks in hospitals throughout the world, as well as in other institutions . No health care worker is immune from exposure to TB . TB control requires education of health care workers, a program of ongoing screening, isolation of patients early in the course of their disease, and administration of multiple anti-TB drug to cover and treat MDR-TB . Newer diagnostic methods are needed as well as more active and less toxic drug therapies . Controlled clinical trials are required to determine optimum therapy.

Exp Toxicol Pathol, 1994 Aug, 46(3), 257 - 62
Frequency of metastasis in Syrian hamster tumor cells selected for low levels of "typical" multidrug resistance; Shtil A et al.; The metastatic capability of cells at the initial stages of selection for "typical" (P-glycoprotein-mediated) multidrug resistance (MDR) was studied . Two independent sublines, 2SC/4-1 and 2SC/4-2 (11-12.4-fold resistant), and their 23- to 23.7-fold resistant 2SC/20-1 and 2SC/20-2 variants were isolated from highly tumorigenic (TrD50 = 10 cells) and highly metastatic Rous sarcoma, virus-transformed Syrian hamster fibroblast HET-SR-2SC-LNM line for resistance to colchicine . 2SC/4 cells were less tumorigenic (TrD50 = 70 cells) but as highly metastatic as parental counterparts . In contrast, both 2SC/20 variants showed a decrease in tumorigenicity (TrD50 = 320 cells) and in the capability to produce spontaneous distant metastases . 2SC/20 cells almost lost the ability to colonize lungs in experimental metastasis assay . The autophosphorylation of pp60src tyrosine kinase in 2SC/20 cells was unaltered . The results suggest that i) selection of tumor cells for low levels of "typical" MDR leads to a decrease in the frequency of spontaneous and experimental metastases, and ii) alterations of malignancy in these cells are not caused by an impairment of function of a transforming oncogene.

Genes Chromosomes Cancer, 1994 Aug, 10(4), 267 - 74
Putative "MDR enhancer" is located on human chromosome 20 and not linked to the MDR1 gene on chromosome 7; Germann UA et al.; The physiologic expression of the human multidrug resistance MDR1 gene product P-glycoprotein is controlled in a tissue- and cell-specific manner, but the regulatory mechanisms have not been characterized in great detail . Studies by Kohno et al . {(1990) J Biol Chem 265:19690-19696} suggested that a tissue-specific enhancer element located approximately 10 kb upstream from the major MDR1 transcription start site may act to increase the levels of transcription in cultured adrenal and kidney cells . Using this putative "MDR enhancer" as a probe, we isolated a 14 kb DNA fragment from a genomic DNA library prepared from human fetal liver . The restriction map and partial nucleotide sequence of this DNA fragment were consistent with the previously described data obtained for a similar piece of genomic DNA derived from human placenta by Kohno et al . (ibid.) . Pulsed-field gel electrophoresis of large genomic DNA fragments, however, showed that the DNA sequences, including the putative "MDR enhancer," were not linked to the MDR1 gene . Fluorescence in situ hybridization analysis revealed that this enhancer-like element is located on chromosome 20 at band q13.1 and is, therefore, distinct from the MDR locus on chromosome 7, band q21.1 . Thus, this putative regulatory element does not modulate the tissue specificity of expression of the MDR1 gene in vivo, but may play a role in the regulation of expression of another, so far unknown gene.

J Clin Oncol, 1994 Aug, 12(8), 1584 - 91
Clinical modulation of multidrug resistance in multiple myeloma: effect of cyclosporine on resistant tumor cells; Sonneveld P et al.; PURPOSE: In multiple myeloma (MM) refractory to doxorubicin (DXR) and/or vincristine (VCR), myeloma cells frequently express the multidrug resistance (MDR) phenotype, associated with overexpression of P-glycoprotein (Pgp), which acts as a drug efflux pump . Recently, studies have shown that clinical resistance can be modulated by drug resistance modifiers . The present study was performed to investigate if MDR modulation in vivo is caused by a direct effect of cyclosporine (CSA) on resistant myeloma plasma cells (PC) . PATIENTS AND METHODS: Eight patients with VAD-refractory MM were treated with DXR, VCR, and dexamethasone (VAD) plus CSA . Pgp expression in PC was determined by flow cytometry/immunocytochemistry before and after clinical treatment . Functional Pgp expression was determined by the effect of CSA on the intracellular accumulation of DXR and VCR . RESULTS: Five of eight patients responded to VAD/CSA . The percentage of Pgp-positive (Pgp+) PC was 30% to 100% (median, 90%) before treatment and 4 to 90% (median, 40%) after treatment . CD56+/- or CD38+/- PC had identical Pgp expression . CSA, as well as SDZ PSC 833, but not dexamethasone, increased pretreatment intracellular accumulation of DXR and VCR in Pgp+ PC in three of four and six of six patients, respectively . After clinical treatment, in vitro drug accumulation in residual Pgp-negative (Pgp-) PC of four of four responding patients was not further modulated by CSA or SDZ PSC 833 . At later relapse, PC of two of four patients remained Pgp- . CONCLUSION: These data indicate that Pgp overexpression is functional in refractory myeloma and that clinical modulation of MDR by CSA is mediated through an inhibition of Pgp-associated drug efflux . Pgp-expressing PC can be eliminated by clinical treatment with VAD/CSA.

Lancet, 1994 Jul 30, 344(8918), 293 - 8
Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study; Heym B et al.; Tuberculosis-control programmes are compromised by the increased frequency of multidrug-resistant strains of Mycobacterium tuberculosis . We used the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis techniques to establish the molecular basis of resistance in 37 drug-resistant isolates of M tuberculosis, and correlated these findings with clinical and antibiotic-sensitivity data . Resistance to isoniazid was found in 36 strains, 16 of which were also resistant to ethionamide . Of the 36 isoniazid-resistant strains, 23 had mutations in the katG gene, and 5 of these also had mutations in the inhA gene . A further 5 strains had alterations in the inhA locus without the katG gene being mutated . Rifampicin resistance was less frequent (13 strains) and usually associated with isoniazid resistance (11 of 13 strains) . Mutations in the rpoB gene were detected for all these rifampicin-resistant isolates . Mutations in the rpsL and rrs genes, associated with streptomycin resistance, were found in 13 of 25 and 2 of 25 streptomycin-resistant strains, respectively . The same chromosomal mutations, or combinations of mutations, were found in strains displaying single or multidrug resistance, from cases of both primary and secondary resistance, and from patients infected with human immunodeficiency virus . Thus, multidrug resistance is not due to a novel mechanism and tuberculosis chemotherapy is not subject to a new threat.

Cancer Lett, 1994 Jul 29, 82(2), 185 - 8
Expression of multidrug resistance-associated protein (MRP) in anaplastic carcinoma of the thyroid; Sugawara I et al.; Although the incidence is very low, the prognosis of anaplastic carcinoma of the thyroid is very poor regardless of the results of various therapeutic trials . We found that the mechanism of anti-cancer drug resistance in anaplastic carcinoma of the thyroid was not explicable only in terms of expression of the mdr1 and its gene product, P-glycoprotein . Therefore, expression of multidrug resistance-associated protein (MRP) mRNA was examined in 11 anaplastic thyroid carcinomas and eight anaplastic thyroid carcinoma cell lines . High MRP mRNA expression was recognized in 7/11 and 8/8, respectively . Our results may contribute to elucidation of the mechanism of anti-cancer drug resistance in this neoplasm.

N Engl J Med, 1994 Jul 21, 331(3), 169 - 73
The use of high-efficiency particulate air-filter respirators to protect hospital workers from tuberculosis . A cost-effectiveness analysis; Adal KA et al.; BACKGROUND . After outbreaks of multidrug-resistant tuberculosis, the Centers for Disease Control and Prevention proposed the use of respirators with high-efficiency particulate air filters (HEPA respirators) as part of isolation precautions against tuberculosis, along with a respiratory-protection program for health care workers that includes medical evaluation, training, and tests of the fit of the respirators . Each HEPA respirator costs between $7.51 and $9.08, about 10 times the cost of respirators currently used . METHODS . We conducted a cost-effectiveness analysis using data from the University of Virginia Hospital on exposure to patients with tuberculosis and rates at which the purified-protein-derivative (PPD) skin test became positive in hospital workers . The costs of a respiratory-protection program were based on those of an existing program for workers dealing with hazardous substances . RESULTS . During 1992, 11 patients with documented tuberculosis were admitted to our hospital . Eight of 3852 workers (0.2 percent) had PPD tests that became positive . Five of these conversions were believed to be due to the booster phenomenon; one followed unprotected exposure to a patient not yet in isolation; the other two occurred in workers who had never entered a tuberculosis isolation room . These data suggest that it will take more than one year for the use of HEPA respirators to prevent a single conversion of the PPD test . Assuming that one conversion is prevented per year, however, it would take 41 years at out hospital to prevent one case of occupationally acquired tuberculosis, at a cost of $1.3 million to $18.5 million . CONCLUSIONS . Given the effectiveness of currently recommended measures to prevent nosocomial transmission of tuberculosis, the addition of HEPA respirators would offer negligible protective efficacy at great cost.

Biochem Pharmacol, 1994 Jul 19, 48(2), 391 - 401
Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100); Sognier MA et al.; A multidrug-resistant Chinese hamster cell line, LZ-8, was subcultured in increasing levels of doxorubicin (DOX) until capable of growth in 100 micrograms/mL DOX . This new derivative, designated LZ-100, is the most DOX-resistant line in the LZ series, based on a comparison of Ki-1 values from cell survival studies . This increased level of drug resistance in LZ-100 cells did not result from (i) higher levels of P-glycoprotein (P-gp) in the plasma membrane compared with LZ-8 cells, since this protein constitutes approximately 20% of the total plasma membrane protein in both cell lines, or (ii) more efficient drug pumping by the same amount of P-gp, since efflux of rhodamine 123 and DOX was comparable in the two cell lines . However, an altered drug distribution was observed in LZ-100 cells compared with wild-type V79 cells; in LZ-100 cells DOX was largely excluded from the nucleus and was sequestered in vesicles in the cytoplasm . The number of vesicles per cell seen after DOX exposure corresponded with the level of drug resistance achieved by the LZ cell lines studied . DOX concentration-response experiments revealed that vesicle formation exhibited a biphasic relationship, with an initial rapid increase followed by a plateau where no further increase was observed . Time-course studies in LZ-100 cells revealed that the maximum number of DOX-containing vesicles per cell occurred 3-4 hr following initiation of DOX treatment . Radiation exposure (10 Gy) immediately preceding DOX treatment decreased the number of vesicles formed in LZ-100 cells by more than one-half and altered the subcellular distribution of DOX from an almost exclusively cytoplasmic to a homogeneous nuclear/cytoplasmic distribution . This redistribution was not a result of radiation inhibition of P-gp efflux . The inhibitory effect of radiation on vesicle formation increased with increasing radiation dose up to 10 Gy . Drug-containing vesicles were also observed in LZ-100 cells following exposure to mitoxantrone or daunorubicin (to which LZ-100 cells are also resistant), but fewer vesicles were observed than with DOX . These studies demonstrate that the drug sequestration phenomenon (i) occurs in cells exhibiting widely different levels of drug resistance, (ii) correlates with the level of drug resistance in LZ cell lines, (iii) occurs rapidly following exposure to DOX, mitoxantrone, or daunorubicin, and (iv) can be inhibited by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Pharmacol, 1994 Jul 19, 48(2), 287 - 92
Antiestrogens and steroid hormones: substrates of the human P-glycoprotein; Rao US et al.; Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp) . Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992) . The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp . In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp . Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil . Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively . Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone . Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil . These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.

Biochem Pharmacol, 1994 Jul 19, 48(2), 277 - 85
Reversal of P-glycoprotein-mediated multidrug resistance by pure anti-oestrogens and novel tamoxifen derivatives; Kirk J et al.; In this study the ability of five novel anti-oestrogens {4-iodotamoxifen, pyrrolidino-4-iodotamoxifen, ethyl bromide tamoxifen (EBTx), ICI 164,384 (ICI 164) and ICI 182,780} to alter drug toxicity to multidrug resistant cell lines have been compared . The effect of these compounds on ATP-dependent vinblastine (VBL) transport was also tested using inside-out vesicles (IOV) prepared from highly P-glycoprotein (Pgp)-expressing CCRF-CEM/VBL1000 cells . The pure anti-oestrogen ICI 164 was most effective, enhancing doxorubicin and VBL toxicity to MCF-7Adr cells 25- and 35-fold, respectively, and was also the best inhibitor of ATP-dependent {3H}VBL accumulation by IOV . Pure anti-oestrogens, tamoxifen and iodotamoxifens completely reversed VBL resistance in the mdr1 transfected lung cancer cell line, S1/1.1, where resistance relative to wild-type cells was mediated solely by Pgp . The membrane impermeant tamoxifen derivative EBTx did not modify drug resistance, yet was as effective an inhibitor of VBL accumulation by inside-out Pgp-positive vesicles as tamoxifen . This indicates an intracellular role for tamoxifen and its derivatives in the modulation of Pgp-mediated drug resistance.

Biochem Biophys Res Commun, 1994 Jul 15, 202(1), 211 - 7
Increased phospholipase D activity in multidrug resistant breast cancer cells; Welsh CJ et al.; An adriamycin-resistant MCF-7 cell line (300-fold increased resistance vs . wild type MCF-7) showed an increased phorbol ester-stimulated phospholipase D activity of approximately 4-6 times over that found in wild type cells . Phospholipase D activity was assessed by monitoring the mass of phorbol ester-induced phosphatidylethanol . The cellular phospholipase D activity was time- and phorbol ester-concentration-dependent and was obvious whether measured as an increase in the mass of PEt or in the production of 3H-labeled phosphatidylethanol in cells prelabeled with {3H}myristic acid . Phorbol ester also stimulated increases in the production of the mass of cellular diacylglycerol and phosphatidic acid in the adriamycin-resistant cells vs . the wild-type cells . Tests with a series of drug-resistant MCF-7 cell lines revealed a positive correlation between increased drug resistance and phorbol ester-stimulated phospholipase D activity.

Biochem Biophys Res Commun, 1994 Jul 15, 202(1), 606 - 12
Tamoxifen aziridine, a novel affinity probe for P-glycoprotein in multidrug resistant cells; Safa AR et al.; In this study for the first time we used an electrophilic analog of tamoxifen, {3H}tamoxifen aziridine, and demonstrated that it covalently and specifically binds to P-glycoprotein in multidrug resistant cells . Tamoxifen and its metabolites, N-desmethyltamoxifen and 4-hydroxytamoxifen, were potent inhibitors of {3H}tamoxifen aziridine binding to P-glycoprotein with 4-hydroxytamoxifen > tamoxifen > N-desmethyltamoxifen . The multidrug resistance-related drugs inhibited {3H}tamoxifen aziridine binding with vinblastine > vincristine > doxorubicin > actinomycin D, while colchicine enhanced the binding . Moreover, the multidrug resistance modulators verapamil, nicardipine, diltiazem, prenylamine, cyclosporin A, FK506, dibucaine, reserpine, monensin and progesterone were all potent inhibitors of {3H}tamoxifen aziridine binding to P-glycoprotein . Our data provide the first evidence that {3H}tamoxifen aziridine directly binds to P-glycoprotein and interacts with the binding sites for multidrug resistance-related drugs and modulators.

Gene, 1994 Jul 8, 144(2), 229 - 36
Utilization of multiple polyadenylation signals in the human RHOA protooncogene; Moscow JA et al.; Little is known regarding the regulation of expression of the RHOA protooncogene, a member of the family of genes encoding Ras-related GTP-binding proteins . We have previously reported that the 3' untranslated region (UTR) of RHOA was contained within a genomic sequence which flanked the 5' end of the human glutathione peroxidase 1-encoding gene {J.A . Moscow et al., J . Biol . Chem . 267 (1992) 5949-5958} . Our previous studies revealed the presence of multiple (1.8 and 1.5 kb) RHOA mRNA species in breast cancer cell lines and of three putative polyadenylation signals in the RHOA 3' UTR . In this report, we have isolated several RHOA cDNAs from a multidrug-resistant MCF-7 human breast cancer cell line . Sequence analyses of these RHOA cDNA clones indicate that multiple polyadenylation signals are used to terminate RHOA transcripts . RNase-protection analysis demonstrated that all three polyadenylation signals are utilized in breast cancer cell lines and RNA stability studies demonstrated that RHOA RNA species with different 3' ends have equivalent stability . Since little is known about the RNA expression of RHOA in human tumors, and since both activated and non-activated RHOA genes possess transformation potential, we analyzed RHOA mRNA in lung and colon tumors by Northern blot and RNase-protection analyses . In all eight lung tumors examined, RHOA RNA levels were decreased relative to the level in normal surrounding tissue, whereas RHOA expression was decreased in only two of six colon tumors . We also found that lovastatin-induced cell cycle arrest resulted in increased RHOA RNA expression in breast cancer cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochem Photobiol, 1994 Jul, 60(1), 61 - 3
Impaired accumulation of a cationic photosensitizing agent by a cell line exhibiting multidrug resistance; Kessel D et al.; Transport and accumulation of copper benzochlorin iminium salt (CDS1), a cationic photosensitizing agent, were examined using the P388/ADR murine leukemia, which exhibits the MDR (multidrug resistance) phenotype, and the wild-type parent cell line, P388 . The recent availability of radioactive CDS1 permitted kinetic studies at drug levels in the submicromolar range . Exclusion of CDS1 by P388/ADR cells could be demonstrated, indicating that this agent is a substrate for the outward transport system associated with MDR . These results have implications with regard to the efficacy of cationic photosensitizers against this common neoplastic phenotype . The CDS1 was readily accumulated by P388 cells and by P388/ADR cells when the outward transport system was inhibited . Under these conditions, CDS1 was tightly bound and could not be washed out even when the outward transport system was reactivated.

Leukemia, 1994 Jul, 8(7), 1191 - 6
Comparative activity of tamoxifen and N-desmethyltamoxifen in human multidrug resistant leukemia cell lines; Berman E et al.; Tamoxifen (Tmx) is one of a number of agents that can reverse the multidrug resistant (MDR) phenotype in vitro; it is unique, however, in that both the parent compound and the main metabolite, N-desmethyltamoxifen (NDMTmx), have long plasma half-lives, 7 and 14 days, respectively . To determine whether NDMTmx was as active as the parent compound in restoring sensitivity to daunorubicin (DNR) in vitro, we performed uptake and retention studies in the MDR-positive human leukemia cell lines CEM-VLB and HL-60/RV+ using laser flow cytometry to quantitate intracellular DNR concentration . Cells incubated with DNR and NDMTmx 10 microM demonstrated increased intracellular DNR levels that were very similar to those obtained with DNR and Tmx 10 microM . Cellular retention of DNR was also measured after incubation with Tmx 10 microM or NDMTmx 10 microM and resuspension in fresh medium containing Tmx or NDMTmx in order to stimulate in vivo conditions . Washout curves in both cell lines were similar with both Tmx and NDMTmx . Finally, clonogenic experiments were performed to determine whether Tmx and NDMTmx demonstrated the same degree of cytotoxicity . The combination of Tmx 10 microM plus DNR and that of NDMTmx 10 microM plus DNR each resulted in > 80% growth inhibition . These results suggest that NDMTmx is as active as Tmx in restoring sensitivity to DNR in vitro, a finding which may have important implications in clinical trials that assess the ability of Tmx to reverse the MDR phenotype.

Leukemia, 1994 Jul, 8(7), 1108 - 12
The immunophenotype of acute promyelocytic leukemia (APL): an ECOG study; Paietta E et al.; In 452 adult patients with de novo acute myeloid leukemia (AML), a series of 22 monoclonal antibodies was used to identify immunophenotypic characteristics of acute promyelocytic leukemia (APL) as compared to other AMLs (groups FAB M1/M2 and M4/M5) . Only those patients with FAB M3 cytology were included in the analysis for which APL was confirmed by the presence of the t(15;17) cytogenetic aberration and the detection of the PML/RAR alpha gene fusion transcript by PCR amplification (35 cases) . Significantly fewer APL blast cells were positive for the stem cell antigen, CD34 (p = 0.0001) as well as for HLA-DR (p < 0.0001) . With respect to myeloid antigens, APLs less frequently expressed the myelomonocytic antigens, CD11b (p = 0.0001) and CD14 (p = 0.0013), whereas expression of CD33, a pan-myeloid marker, was more frequent in APL (p = 0.0001) . CD15, the X-hapten carbohydrate structure (lacto-N-fucopentaose-III), typically expressed at the maturation stage of normal promyelocytes, was found to be sialylated on APL blasts as recognized by differential binding of the anti-CD15 antibodies, VIM-D5 (non-sialylated CD15) and VEP-9 (sialylated CD15) . Expression of the T-cell associated CD7 antigen was rarer on APL than non-APL cells (p = 0.0001), as was that of the multidrug resistance P-glycoprotein (p = 0.0038) . Marginal correlations existed between antigen profile (particularly CD2) and the type of PML/RAR alpha transcripts . In addition to its unique genotypic features, these data establish APL as a distinct immunophenotypic entity.

J Nucl Med, 1994 Jul, 35(7), 1179 - 84
In vivo uptake of carbon-14-colchicine for identification of tumor multidrug resistance; Mehta BM et al.; A major limitation in the treatment of cancer with natural product chemotherapeutic agents is the development of multidrug resistance (MDR) . Multidrug resistance is attributed to enhanced expression of the multidrug resistance gene MDR1 . Colchicine (CHC) is known to be one of the MDR drugs . We have previously demonstrated that it is possible to distinguish multidrug-resistant tumors from multidrug-sensitive tumors in vivo on the basis of tritium (3H) uptake following injection of 3H-CHC . METHODS: The present studies were carried out in xenografted animals using 14C-CHC which may be more indicative of 11C-labeled CHC distribution with regard to circulating metabolites, since metabolic processes following injection of (ring C, methoxy-11C)-CHC may produce significant amounts of circulating 1-carbon fragments (i.e., methanol and/or formaldehyde) . Experiments were carried out at a dose of 2 mg/kg . RESULTS: Activity concentration per injected dose was approximately twice as great in sensitive as in resistant tumors (p < 0.05) at 60 min following intravenous injection of 14C-CHS . About 75% of total activity was CHC in the sensitive tumors . The findings are further confirmed by the quantitative autoradiographic evaluation of resistant and sensitive tumors . CONCLUSIONS: These studies confirm our previous observations that it is possible to noninvasively distinguish multidrug-resistant tumors from sensitive tumors in vivo based on uptake of an injected MDR drug using a 14C-labeled CHC at the same position and of comparable specific activity to a 11C-CHC tracer used for PET imaging.

J Infect Dis, 1994 Jul, 170(1), 151 - 6
Multidrug-resistant tuberculosis in the New York State prison system, 1990-1991; Valway SE et al.; Three epidemiologically linked multidrug-resistant (MDR) tuberculosis (TB) outbreaks in 1990-1991 involving New York State (NYS) inmates suggested MDR-TB was widespread in NYS prisons . Inmate lists were linked to 1990-1992 TB registries, medical records were reviewed, and movement histories for inmates with MDR-TB were examined within and between prisons and hospitals . In 1990-1991, 171 inmates were diagnosed with TB . This rate (156.2/100,000) was significantly higher than the 1990-1991 US rate (10.4/100,000) and the 1986 rate among NYS inmates (105.5/100,000) . Of 171 cases, 155 were cultured-confirmed; 37 (32%) of 116 with drug susceptibilities determined had MDR-TB . Two other inmates with TB before 1990 were diagnosed with MDR-TB in 1990-1991 . Of 39 inmates with MDR-TB, 38 (97%) were infected with the human immunodeficiency virus and 34 (87%) have died . These 39 lived in 23 of the 68 NYS prisons while potentially infectious; 12 were transferred through 20 prisons while ill with MDR-TB . Policies of correctional systems on infection control and inmate transfers need to be reevaluated to prevent spread of TB.

Int J Cancer, 1994 Jul 1, 58(1), 85 - 94
Hydroxyrubicin, a deaminated derivative of doxorubicin, inhibits mammalian DNA topoisomerase II and partially circumvents multidrug resistance; Solary E et al.; In vivo effectiveness of doxorubicin remains restricted due to toxicity and drug resistance . Hydroxyrubicin is a synthetic analog of doxorubicin in which the basic amino group at the C-3' has been replaced by a hydroxyl group in order to overcome recognition by the multidrug resistant (MDR) P-glycoprotein and limit cardiotoxicity . The present study shows that hydroxyrubicin is a less potent intercalator than doxorubicin . Induction of topoisomerase II-mediated DNA cleavage in the human c-myc origin by the two drugs was similar, reaching a maximum at 0.5 microM . Results from the NCI Cell Screening program indicate a relatively good correlation between the cytotoxicity of the 2 drugs on 55 cell lines of various origins (r = 0.723) . Using a clonogenic assay, we observed that hydroxyrubicin was 20-fold more cytotoxic against the MDR KB-V1 cell line than doxorubicin and was slightly more cytotoxic than doxorubicin in the sensitive KB3.1 cell line . Uptake studies showed that doxorubicin was retained up to 1 hr in KB3.1 cells and rapidly eliminated from resistant KB-V1 cells . In contrast, hydroxyrubicin was rapidly eliminated from both sensitive KB3.1 and MDR-positive KB-V1 cells . Both drugs induced protein-linked DNA single-strand breaks (SSBs) in both KB3.1 and KB-V1 cells, which is consistent with topoisomerase inhibition . However, the kinetics of DNA SSBs induced by both drugs was very different . DNA breaks disappeared quickly in both KB3.1 and KB-V1 cell lines after hydroxyrubicin removal while DNA breaks induced by doxorubicin disappeared very slowly in KB3.1 cells and rapidly in KB-V1 cells . We conclude that removal of the basic amino group at the C-3' of doxorubicin modifies drug transport and partially circumvents MDR without changing topoisomerase II inhibition when compared with doxorubicin.

Hematol Oncol, 1994 Jul-Aug, 12(4), 155 - 61
Multiple drug resistance, the MDR gene, and the law of maximum perversity as it applies to oncology: an hypothesis; Spiers AS; The so-called Law of Maximum Perversity, generally stated, says that when more than one outcome is possible, that which is the more adverse is the outcome most likely to occur . In medical oncology, the most obvious expression of this law is the fact that all the neoplasms that are most sensitive to cytotoxic drugs and are most curable by chemotherapy, are rare and in numerical terms are not important as causes of cancer-related deaths . Conversely, the most commonly encountered neoplasms that make up the bulk of oncologic practice and that cause over 90 per cent of cancer-related deaths, are all relatively resistant to cytotoxic agents and are virtually never curable by chemotherapy administered in standard (i.e . non-transplant) doses . It is postulated that the biological properties and the normal tissue distribution of the multidrug resistance (MDR) gene and its product p-170 glycoprotein explain the observed incidences and distribution of tumours that are sensitive or insensitive to cytotoxic agents . The normal role of MDR is to protect cells from environmental carcinogens, and the tissues that are most at risk, and most richly supplied with MDR, will produce drug-resistant neoplasms . Current attempts at MDR reversal may facilitate the treatment of some tumours that are resistant to multiple drugs but may cause severe toxic effects as a consequence of abrogating the largely unknown physiologic functions of P-170.

Hum Gene Ther, 1994 Jul, 5(7), 845 - 52
Retroviral delivery of DNA into the livers of transgenic mice bearing premalignant and malignant hepatocellular carcinomas; Kimura O et al.; To develop gene therapy for hepatocellular carcinoma (HCC), we infused mice through the portal vein with retrovirus carrying the Escherichia coli beta-galactosidase reporter gene under the transcriptional control of the viral long terminal repeat (LTR) and the promoter from the mouse multidrug resistance gene mdr1b . Two transgenic mouse HCC models were used, one bearing the human hepatitis B viral envelope protein and the other SV40 T antigen . These animals develop HCC with predictable pathological manifestations . The viral transduction efficiency appeared to depend upon the stage of the disease in the animals . The most efficient transduction occurred when the livers had developed microscopic nodular hyperplasia; in some cases as many as 0.01-0.1 copies/cell were transduced . The transduction efficiency was lower in the late stage of the disease when livers had a heavy tumor burden and in the early stage when no lesion was evident . Low viral transduction efficacy was also seen in nontransgenic animals but was significantly increased by partial hepatectomy . The expression of the reporter gene in these animals was very low, as determined by histological staining . These results suggest that hepatocarcinogenesis can enhance retroviral delivery of foreign genes into the liver . Further development by increasing the viral transducing efficiency and the level of expression of transduced gene is required.

Anticancer Res, 1994 Jul-Aug, 14(4A), 1541 - 8
Conformational changes in membrane proteins of multidrug-resistant K562 and primary rat hepatocyte cultures as studied by Fourier transform infrared spectroscopy; Le Gal JM et al.; The multidrug resistance (MDR) phenotype has been investigated by means of Fourier transform infrared spectroscopy (FT-IR/S) on cell smears . We investigated K562 cell lines (sensitive and doxorubicin-resistant, the latter being MDR too) and primary cultures of rat hepatocytes (HEP) . HEP displayed elevated levels of P-glycoprotein (P-gp) with time in 2-4 day-old culture, thus developing in the same time a MDR phenotype . No functional P-gp activity could be detected in HEP at day 1 after cell seeding . Given the sensitivity of FT-IR/S and using computational treatment of FT-IR data, we found that spectra of MDR-K562 and HEP from day 2 to day 4 displayed close protein conformational changes involving beta-sheets . These changes might be in close relationship with the MDR-phenotype and P-gp overexpression.

J Lipid Res, 1994 Jul, 35(7), 1232 - 40
Effects of the glucosphingolipid synthesis inhibitor, PDMP, on lysosomes in cultured cells; Rosenwald AG et al.; The glucosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) has a wide range of effects on cell physiology and morphology . Here, we studied the effects of high concentrations of PDMP on cells in culture and found that fluorescent analogs of PDMP targeted to the lysosomes of Chinese hamster ovary (CHO) cells . Overnight incubation of the cells in the presence of drug induced enlargement ("vacuolization") of the lysosomes . PDMP was toxic at high concentrations (> 30 microM); this finding was used to select CHO cells that exhibited increased resistance to PDMP (PDMPR cells) . The PDMPR cells were approximately 2-fold more resistant to PDMP than the parental cells (CHO-P) . PDMPR cells were resistant to a number of other drugs that are also lipophilic and possess a titratable amino group . The multidrug resistance exhibited by the PDMPR cells was distinct from that observed in cells (MDR cells) that overproduce the plasma membrane drug pump, P-glycoprotein . In addition, MDR cells were extremely sensitive to PDMP.

Infect Control Hosp Epidemiol, 1994 Jul, 15(7), 497 - 9
Pros and cons of BCG vaccination in countries with low incidence of tuberculosis; Tala EO et al.; Preventive bacille Calmette-Guerin (BCG) vaccination, together with case finding and effective chemotherapy, has formed an integral part of the tuberculosis (TB) control program in most countries . In some low-incidence countries the balance of prevention has been more on the side of chemoprophylaxis than of BCG vaccination . The time clearly has come when the strategy of mass BCG vaccination no longer is indicated medically, nor is it cost-effective . The pros and cons of the programs need to be critically evaluated against the present epidemiological background, taking into account the facts that TB, the killer disease, is recovering strength, human immunodeficiency virus infection is on the increase, and multidrug-resistant TB has changed the outcome of this previously fully curable disease . Although no longer appropriate for mass programs, BCG vaccination still should be considered for the protection of selected risk groups in low-incidence countries . The overall efficacy may be of the order 50% to 80%, but the variation is great . Therefore, further research urgently is needed on the effectiveness of BCG as an intervention in local TB programs.

Zentralbl Pathol, 1994 Jul, 140(2), 149 - 53
{Expression of P-glycoprotein as a multidrug resistance gene product in human reactive astrocytes and astrocytoma}; Dietzmann K et al.; The immunohistochemical detection of multidrug resistance (MDR1) gene products and their mRNA within brain tumor cells has already been described by Fojo et al . 1987 . 63 specimens of astrocytomas and glioblastomas were analysed in the present study (Grading type 1 to 4) by means of the monoclonal antibody JSB1 . The endothelial cells were positive only in astrocytic tumors with a grading of 1 . Increasing tumor grading resulted in more positive immunological reactions in tumor cells . The most impressive reaction could be found in anaplastic astrocytoma and glioblastoma (G3 and G4) . Overexpression of this P-glycoprotein, a plasma membrane component of a relative molecular mass of 170 kDa was not only found in tumor cells of anaplastic astrocytomas, but also in endothelial cells and some non-neoplastic brain diseases . Positive immunological reactions in protoplasmatic astrocytes could be demonstrated in cases of phenylketonuria (1/1), tuberculous leptomeningitis (2/2), SSPE (3/4), X-ray necrosis (1/1) and necrotizing viral encephalitis (1/4) . According to this, it seems that astrocytes are able to express P-glycoprotein under the influence of some special metabolic conditions . This underlines the detoxicating function of reactive astrocytes within the total number of cells in the CNS.

Trends Pharmacol Sci, 1994 Jul, 15(7), 260 - 3
A mechanism for P-glycoprotein action in multidrug resistance: are we there yet?
Ruetz S, Gros P.
Multidrug resistance is associated with the overexpression of P-glycoprotein, a membrane glycoprotein . The mechanism by which P-glycoprotein confers to cells the capacity to resist cytotoxic attack by structurally unrelated drugs has remained difficult to decipher . However, the recent functional expression of this group of proteins in the membrane of secretory vesicles from yeast mutants has allowed the systematic analysis of the parameters of drug transport by this protein . Proposed mechanisms of action of P-glycoprotein are discussed in this review by Stephan Ruetz and Philippe Gros.

Chem Pharm Bull (Tokyo), 1994 Jul, 42(7), 1459 - 62
Structure-activity relationships of diamines, dicarboxamides, and disulfonamides on vinblastine accumulation in P388/ADR cells; Sawanishi H et al.; Diamines, dicarboxamides, and disulfonamides that have terminal benzene, methyl- or chloro-substituted benzene rings were synthesized and evaluated for the activity of {3H}vinblastine accumulation in multidrug-resistant P388/ADR cells . The efficacy of these compounds was generally in the order of dicarboxamides < diamines < disulfonamides . N-Methylated diamine and disulfonamide compounds having terminal methyl- or chloro-substituted benzene rings in their structure also showed rather potent efficacy . From these findings, we synthesized a novel disulfonamide compound, 1,2,3,4,5,6-hexahydro-2,5-bis(p-toluenesulfonyl)benzo{2,5}diazocine++ + (22) . Compound 22 suppressed the efflux of vinblastine from P388/ADR cells and increased its intracellular accumulation, while it barely increased the vinblastine accumulation in sensitive cells (P388/S) . Compound 22 significantly potentiated the growth-inhibitory effects of vinblastine, vincristine, colchicine and Adriamycin against P388/ADR cells in vitro.

J Clin Pathol, 1994 Jul, 47(7), 619 - 24
Specificity and sensitivity of immunocytochemistry for detecting P-glycoprotein in haematological malignancies; Gala JL et al.; AIMS--To determine the optimal working conditions of the alkaline phosphatase-antialkaline phosphatase (APAAP) method to establish a specific and sensitive assay for the detection of low numbers of MDR positive cells in patients with hematological malignancies . METHODS--Three monoclonal antibodies (C-219, JSB-1, MRK-16) were used for the detection of P-glycoprotein (P-gp) in cell lines and in samples from 43 patients with haematological malignancies . The results of the APAAP method were compared with western blotting for specificity and sensitivity . RESULTS--Excellent correlation was obtained between optimised APAAP and western blotting, except in the case of multiple myeloma . JSB-1 seemed to be the more useful monoclonal antibody for the APAAP which was more sensitive than western blotting in its ability to detect single P-gp positive cells . CONCLUSIONS--Methods for P-gp detection, as defined by multidrug resistant (MDR) cell lines, are not necessarily optimal and specific for clinical samples and may lead to higher false positive and negative results, according to the conditions and the monoclonal antibodies used.

Chemotherapy, 1994 Jul-Aug, 40(4), 265 - 71
Intrinsic overexpression of two different mechanisms of resistance to chemotherapy (P-glycoprotein and GST-pi) in human endometrial carcinoma; Schneider J et al.; Endometrial carcinoma is considered a tumor which does not respond well to chemotherapeutic treatment . Among the various mechanisms of resistance to chemotherapy which are under investigation, two of them (multidrug resistance, mediated by P-glycoprotein, and glutathione-S-transferase-pi {GST-pi} overexpression) are of great interest for gynecologic oncologists, because they involve several drugs commonly used in practice, among which Adriamycin and cisplatin are probably the most important ones . We have studied 23 human endometrial carcinomas of different histological varieties and 3 normal endometrial samples for the overexpression of both P-glycoprotein and GST-pi by means of immunohistochemistry . Both resistance markers were detectable in all tumor samples, and in normal endometrial tissue as well . The concomitant intrinsic overexpression of these two resistance mechanisms may in part explain why these tumors tend to be extremely resistant to chemotherapy.

Leukemia, 1994 Jul, 8(7), 1116 - 23
Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs . II . Potent cytotoxicity of 2-chloro-deoxyadenosine in acute lymphoblastic leukemia; Kumagai M et al.; We used a recently established stroma-supported tissue culture technique that allows long-term culture of acute lymphoblastic leukemia (ALL) cells to study 2-chloro-2'-deoxyadenosine (2CdA) cytotoxicity to leukemic lymphoblasts . In the 20 cases of ALL studied, the number of cells recovered after 7 days of culture on allogeneic stromal layers were 58-192% (median, 95.5%) of those originally seeded . In parallel cultures with 2CdA (100 nM), 74- > 99% (median, 97.5%) of leukemic lymphoblasts were killed . The cytotoxicity of 2CdA extended to all ten samples with either the t(9;22) (q34;q11) or 11q23 chromosomal abnormalities, karyotypes associated with an extremely poor outcome, as well as to two samples collected at the time of relapse . The effects of 2CdA were dose-dependent, and were due to triggering of apoptosis as shown by typical morphologic changes and occurrence of DNA fragmentation . Stromal layers were apparently not affected by 2CdA treatment, even when used at 1000 nM . We also tested 2CdA cytotoxicity to multidrug resistant subclones of the CCRF-CEM ALL cell line . CEM/VLB100 expresses P-glycoprotein, whereas CEM/VM-1 and CEM/VM-1-5 have topoisomerase II mutations that are associated with resistance to topoisomerase II inhibitors . Overexpression of P-glycoprotein or alterations in topoisomerase II did not protect cells from 2CdA cytotoxicity . We conclude that 2CdA is cytotoxic in most cases of ALL . The method used in this study may be applied to evaluate leukemic blast cell sensitivity to compounds with potential anti-leukemic activity, and to select patients for entry into clinical trials.

J Pharmacol Exp Ther, 1994 Jul, 270(1), 1 - 7
Enhanced transepithelial flux of cimetidine by Madin-Darby canine kidney cells overexpressing human P-glycoprotein; Pan BF et al.; Cimetidine has been used as a relatively selective inhibitor of renal organic cation secretion, analogous to the use of probenecid to inhibit organic anion secretion . Many of the substrates for the multidrug transporter P-glycoprotein, which is overexpressed in multidrug-resistant tumor cells, are organic cations . Furthermore, the protein is normally expressed on the apical membranes of proximal tubule cells, the postulated site for active organic cation secretion . To test directly whether P-glycoprotein might serve as a carrier for cimetidine, we measured cimetidine transepithelial movement across Madin-Darby canine kidney cells grown as monolayers on membrane filters . A retrovirally transduced Madin-Darby canine kidney cell line (Madin-Darby canine kidney cells transfected with the human multiple drug resistance 1 cDNA for P-glycoprotein), that expresses the human form of P-glycoprotein on its apical membrane, had an increased capacity to transport cimetidine from the basolateral to apical medium (b-->a) but not in the reverse direction (i.e., a-->b) . Qualitatively similar results were observed with daunomycin, a well established substrate for P-glycoprotein . Cellular uptake and energy-dependent efflux experiments further established cimetidine to be a substrate for the human P-glycoprotein . Thus, P-glycoprotein may play a role in the renal secretion of cimetidine and perhaps other organic cations.

Leuk Res, 1994 Jul, 18(7), 475 - 84
Multidrug resistance phenotype in patients with chronic lymphocytic leukemia as detected by immunofluorescence (FACS) and northern blot analysis; Wulf G et al.; The multidrug resistance (MDR) phenotype has been demonstrated to be related to the overexpression of P-glycoprotein, a 170 kDa transmembrane efflux pump . We studied P-glycoprotein expression in 40 patients with chronic B-cell leukemias by FACS analysis using MoAb c219, which recognizes both the MDR1 and the MDR3 gene product . We found significantly elevated P-glycoprotein expression in these patients as compared with normal controls . Patients who had received previous chemotherapy regimens containing MDR-related drugs showed significantly higher P-glycoprotein expression with MoAb c219 than those patients who had been untreated . Northern blot analysis of MDR1 and MDR3 gene expression in 32 of the patients gave a similar result: in the analysis of total RNA four of six patients (66%) pretreated with either vinca alkaloids or anthracyclines were MDR1 positive as opposed to 6 of 26 (23%) who had no treatment or treatment without these agents . In contrast, MDR3 expression was found more frequently (63%), but was randomly distributed in the differently treated groups . Increasing the sensitivity level by analysis of enriched mRNA (polyA+RNA) led to the detection of MDR1 and MDR3 expression all B-CLL patients . We conclude that a basic elevated P-glycoprotein expression is intrinsic in CLL cells, which is possibly upregulated under chemotherapy . This might be responsible for initial and acquired chemotherapy resistance in CLL patients . Follow-up of the B-CLL patients over 46 months showed that the median survival time for MDR1+ patients was 19 months as opposed to 46 months for MDR1- patients (p < 0.01) . There was no statistical difference in survival between MDR3+ and MDR3- patients . In the MDR1+ group, eight of nine patients had developed resistance to the therapy with MDR-related drugs . The expression of MDR1 might, therefore, predict treatment failure with MDR-related drugs and be a negative prognostic factor.

Hepatology, 1994 Jul, 20(1 Pt 1), 170 - 6
Bile acid inhibition of P-glycoprotein-mediated transport in multidrug-resistant cells and rat liver canalicular membrane vesicles; Mazzanti R et al.; To study the effect of bile acids on P-glycoprotein-mediated drug transport, we performed experiments using multidrug resistant cells and rat canalicular membrane vesicles . Cellular accumulation and efflux of rhodamine 123 were measured in drug-resistant cells by means of computerized quantitative image analysis and fluorescence microscopy . ATP-dependent {3H}daunomycin transport was studied by means of rapid filtration in canalicular membrane vesicles prepared from normal rats . Doxorubicin-sensitive (PSI-2) and -resistant (PN1A) 3T3 cells and human-derived hepatocellular carcinoma doxorubicin-sensitive and -resistant cells were used . Taurochenodeoxycholate and glycochenodeoxycholate, taurolithocholate and ursodeoxycholate (50 to 200 mumol/L) inhibited rhodamine 123 and {3H}daunomycin transport in multidrug-resistant cells and canalicular membrane vesicles, respectively, whereas taurocholate, taurodeoxycholate and tauroursodeoxycholate did not . Primary and secondary unconjugated bile acids had no effect . These results reveal that taurolithocholate, taurochenodeoxycholate and glycochenodeoxycholate and ursodeoxycholate inhibit P-glycoprotein-mediated drug transport function in multidrug resistant cell lines and in canalicular membrane vesicles . These results suggest possible interaction between P-glycoprotein function and bile acids in cholestasis and after treatment of patients with ursodeoxycholic or chenodeoxycholic acid.

Cell, 1994 Jul 1, 77(7), 1071 - 81
Phosphatidylcholine translocase: a physiological role for the mdr2 gene; Ruetz S et al.; P-glycoproteins (P-gps) encoded by the mouse mdr2 and mdr3 genes were expressed in secretory vesicles (SVs) from the yeast mutant sec6-4, and their capacity to function as a lipid translocase/flippase was tested . An assay that uses a fluorescent phosphatidylcholine (PC) analog was developed to quantitate asymmetric lipid distribution in the outer and inner leaflets of the lipid bilayer of these vesicles . Mdr2 expression in SVs caused a time- and temperature-dependent enhancement of PC translocation to the inner leaflet of the membrane . The Mdr2-mediated effect was specific since expression of Mdr3 in these vesicles was without effect on the membrane distribution of PC . Increased Mdr2-mediated PC translocation was strictly ATP and Mg2+ dependent, was abrogated by the ATPase inhibitor vanadate and the P-gp modulator verapamil, but was insensitive to the presence of excess of the multidrug resistance drugs colchicine and vinblastine.

Blood, 1994 Jul 1, 84(1), 262 - 9
Comparative evaluation of S9788, verapamil, and cyclosporine A in K562 human leukemia cell lines and in P-glycoprotein-expressing samples from patients with hematologic malignancies; Merlin JL et al.; The activity of S9788, recently synthetized as a modulator of multidrug resistance (MDR), was compared with verapamil and cyclosporine A in normal sensitive and MDR K562 cell lines, then in samples from 33 patients with hematological malignancies, using flow cytometry with simultaneous detection of P-glycoprotein and determination of intracellular daunorubicin fluorescence . This technique was compared and correlated with a tritiated daunorubicin accumulation method . In K562 cell lines, S9788 exhibited a significantly higher reversing activity than verapamil and cyclosporine A, and allowed a complete restoration of the accumulation of daunorubicin when used at 5 mumol/L . In the clinical samples, the three compounds were evaluated at equimolar concentration (5 mumol/L) using concomitant exposure to daunorubicin and to the reversing agent . In P-glycoprotein-negative samples, no significant effect on intracellular daunorubicin fluorescence of any of the reversing agents was noted . In the 15 P-glycoprotein-positive samples, a significant increase in daunorubicin fluorescence, by at least one reversing agent, was seen in 10 cases, among which S9788 reversing activity was higher than that of the two other agents in seven cases . Complete reversal was only achieved in one case with S9788.

Blood, 1994 Jul 1, 84(1), 229 - 37
Effect of the protein kinase C inhibitor staurosporine on chemosensitivity to daunorubicin of normal and leukemic fresh myeloid cells; Laredo J et al.; The effect of the protein kinase C (PKC) inhibitor staurosporine (ST) on the chemosensitivity of normal (colony-forming unit granulocyte-macrophage {CFU-GM}) and leukemic (acute myeloid leukemia-CFU {AML-CFU}) myeloid progenitors to daunorubicin (DNR) was evaluated . Primary colony inhibition assays allowed us to characterize two distinct groups of AML, a DNR-resistant group (patients no . 1 through 6), which displayed significantly lower DNR sensitivity than normal CFU-GM (D50 = 11.3 +/- 1.4 ng/mL v 1.8 +/- 0.5 ng/mL, after 7 days of exposure, respectively; P < 0.01) and a DNR-sensitive group (patients no . 7 through 12) with D50 = 2.7 +/- 0.4 ng/mL . This classification remained unaltered when assessed by secondary colony inhibition assay (evaluating the self-renewal fraction of AML-CFU) or by viability assay (evaluating the ultimately differentiated blast cell population), suggesting that the DNR sensitivity profile in maintained throughout AML-CFU differentiation . DNR resistance of the differentiated blast cell population was not correlated with the level of P-glycoprotein (P-gp) expression but rather with the ability to extrude rhodamine 123 (Rh123) . ST used at subtoxic concentrations induced a twofold to threefold enhancement of DNR cytotoxicity, increased Rh123 accumulation, and decreased Rh123 efflux kinetics in resistant AML cells . These effects were observed for ST concentrations much lower than those required to displace the P-gp-binding probe azidoprazosin, suggesting that ST might act through its PKC inhibitory effect and not through P-gp binding . Finally, this study provides evidence that DNR resistance in AML cells is, at least in part, related to the multidrug-resistance (MDR) phenotype . Because P-gp function can be downregulated by ST, it seems likely that the MDR pheno-type can be functionally regulated by cellular signalization in AML cells.

Br J Cancer, 1994 Jul, 70(1), 60 - 6
Modification by brefeldin A, bafilomycin A1 and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) of cellular accumulation and intracellular distribution of anthracyclines in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R; Rhodes T et al.; We have investigated the effects of H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitro-benz-2-oxa-1,3 diazole (NBD), and the Golgi inhibitor, brefeldin A, on daunorubicin accumulation and doxorubicin intracellular distribution in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R . This cell line overexpress a 190 kDa protein which is probably the product of the MRP gene and shows an anthracycline accumulation defect and a drastically altered intracellular anthracycline distribution from the parental cell line COR-L23/P . We found that all three agents could selectively increase the cellular accumulation of daunorubicin in resistant cells . However, these effects were only seen at doses of the modifiers which were equal to or greater than the IC50 of the modifier alone . Effects of the modifiers on the intracellular distribution of doxorubicin fluorescence could, however, be seen at doses lower than those required to produce significant effects on daunorubicin accumulation . However, when used in a continuous MTT chemosensitivity assay none of the agents, used at maximum non-toxic doses, was able to sensitise COR-L23/R cells to doxorubicin or to colchicine . Although these lead compounds are unlikely to be useful as clinical modifiers, development of more selective analogues may prove useful in the modification of non-P-glycoprotein-mediated multidrug resistance.

Indian Pediatr, 1994 Jul, 31(7), 813 - 9
Enteric fever: a changing perspective; Biswal N et al.; All the cases of enteric fever admitted between 1988-1992 were studied . There was a gradual rise in the number of admitted cases . Central nervous system (CNS) complications like encephalopathy (14.9%), meningitis (8.8%), seizures (8.5%) and cerebellitis (3.4%) were noted more during 1991 and 1992 . Other complications like myocarditis (4.6%), hepatitis (9.5%) and gastrointestinal bleeding were noted in increasing numbers during 1991-1992 . Multidrug resistant (MDRT) cases were 46.3% in 1991 and 33.5% in 1992 . There was a significant difference in the time taken for defervescence (a gradual rise) between the years but between the individual drugs there was no such significant difference . Deaths were noted only in 1991 and 1992 in cases of MDRT with complications . There has been an increase in resistance of S . typhi to commonly used drugs like ampicillin, chloramphenicol and cotrimoxazole . S . typhi resistant to ciprofloxacin was cultured in 2 cases each from 1990-1992 . Further, the time taken for defervescence with ciprofloxacin also showed a gradual rise from 3.5 days in 1990 to 6.2 days in 1992 . Nevertheless, ciprofloxacin is still the drug of choice for treatment of complicated cases of MDRT.

Indian Pediatr, 1994 Jul, 31(7), 807 - 11
Hepatic manifestations in typhoid fever; Jagadish K et al.; Thirty one children with typhoid fever aged 2 months to 12 years and blood culture positive for multidrug resistant S . typhi were prospectively studied for their hepatic functions at the time of hospitalization and 2-3 weeks after completion of antibiotic therapy . Hepatic manifestations included hepatomegaly (51.6%); jaundice (16.1%); raised levels of serum glutamic oxaloacetic transaminase (SGOT) (61.3%), serum glutamic pyruvic transaminase (SGPT) (48.4%), alkaline phosphatase (AP) (22.6%) and serum bilirubin (SB) (6.1%); reduced levels of serum albumin (SA) (41.9%); prolonged prothrombin time (PT) (9.7%) and abnormal ultrasound abdomen (19.3%) . Hepatic dysfunction was a notable feature even in those cases without hepatomegaly, with raised levels of SGOT (60%), SGPT (40%), AP (20%), SB (6.7%), decreased SA (53.3%) and prolonged PT (6.7%) . There was no correlation between the degree of hepatic enlargement or hyperbilirubinemia with abnormalities in liver functions . Hepatic dysfunction was noticed to be transient, as all these parameters returned to normal within 2-3 weeks after successful antibiotic therapy.

J Med Assoc Thai, 1994 Jul, 77(7), 363 - 7
Prevalence of drug resistance in Thai human immunodeficiency virus seropositive tuberculosis patients; Hongthiamthong P et al.; The emergence of drug resistant tuberculosis has been reported from many countries which have had epidemics of human immunodeficiency virus (HIV) infection . This study was conducted at the Central Chest Hospital, Thailand in order to determine the prevalence of drug resistance before treatment in Thai HIV-infected tuberculosis patients . From the Statistics and Registration Unit, pulmonary tuberculosis patients with HIV seropositivity were matched in terms of age and gender with control cases who attended the tuberculosis clinic on the same day . Results of sensitivity test were obtained from record cards in the Microbiology Section . The method for determining the sensitivity test was absolute concentration . During the study period from January 1988 to December 1993, 798 patients were registered as having tuberculosis and HIV infection . Only 406 sensitivity tests were available before treatment and resisted to Isoniazid 56 (13.8%), rifampicin 36 (8.9%), ethambutol 6 (1.5%), streptomycin 64 (15.8%) and Multidrug resistant (MDR)- TB 11 (2.7%) . In the control group, 475 tests were available and resisted to isoniazid 61 (12.8%), rifampicin 52 (10.9%), ethambutal 2 (0.4%), streptomycin 46 (9.7%) and MDR-TB 13 (2.7%) . The prevalence of resistance to each drug was not significantly different except for streptomycin . We concluded that the prevalence of antituberculous drug resistance among Thai HIV-infected tuberculosis patients was not higher than among general tuberculosis patientsPIP: This study was conducted at the Central Chest Hospital, Thailand, in order to determine the prevalence of drug resistant tuberculosis before treatment in Thai HIV-infected tuberculosis patients . From the Statistics and Registration Unit, pulmonary tuberculosis patients with HIV seropositivity were matched in terms of age and gender with control cases who attended the tuberculosis clinic on the same day . Results of the sensitivity test were obtained from record cards in the Microbiology Section . The method for determining the sensitivity test was absolute concentration . During the study period, from January 1988 to December 1993, 798 patients were registered as having tuberculosis and HIV infection . Only 406 sensitivity tests were available before treatment; 56 were resistant to isoniazid (13.8%), 36 to rifampicin (8.9%), 6 to ethambutol (1.5%), 64 to streptomycin (15.8%), and 11 were multidrug resistant (MDR) (2.7%) . In the control group, 475 tests were available; 61 were resistant to isoniazid (12.8%), 52 to rifampicin (10.9%), 2 to ethambutol (0.4%), 46 to streptomycin (9.7%), and 13 were MDR (2.7%) . The prevalence of resistance to each drug was not significantly different, except for streptomycin . The authors concluded that the prevalence of antituberculous drug resistance among Thai HIV-infected tuberculosis patients was not higher than among general tuberculosis patients . author's modified

Pharmacol Res, 1994 Jul, 30(1), 81 - 90
Effects of 8-chloro-cyclic adenosine monophosphate on the growth and sensitivity to doxorubicin of multidrug-resistant tumour cell lines; Borsellino N et al.; We examined the in vitro effects of 8-chloro-adenosine 3':5'-monophosphate (8-Cl-cAMP), a reportedly stable, potent and site-selective analogue of cAMP, on the proliferation and sensitivity to doxorubicin (DXR) of two mouse cell lines, the B16 melanoma and Friend leukaemia, both as wild-type (B16, FLC) and DXR-resistant (B16/DXR, FLC/DXR) variants . The latter strains had characteristics of 'typical' multidrug resistance (MDR), including the over-expression of P-glycoprotein . Encouragingly, 8-Cl-cAMP affected almost equally the growth of the chemosensitive and chemoresistant variants of both cell lines . Its activity proved to be much more elevated on cells cultivated with fresh rather than heat-inactivated calf serum . In fact, the IC50 values for B16 and B16/DXR were about 4.7 microM in fresh serum and 215 microM in heat-inactivated serum; the IC50 values for FLC and FLC/DXR were about 12 microM in fresh serum and 70 microM in heat-inactivated serum . Furthermore, experiments with B16 showed that cotreatments with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, or adenosine deaminase (ADA) greatly reduce the activity of 8-Cl-cAMP bringing it to comparable levels in fresh and heat-inactivated serum . These results indicate that the antiproliferative effects of 8-Cl-cAMP may be due principally to metabolites formed by the enzymic activities of the serum, most probably including 8-chloro-adenosine (8-Cl-adenosine), as suggested by other authors . Moreover, the dose-response curves and the IC50 values of the latter compound for the various cell lines were compatible with those observed for 8-Cl-cAMP in fresh serum . Finally, there was no evidence that 8-Cl-cAMP, either in the presence of fresh or heat-inactivated serum, or 8-Cl-adenosine may increase the sensitivity to DXR of the MDR variants of B16 melanoma and Friend leukaemia.

Haematologica, 1994 Jul-Aug, 79(4), 328 - 33
Effects of R-enantiomer (GR66234A) and L-enantiomer (GR66235A) of telupidine, a new dihydropyridine derivative, on cell lines displaying the multidrug resistant phenotype; Tolomeo M et al.; BACKGROUND . Many dihydropyridine analogues with calcium channel blocker activity are able to reverse multidrug resistance (MDR) . We studied the daunorubicin resistance reversing activity of the R enantiomer (GR66234A) and the L-enantiomer (GR66235 A) of teludipine, a new lipophilic calcium channel blocker synthesized by Glaxo . METHODS . The daunorubicin resistance reversing activity of the enantiomers of teludipine was evaluated in two MDR cell lines: ARNII, an erythroleukemia cell line which expresses p-glycoprotein, and MCF 7/R, a breast cancer cell line with p-glycoprotein and high levels of glutathione S transferase (GST) and glutathione peroxidase (GSH Px) . RESULTS . GR66234A and GR66235A show the same activity in reversing daunorubicin resistance and are more effective than verapamil . The difference in activity between verapamil and the enantiomers of teludipine is greater in ARNII cells than in MCF 7/R cells . Nevertheless, there are no significative differences in cellular daunorubicin accumulation between ARNII and MCF 7/R following exposure to teludipine, nor are there differences in intracellular daunorubicin distribution in the presence of either MDR reversing agent . CONCLUSIONS . The low calcium channel antagonistic activity of GR66234A suggests that this compound may be useful in combination with chemotherapy in MDR malignancies.

Hum Gene Ther, 1994 Jul, 5(7), 891 - 911
Retroviral mediated transfer of the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation after intensive chemotherapy for metastatic breast cancer; O'Shaughnessy JA et al.; Patients with metastatic breast cancer will receive 4-5 cycles of induction chemotherapy on one of the ongoing Medicine Branch protocols . Patients achieving at least a partial response, and who do not have evidence of bone marrow involvement and who do not have metastatic bone disease, will undergo PBSC and bone marrow harvest when hematologic recovery has occurred . Patients who have not achieved a PR, but who are responding to therapy, may be treated with additional cycles of therapy in an attempt to achieve a PR . Such patients will be eligible for transplant if a PR is obtained . 70% of the bone marrow and PBSC will be cryopreserved . The CD34+ subpopulation from the remaining 30% of the bone marrow and PBSC harvest will be obtained using an anti-CD34+ antibody and immunoabsorption column . The bone marrow and peripheral blood CD34 cells will be transduced with a retroviral vector expressing the human MDR-1 cDNA . Patients with positive bone scans or histologic evidence of bone marrow involvement will be excluded from the gene transfer component of the protocol . The MDR-1 transduced CD34 cells will be reinfused along with the non-transduced bone marrow and PBSC into patients following high dose ICE chemotherapy . Serial peripheral blood and bone marrow samples will be obtained to study hematopoietic reconstitution with MDR-1 transduced cells . Patients with residual or progressive disease after ABMT will be treated with taxol or vinblastine . In these relapsed patients, peripheral blood and bone marrow samples will be obtained to study whether chemotherapy amplifies the proportion of hematopoietic cells containing the MDR-1 provirus . We will monitor the nadir blood counts of each patient receiving salvage chemotherapy for evidence of myeloprotection and correlate this data with changes in the mean proviral copy number . Sites of relapsed tumor will be biopsied to test for the presence of the MDR-1 provirus.

Eur J Biochem, 1994 Jul 1, 223(1), 125 - 33
Mobile ionophores are a novel class of P-glycoprotein inhibitors . The effects of ionophores on 4'-O-tetrahydropyranyl-adriamycin incorporation in K562 drug-resistant cells; Borrel MN et al.; The decrease of the intracellular concentration of drug in resistant cells compared to sensitive cells is, in most cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein responsible for an active efflux of the drug . In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, we have examined the cellular accumulation of 4'-O-tetrahydropyranyl-adriamycin, alone and in conjunction with various ionophores on the one hand and with cyclosporin A on the other hand . The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds . Erythroleukemia K562 cell lines were used . Using experimental conditions in which these ionophores were unable to modify either the intracellular pH, or the transmembrane potential, or to induce an intracellular ATP depletion, we have shown that mobile ionophores as well as cyclosporin inhibit the P-glycoprotein-mediated efflux of 4'-O-tetrahydropyranyl-adriamycin in K562 resistant cells, whereas gramicidin, a channel-forming ionophore, does not . The concentration that must be used to inhibit 50% of the efflux was 0.7 microM for valinomycin, 0.4 microM for nonactin, 0.2 microM for nigericin, 1.1 microM for monensin, 0.4 microM for lasalocid, 1.2 microM for calcimycin and 0.4 microM for cyclosporin . Due to the high toxicity of the ionophores, the observation that they increased 4'-O-tetrahydropyranyl-adriamycin accumulation in the multidrug-resistant cells is not correlated with an effect of these compounds on drug resistance . However, the correlation exists in the case of cyclosporin . From our data showing that lipophilic neutral complexes, formed between carboxylic ionophores and metal ions, are both able to inhibit the P-glycoprotein-mediated efflux of anthracycline we can infer that the lipophilicity but not the cationic charge is an important physical property.

Biochem Biophys Res Commun, 1994 Jun 30, 201(3), 1424 - 32
Inhibition of 3'azido-3'deoxythymidine-resistant HIV-1 infection by dehydroepiandrosterone in vitro; Yang JY et al.; Human immunodeficiency virus type 1 (HIV-1) isolated from patients with acquired immunodeficiency syndrome (AIDS) shows resistance to 3'azido-3'deoxythymidine (AZT) after one or two years of treatment . AZT also has significant toxic side effects, further limiting its use in the therapy of HIV-1-infected individuals . Dehydroepiandrosterone (DHEA) has been shown to have a broad spectrum of biological functions, to be bioavailable orally and to be relatively nontoxic . Epidemiological studies provide evidence that reduced serum levels of DHEA are related to the progression of AIDS in HIV-1 infection . DHEA has also been shown to inhibit HIV-1 replication in vitro and block HIV-1 reactivation from chronically infected cell lines . However, there have been no reports on the ability of DHEA to inhibit the replication of AZT-resistant strains of HIV-1 . We investigated whether DHEA treatment could inhibit replication of AZT-resistant strains of HIV-1 . Addition of DHEA to MT-2 cell cultures infected with either AZT-sensitive or AZT-resistant isolates of HIV-1 resulted in dose-dependent inhibition of HIV-1-induced cytopathic effect and suppression of HIV-1 replication as measured by accumulation of reverse transcriptase activity . At a concentration as low as 50 microM, DHEA reduced AZT-resistant HIV-1 replication over 50 percent as measured by cytopathic effect and accumulation of reverse transcriptase activity . This study provides evidence that DHEA can inhibit the replication of AZT-resistant as well as wild-type HIV-1 . Since the main targets for DHEA are metabolic and cellular signaling pathways leading to HIV-1 replication-activation, DHEA should be effective against multidrug-resistant strains of HIV-1 . Combined with recently discovered immunoregulatory properties, the finding that DHEA is able to inhibit replication of both wild-type and AZT-resistant HIV-1 suggests that in vivo DHEA may have a much broader spectrum of action than originally anticipated.

J Med Chem, 1994 Jun 24, 37(13), 1918 - 28
Derivatives of a novel cyclopeptolide . 2 . Synthesis, activity against multidrug resistance in CHO and KB cells in vitro, and structure-activity relationships; Emmer G et al.; A series of derivatives of the novel cyclopeptolide 1 was prepared, and their ability to chemosensitize multi drug resistant CHO and KB cells in vitro was evaluated . In contrast to the parent compound, several of the derivatives were found to be highly active . In particular, conversion of the R-lactic acid residue of 1 into its S-isomer via lactone ring cleavage and recyclization with inversion resulted in a marked enhancement of activity . Some of these derivatives (e.g., 15a, SDZ 280.446) belong to the most potent resistance modulating compounds known so far.

Lancet, 1994 Jun 18, 343(8912), 1531 - 4
Deletion of gene for multidrug resistance in acute myeloid leukaemia with inversion in chromosome 16: prognostic implications; Kuss BJ et al.; Acute myeloid leukaemia (AML) associated with an inversion in chromosome 16 has a relatively favourable prognosis . The AML subclass most commonly associated with this chromosomal abnormality is acute myelomonocytic leukaemia with abnormal eosinophils . In some AML patients with inversion 16 the chromosomal lesion results in deletion of MRP, the gene for multidrug resistance associated protein . This gene is proximal to the primary breakpoint and loss of its function may play a key role in determining the favourable outcome in inversion 16 AML . We have demonstrated deletion of MRP by in situ hybridisation, by gene dosage studies and by studying loss of heterogeneity of a flanking microsatellite marker . Among 13 AML patients with inversion 16 MRP deletion was detected in 5 while 7 had no deletion . Deletion of MRP gene was associated with longer time from diagnosis until death or relapse from complete remission (p = 0.007) . These findings provide important insight into the biology of inversion 16 leukaemia and suggest that MRP deletion, as detected by molecular analysis, may have a key role in determining outcome in patients with inversion 16 AML.

Cancer Res, 1994 Jun 15, 54(12), 3210 - 7
CGP 48664, a new S-adenosylmethionine decarboxylase inhibitor with broad spectrum antiproliferative and antitumor activity; Regenass U et al.; Inhibitors of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (SAMDC), derived from methylglyoxal-bis(guanylhydrazone) (MGBG), have been shown to have significant antitumor activity in several human solid tumor systems (U . Regenass et al., Cancer Res., 52:4712-4718, 1992) . From an ongoing effort to synthesize derivatives with increased enzyme specificity and potency and improved antitumor efficacy, we have now identified CGP 48664, a 4-amidinoindan-1-one 2'-amidinohydrazone (J . Stanek et al., J . Med . Chem., 36:2168-2171, 1993) . The compound displays potent inhibition of SAMDC (50% inhibitory concentration, 5 nM), modest inhibition of diamine oxidase (50% inhibitory concentration, 4 microM), and no detectable inhibition of ornithine decarboxylase . CGP 48664 inhibits the growth of a panel of human and mouse tumor cell lines, including one which expresses the multidrug resistance phenotype, with 50% inhibitory concentrations ranging between 0.3 and 3 microM . CGP 48664 does not seem to utilize the polyamine transport carrier system since it competes poorly with spermidine for uptake into L1210 cells (Ki 161 microM) and inhibits the growth of polyamine transport-deficient Chinese hamster ovary cells . Relative to MGBG or previously described MGBG analogues, CGP 48664 accumulates to much lower intracellular concentrations . Treatment of the L1210 cell for 48 h with 3 microM CGP 48664 decreases SAMDC activity to < 10% of control and initiates a compensatory 3-fold rise in ornithine decarboxylase . Consistent with SAMDC inhibition, putrescine pools increase 10-fold, whereas spermidine and spermine pools fall to < 10% of control . In contrast to MGBG, CGP 48664 displays attenuated antimitochondrial activity as indicated by a lack of effect on pyruvate oxidation and mitochondrial DNA levels under treatment conditions which inhibit cell proliferation . Specificity of drug action was indicated further by prevention of L1210 cell growth inhibition by exogenous spermidine or spermine . More convincingly, Chinese hamster ovary cells made approximately 1000-fold resistant by chronic exposure to the analogue were found to selectively overexpress SAMDC mRNA due to gene amplification . The new SAMDC inhibitor showed potent antitumor activity against syngeneic tumors (B16 melanoma and Lewis lung carcinoma) and nude mouse human tumor xenografts (T-24 bladder carcinoma, SK MEL-24 melanoma, and MALME-3M melanoma) . On the basis of its novel structure, its apparent specificity of action, and its potent antitumor activity, CGP 48664 is the candidate drug for further preclinical development.

Biochem Pharmacol, 1994 Jun 15, 47(12), 2302 - 6
P-glycoprotein expression and function in rat hepatocytes in culture; Le Bot MA et al.; Expression of P-glycoprotein, which confers multidrug resistance to a broad range of anticancer drugs, was studied in rat hepatocytes in culture . P-glycoprotein was localized in the plasma membrane by immunohistochemical staining and was evaluated by western blotting with C219 as primary antibody and quantification of the coloured spots . The conditions of culture (time in culture, cell density at seeding) had a strong effect on the expression of P-glycoprotein . Expression increases with time in culture . At 10 x 10(6) cells/75 cm2 flasks, which is the normal density seeding for hepatocytes in culture, the increase of P-glycoprotein was 17% between 4 and 24 hr in culture, 52% between 24 and 48 hr and 37% between 48 and 96 hr . At low density cell seeding (2 x 10(6) cells/75 cm2), the expression of P-glycoprotein was higher than at normal density from the first day in culture (+20%) . This difference of expression was maintained until 96 hr of culture and was maximum at 48 hr (+44%) . This P-glycoprotein was functional and this overexpression was correlated with a decrease of doxorubicin retention in hepatocytes.

Biochem Pharmacol, 1994 Jun 15, 47(12), 2125 - 35
Kinetics of daunorubicin transport in Ehrlich ascites tumor cells with different expression of P-glycoprotein; Nielsen D et al.; The classical multidrug resistance (MDR) phenotype is characterized by a decrease in the intracellular drug concentration in resistant cells as compared to sensitive cells . P-glycoprotein (P-gp) is thought to be responsible for an active efflux of lipophilic drugs . Four Ehrlich ascites tumor cell lines selected in vivo for resistance to daunorubicin (DNR) and their sensitive counterpart were investigated . The resistant sublines EHR2/0.1, EHR2/0.2, EHR2/0.4, and EHR2/0.8 were developed by treatment of tumor bearing mice with DNR 0.1, 0.2, 0.4, and 0.8 mg/kg x 4 weekly, respectively . One passage from EHR2/0.1, EHR2/0.2, and EHR2/0.4 and two passages from EHR2/0.8 were investigated . Western blot analysis showed significantly different amounts of P-gp (a 6-fold variation) . Efflux of DNR in a drug free medium was investigated with and without presence of verapamil (VER) . Efflux from sensitive and resistant cells was described by mono- and bi-exponential kinetics, respectively . In all cases but one, a correlation between resistance, expression of P-gp, P-gp mediated efflux capacity and effect of VER was established . In passage No . 12 of EHR2/0.8, however, a high expression of P-gp was found in spite of a low degree of resistance and a low efflux capacity . In this subline the effect of VER did not correlate to the expression of P-gp . Active efflux seemed to be saturable and was suggested to constitute the major route of efflux in MDR cells . A dose-response relationship was established for the effect of VER on efflux . In conclusion, the results support that P-gp acts as a drug efflux pump . No simple correlation, however, could be established between P-gp and drug transport in all the investigated cell lines . Other factors which might influence transmembranous transportation of DNR are suggested . The active efflux capacity of the cell lines seemed to determine the degree of resistance and the sensitivity to circumvention by VER.

Int J Cancer, 1994 Jun 15, 57(6), 841 - 6
Immunohistochemical evaluation of P-glycoprotein in human malignancies by monoclonal antibody MC57; Leonardo E et al.; P-glycoprotein expression was analyzed on 137 formalin-fixed, paraffin-embedded human tumours by monoclonal antibody (MAb) MC57 . This MAb recognizes an extracellular human specific P-glycoprotein epitope and defines their multidrug resistance (MDR) phenotype by its binding on cells . Immunohistochemistry indicated that this MAb reacted in human cells and tissues in the same pattern as that found with other MAbs to P-glycoprotein . However, the present extensive study demonstrated that MAb MC57 is a highly specific reagent for the evaluation of an extracellular P-glycoprotein epitope preserved after fixation procedures and that this MAb is available to assess P-glycoprotein expression in routinely processed human tumour specimens.

Cancer Res, 1994 Jun 15, 54(12), 3202 - 9
Transmembrane orientation and topogenesis of the third and fourth membrane-spanning regions of human P-glycoprotein (MDR1); Skach WR et al.; Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require insight into the mechanism of assembly, transmembrane topology, and intracellular trafficking of human P-glycoprotein (MDR1) . Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected interaction between novel topogenic sequence subtypes and that transmembrane topology of corresponding amino and carboxy halves was not equivalent . We now investigate topology and topogenic activities of the third and fourth transmembrane regions (TM3 and TM4) of human MDR1 using protease protection of defined reporter epitopes expressed in Xenopus laevis oocytes . As was previously observed for TM1 and TM2, determinants in TM3 and TM4 exhibited cooperativity in directing proper assembly and transmembrane orientation . The signal sequence encompassing TM3 required residues from TM4 to reinitiate translocation of the MDR1 chain into the endoplasmic reticulum (ER) lumen . Remaining residues from TM4 terminated translocation and established a polytopic transmembrane topology in which TM3 and TM4 both spanned the membrane in the orientation predicted by hydropathy-based models . Remarkably, when translocating sequentially into the ER lumen, neither TM4 alone nor TM4 together with TM3 efficiently terminated translocation . Thus, MDR1 biogenesis required both the presence of these sequences and their proper orientation with respect to the ER translocation apparatus . This conclusion was supported by experiments in which TM3 and TM4 topology was reproduced in a defined chimeric protein which mimicked native MDR1 presentation . These additional variations on simple themes of protein topogenesis utilized by MDR1 demonstrate that events of complex protein biogenesis may be dissected and studied using protein chimeras with defined translocation properties.

Cancer Res, 1994 Jun 15, 54(12), 3088 - 91
Reversal of multidrug resistance by RU 486; Gruol DJ et al.; P-Glycoproteins represent a family of drug efflux proteins that convey multidrug resistance to cells in which they are expressed . This phenomenon can lower the efficacy of drugs used in chemotherapy . The steroid progesterone has been shown to bind P-glycoproteins and inhibit their drug efflux . We report that the antiprogestin RU 486 can reverse multidrug resistance in cells overexpressing the mouse mdr1 gene . Using flow cytometry to measure inhibition of P-glycoprotein-dependent efflux of rhodamine 123, RU 486 was found to be considerably more effective than progesterone and one-half as effective as verapamil . The results suggest a valuable new use for RU 486.

J Natl Cancer Inst, 1994 Jun 15, 86(12), 913 - 20
Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells; Dong Z et al.; BACKGROUND: The major cause of death from cancer is metastases that are resistant to conventional therapies . The resistance of metastatic tumor cells to chemotherapy can be caused by their intrinsic properties, such as increased expression of the mdr genes . PURPOSE: The purpose of our present study was to determine some of the mechanisms by which the organ microenvironment influences the response of tumor cells to chemotherapy . METHODS: Murine CT-26 colon cancer cells growing in continuous culture (parental cells) were harvested and injected subcutaneously into the lateral flank (to produce subcutaneous tumors) or the lateral tail vein (to produce experimental lung metastases) of 10 8-week-old syngeneic male BALB/c mice . Seven days after tumor-cell injection, the mice were given intravenous injections of either doxorubicin (10 mg/kg) or 0.9% NaCl (controls) . This in vivo injection was repeated 7 days later . Mice with subcutaneous tumors and lung metastases were killed by cervical dislocation on day 21, and tumor samples from control mice were harvested and adapted to culture . The sensitivity of the cultured cells to doxorubicin and fluorouracil (5-FU) was determined at multiple time points . Levels of mdr-1 DNA were measured by slot-blot and Southern-blot analyses . mdr mRNA expression levels were measured by Northern-blot analysis using mdr-1- and mdr-3-specific hybridization probes, and P-glycoprotein level was determined by fluorescence-activated cell sorting using different monoclonal antibodies . RESULTS: Treatment with doxorubicin produced 80% growth inhibition of CT-26 subcutaneous tumors but had little effect on the number (and size) of experimental lung metastases . Collectively, the results suggest that the multidrug-resistant phenotype developed in CT-26 cells growing in the lung environment . Cultures established from lung metastases were initially resistant to doxorubicin (but not to 5-FU) and showed elevated expression of mdr-1 mRNA transcripts and P-glycoprotein . This resistance could be overcome by verapamil and disappeared after 21 days in culture . No mdr gene amplification was detected . The expression level of mdr-specific mRNA (predominance of mdr-1) and P-glycoprotein was directly associated with resistance to doxorubicin . CONCLUSIONS: Results of this study have demonstrated that the in vivo sensitivity of murine CT-26 colon carcinoma cells to doxorubicin depends on the organ environment . The organ environment can influence the P-glycoprotein-mediated multidrug-resistant phenotype in tumor cells, and the increased expression of P-glycoprotein is transient; once removed from the environment (lung), the cell's resistance reverts to that of the sensitive parent cells.

Eur J Biochem, 1994 Jun 15, 222(3), 813 - 24
Probing the interaction of the multidrug-resistance phenotype with the polypeptide ionophore gramicidin D via functional channel formation; Assaraf YG et al.; It has been proposed that the multidrug resistance (MDR) transporter, P-glycoprotein (P-170), may be physiologically involved in the transport of polypeptides . As a step towards understanding the interaction of P-170 with polypeptides, we isolated various gramicidin-D-resistant mammalian cell lines . Gramicidin D is a hydrophobic pentadecapeptide ionophore that forms proton and alkali metal cation-permeable channels in lipid bilayers . Gramicidin-D-resistant cells displayed a prominent MDR gene amplification, P-170 overexpression, reduced drug accumulation, and consequent resistance to MDR-type cytotoxic agents . Modulators of the MDR phenotype, including verapamil, reserpine and quinidine, rendered these cells sensitive to gramicidin D . Using these cell lines, we established an assay that probes for the intra-membranal interaction between P-170 and gramicidin D . Gramicidin-D channel formation was followed by cellular accumulation of 86Rb+ . Ionophore-resistant cells, and other MDR cells, did not show an appreciable increase in 86Rb+ influx rates, in the presence of increasing gramicidin-D concentrations . In contrast, parental cells displayed a dose-dependent increase in the 86Rb+ influx rates . Interestingly, in the absence of serum, gramicidin-D-resistant cells resumed the wild-type, ionophore-dose-dependent increase in 86Rb+ influx rates . MDR modulators caused a resumption of channel formation in ionophore-resistant cells . We conclude that acquisition of the MDR phenotype is an efficient means of cellular protection against gramicidin D . Hence, a new approach is offered in which P-170 interaction with gramicidin D is quantitatively followed by a rapid assessment of the biological activity (i.e . channel formation) of the substrate itself . Possible mechanisms of P-170 interaction with free ionophore monomers, and membrane-associated gramicidin D are discussed.

Biochemistry, 1994 Jun 14, 33(23), 7229 - 38
Low external pH and osmotic shock increase the expression of human MDR protein; Wei LY et al.; We have studied the effects of extracellular pH (pHo) and osmotic strength on the expression of the human multidrug resistance (MDR) protein . Both lowered pHo and hypertonic shock increase the level of hu MDR protein 5-10-fold in membranes isolated from the human colon carcinoma cell lines SW620 and HCT15 and the human kidney carcinoma line SKRC-39 . Increased protein expression is dependent on the duration of acid or osmotic shock and is reversed within several days when normal growth conditions are restored . Quantitative northern blot analysis with a hu MDR 1 specific probe reveals increased MDR mRNA in the acid and hypertonically shocked cells . Interestingly, we find a greater increase in mRNA levels for hypotonically shocked colon cells, without an apparent increase in protein levels . Overexpressing cells are found to retain less {3H}vinblastine relative to cells cultured under normal conditions, and they are resistant to the cytotoxic effects of doxorubicin, vinblastine, and colchicine, but not methotrexate . This resistance appears to be reversed by treatment with verapamil . In contrast, SW620 cells previously induced to overexpress MDR protein via the administration of differentiation agents {Mickley et al . (1989) J . Biol . Chem . 264, 18031-18040} did not exhibit decreased retention of {3H}vinblastine; thus low-pHo-induced overexpression of MDR protein in these cells may provide additional factors that promote the full expression of the MDR phenotype . These data may help to explain why many solid tumors (e.g., of colon and kidney origin) develop MDR prior to chemotherapy, since they usually grow under similar acidic conditions . These data also support the contention that MDR protein may play a role in intracellular pH and volume homeostasis.

Biochemistry, 1994 Jun 14, 33(23), 7069 - 76
Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein; Urbatsch IL et al.; A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E . (1993) J . Biol . Chem, 268, 4197-4206) . The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose . The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM) . A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity . Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors . There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection . Vanadate was also an effective inhibitor . Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp . The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.

FEBS Lett, 1994 Jun 13, 346(2-3), 141 - 5
Cooperative P-glycoprotein mediated daunorubicin transport into DNA-loaded plasma membrane vesicles; Guiral M et al.; Most of the multidrug resistant human tumor cell lines overexpress the MDR1 gene product P-glycoprotein (P-gp) which is believed to function as an energy-dependent drug efflux pump . Here we describe a novel method that allows the kinetic characterization of P-gp-mediated active drug transport . This method is based on the fluorescence quenching of anthracyclines transported into DNA-loaded plasma membrane vesicles . The uptake of daunorubicin (DNR) into the plasma membrane vesicles was saturable in terms of the extravesicular DNR concentration with a Km of 1.5 +/- 0.1 microM . This transport occurred by a cooperative process with a Hill coefficient close to 2 for DNR . A model is discussed in which P-gp pumps two molecules of drug per turnover.

J Biol Chem, 1994 Jun 10, 269(23), 16397 - 402
Involvement of Jun and Fos proteins in regulating transcriptional activation of the human pi class glutathione S-transferase gene in multidrug-resistant MCF7 breast cancer cells; Moffat GJ et al.; Elevated levels of the human pi class glutathione S-transferase (GSTP1-1) have been implicated in the development of antineoplastic drug resistance . Using GSTP1 promoter deletion constructs we have shown that enhanced GSTP1 transcription (up to 18-fold) is the predominant mechanism responsible for increased GSTP1-1 levels in a multidrug resistant derivative (VCREMS) of the human mammary carcinoma cell line MCF7 . Furthermore, disruption of a putative AP-1 response element within the GSTP1 promoter (nucleotides -69 to -63) abrogated GSTP1 transcription in both cell lines . In addition, band shift assays demonstrated binding of a VCREMS nuclear complex to the promoter region C1 (-73 to -54) which could be competed for by a DNA fragment containing a known AP-1 binding site from the human collagenase promoter . However, no such competition was observed for the major MCF7 C1 complex . The role of a Fos-Jun-like complex in regulating GSTP1 transcription in VCREMS cells was further emphasized by the introduction of point mutations within the C1 region which were known to inhibit AP-1 binding and the interaction of antisera raised against human c-Jun and c-Fos with the major C1 complex in VCREMS cells . These studies therefore highlight cell-specific differences in the binding pattern of Jun and Fos proteins to the GSTP1 promoter which are likely to play an important role in regulating transcriptional activation of the GSTP1 gene in drug-resistant breast cancer cells.

MMWR Morb Mortal Wkly Rep, 1994 Jun 10, 43(22), 417 - 9
Multidrug-resistant tuberculosis in a hospital--Jersey City, New Jersey, 1990-1992; Interaction of prenylcysteine methyl esters with the multidrug resistance transporter; Department of Molecular Cancer Biology and Biochemistry, Durham Veterans Administration Medical Center, North CarolinaThe multidrug resistance transporter is an integral membrane protein, termed P-glycoprotein, which can function as an ATP-dependent drug efflux pump to reduce intracellular drug accumulation in treated cells . The physiologic function of this protein in normal cells, however, is not completely understood . We report here that prenylcysteine methyl esters, which represent the C-terminal structures of prenylated proteins, both stimulate the transporter's intrinsic ATPase activity and compete for drug binding . The structural elements of prenylcysteine methyl esters involved in their interaction with P-glycoprotein include the isoprenoid moiety, the carboxyl methyl group, and the free amino group . These findings indicate that these molecules are potential physiologic ligands of the transporter . Furthermore, as the structures of the active prenylcysteines are distinct from the known substrates of P-glycoprotein, this information may facilitate design of novel inhibitors of the transporter.

Br J Cancer, 1994 Jun, 69(6), 988 - 94
Selective toxicity of TGF-alpha-PE40 to EGFR-positive cell lines: selective protection of low EGFR-expressing cell lines by EGF; Kirk J et al.; The sensitivity of human breast and lung cancer cell lines to TGF-alpha-PE40, a novel chimeric recombinant cytotoxin composed of two independent domains, (i) TGF-alpha and (ii) a 40 kDa segment of the Pseudomonas exotoxin protein, PE-40, was investigated . Toxicity varied widely, correlated with epidermal growth factor receptor (EGFR) levels (P = 0.01) and was greatly reduced by EGF, indicating that binding of TGF-alpha-PE40 to EGFR is important in mediating toxicity . Cell lines expressing low EGFR levels were most highly protected by EGF, indicating that normal (low EGFR-expressing) tissue may be selectively protected by EGF in vivo . P-glycoprotein did not confer resistance to TGF-alpha-PE40, and toxicity was unaffected by multidrug resistance-modulating agents (cyclosporin A, tamoxifen, verapamil), indicating a role for TGF-alpha-PE40 in the clinical management of drug-resistant tumours.

Exp Hematol, 1994 Jun, 22(6), 517 - 20
Limiting dilution analysis of a novel tripeptide anticancer agent Ambamustine (PTT-119): effect on K-562, CCRF-SB and multidrug resistant LoVo-Dk cell lines; Manna A et al.; Cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SB or K-562 lines . After incubating the cell mixtures in vitro with different dose levels of Ambamustine (PTT-119), a quantity of 10(4) treated cells were dispensed into microculture plates, and graded cell numbers of the lines used to contaminate the normal marrow were added . Limiting dilution analysis (LDA) was used to estimate the frequency of leukemic cells persisting after treatment . Incubation with 50 micrograms/mL of PTT-119 produced a total elimination of K-562 acute myelogenous blasts, whereas nearly 0.17 and 0.27 leukemic cells were still present in the cell mixtures after treatment with 5 and 25 micrograms/mL, respectively . When normal bone marrow was contaminated with CCRF-SB lymphoblastic cells, incubation with either 50 or 25 micrograms/mL of PTT-119 produced a complete clearing of leukemic cells, whereas with 5 micrograms/mL the leukemic cells in each well were 0.18 . When PTT-119 was incubated with LoVo-DX, a colon cancer cell line which expresses the pleiotropic drug resistance MDR phenotype, virtually complete inhibition of clonogenic colonies was observed with as little as 5 micrograms/mL . This suggests that PTT-119 could be used in clinical trials as a non-cross-resistant agent in multidrug protocol.

JAMA, 1994 Jun 1, 271(21), 1677 - 9
Infectious diseases; Stoeckle MY et al.; Unlike the initial H influenzae type b vaccine, which contained polysaccharide alone, the conjugate vaccines effectively induce immunity in infants as young as 2 months . Multidrug-resistant strains of S pneumoniae are emerging as important pathogens in the United States.

Cell Mol Biol (Noisy-le-grand), 1994 Jun, 40(4), 551 - 60
Drug resistance and its counteraction by cyclosporin A in function of metastatic potential in the Lewis lung carcinoma system; Michowitz M et al.; Tumor progression (TP) is often accompanied by evolution of drug resistant clones . Decreased intracellular accumulation of cytotoxic agents is probably the major mechanism of drug resistance . In the present study, we tried to examine the possibility to overcome the resistance to adriamycin (ADR) treatment, by cyclosporin A (CS) in two models of TP in the Lewis lung carcinoma (3LL) system . The first model consisted in the comparison of primary tumor cells (3LL-PT) to metastatic cells (3LL-MT) and the second consisted in comparison of lung metastases of the highly malignant variant D122 to those of the parental 3LL tumor . Cyclosporin had a weak augmenting effect on ADR uptake, in the two more malignant cell variants and no influence on the 3LL-PT cells, according to FACS analysis . Cytofluorometry also showed practically no effect of CS on cell size, unlike the effect of other chemosensitizers, such as membrane active agents . In order to find out whether CS counteracts resistance to ADR despite the fact that it does not increase cytotoxic agent uptake, we examined its effect on in vitro proliferative capacity of the 3LL-PT cells . CS in combination with ADR had a more pronounced effect, as compared to single treatments on cell proliferation . The low effect of CS on ADR uptake according to FACS analysis, and by contrast, its efficiency to overcome resistance to ADR according to the in vitro growth results, suggest that the mechanism of the CS action as a chemosensitizer is not related to the p-glycoprotein (P-G-P), known to be overexpressed in the typical multidrug-resistance (MDR) phenotype . A better understanding of the complexity of MDR mechanisms may contribute to the design of new modalities to overcome this phenomenon, which still limits effectiveness of cancer cure, to the early stages of the disease.

Trans R Soc Trop Med Hyg, 1994 Jun, 88 Suppl 1, S27 - 9
Chemistry of artemisinin: an overview; Webster HK et al.; The endoperoxide sesquiterpene lactone, artemisinin, and its derivatives have become increasingly important as antimalarial drugs with impressive activity against multidrug resistant forms of Plasmodium falciparum . Artemisinin has a novel structure among known antimalarial compounds with a unique 1,2,4-trioxane ring that is essential for activity . This paper gives an overview of the chemistry of artemisinin and comments on future prospects for artemisinin and its derivatives.

Am J Trop Med Hyg, 1994 Jun, 50(6), 790 - 5
Comparison of micronized halofantrine with chloroquine-antibiotic combinations for treating Plasmodium falciparum malaria in adults from Gabon; Kremsner PG et al.; Multidrug resistance of Plasmodium falciparum is spreading throughout Africa . In Lambarene, Gabon where chloroquine-resistant malaria is prevalent, a randomized comparative trial with three regimens for treating P . falciparum malaria in adults was performed . One hundred two patients evaluated received either a new micronized formulation of halofantrine (8 mg/kg every 6 hr in three doses) (group H) or chloroquine (25 mg/kg for a 48-hr period) plus clindamycin (5 mg/kg every 12 hr in six doses) (group CC1), or chloroquine (as above) plus doxycycline (2 mg/kg every 12 hr in six doses) (group CD) . All treatment regimens were well-tolerated . In group H, 100% of the patients were cured, and in group CC1, 97% of the patients were cured by day 28 of follow-up . In group CD, a significantly lower cure rate of 75% (P < 0.01) and a slower parasite clearance was observed, but only low grade (RI) resistance occurred.

Gan To Kagaku Ryoho, 1994 Jun, 21(7), 936 - 44
{Multidrug resistance (MDR)}; Wada M et al.; Multiple drug resistance (MDR) is a major problem of current chemotherapy . Establishment of multiple drug resistant cell lines in culture and isolation of P-glycoprotein-coding MDR genes have promoted understanding of the molecular mechanisms underlying drug resistance in tumors . Another gene, MRP, has been recently isolated from a multiple drug resistant cell line which did not express P-glycoprotein . Both genes have DNA sequence homology for each other and have been identified as members of ATP binding cassette (ABC) transporter superfamily . This review refers to recent progress in MDR and MRP study, and focuses on involvement of these two drug-resistance-related genes in acquiring drug resistance and their physiological functions.

Bull Acad Natl Med, 1994 Jun, 178(6), 1177 - 88; discussion 1188-9
{Potentiation of the photocytotoxic effect of photofrin II: synergistic action of verapamil and lovastatin}; Maziere JC et al.; Photofrin II (P2) is at the present time the most used drug in the photochemotherapy of tumors . As previous studies from our group have demonstrated that P2 is taken up by cells mainly via the low density lipoprotein (LDL) receptor pathway, we tried to increase the amount of drug delivered to cells by enhancing the LDL receptor expression . For this purpose, we used hydroxy methyl glutaryl Coenzyme A (HMG-CoA) reductase inhibitors such as lovastatin . In the present work, we show that the calcium antagonist verapamil, which is currently used in human chemotherapy to overcome multidrug resistance, enhances in a synergistic manner the potentiating action of lovastatin on the photocytotoxic effect of P2.

Semin Respir Infect, 1994 Jun, 9(2), 104 - 12
Drug-resistant tuberculosis: etiology, management and prevention; O'Brien RJ; Drug-resistant Mycobacterium tuberculosis inevitably arises from inadequate or inappropriate drug taking or drug prescribing, effectively resulting in monotherapy . This may occur in the patient being treated (acquired resistance) or in a patient who has been infected by another patient with drug resistant tuberculosis (primary resistance) . There is some evidence that multidrug-resistant tuberculosis, ie, resistance to both isoniazid and rifampin, is increasing in the United States and in other countries where unsupervised treatment with rifampin has been common . Human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS), although not causing drug-resistant tuberculosis, have certainly magnified the problem, especially in New York City . Treatment of drug-resistant tuberculosis must be based on results of drug susceptibility studies . Patients with isolated isoniazid-resistant tuberculosis respond well to modified short-course therapy with rifampin, ethambutol, and pyrazinamide . Multidrug-resistant disease is more difficult to treat, although most patients will respond to regimens of second-line drugs if the infecting organisms are susceptible to these agents . Drug-resistant tuberculosis can be prevented by accurate identification of patients with newly diagnosed tuberculosis who may be at increased risk of primary drug resistance, the administration of an appropriate treatment regimen to all newly diagnosed patients, the application of fully supervised therapy during at least the initial phase of treatment, the use of combination preparations of drugs, and the proper management of failure and relapse cases.

Cancer Metastasis Rev, 1994 Jun, 13(2), 223 - 33
P-glycoprotein, multidrug resistance and tumor progression; Bradley G et al.; P-glycoprotein (Pgp) is a plasma membrane protein that was first characterised in multidrug resistant cell lines . The occurrence of Pgp in clinical tumors has been widely studied . Recent investigations have begun to focus on the relationship between Pgp detection in tumors and treatment outcome . In several types of tumors, detection of Pgp correlates with poor response to chemotherapy and shorter survival . P-glycoprotein over-expression often occurs upon relapse from chemotherapy but may also occur at the time of diagnosis . Studies of experimental rat liver carcinogenesis have shown that Pgp expression increases in late stages of carcinogenesis, suggesting that Pgp may be involved in tumor progression . While some of the Pgp isoforms are known to transport hydrophobic chemotherapeutic drugs out of tumor cells, the biologic effects of Pgp overexpression in tumor cells are not fully understood, because the spectrum of substrates for Pgp-mediated transport has not been determined . In the rat liver carcinoma model, strong expression of Pgp is associated with a highly vascular stroma, suggesting that Pgp in tumor cells may affect the connective tissue stroma . The regulation of Pgp appears to be complex, and little is known about how it is up-regulated during carcinogenesis . Further studies of the role of Pgp in malignancy may contribute to our understanding of molecular mechanisms which underlie tumor progression.

Cancer Metastasis Rev, 1994 Jun, 13(2), 209 - 22
Modulation of tumor cell response to chemotherapy by the organ environment; Fidler IJ et al.; The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors . The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential . While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes . The organ microenvironment can also influence the response of metastases to chemotherapy . It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy . We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma . The tumor cells were implanted subcutaneously or into different visceral organs . Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not . In contrast, sensitivity to 5-FU did not differ between these sites of growth . The differences in response to DXR between s.c . tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution . The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression of mdr1 mRNA . However, the organ-specific mechanism for upregulating mdr1 and P-glycoprotein has yet to be elucidated.

Cancer Metastasis Rev, 1994 Jun, 13(2), 191 - 207
Reverse transformation of multidrug-resistant cells; Biedler JL et al.; Spontaneously transformed Chinese hamster lung cells with high levels of resistance (approximately 100-fold to 70,000-fold) to actinomycin D, daunorubicin, or vincristine exhibit morphology and growth patterns characteristic of normal cells in vitro and reduced tumorigenicity in vivo . These reverse transformed, multidrug-resistant cells amplify and highly overexpress one or more genes encoding P-glycoprotein . Similarly, hydrocarbon-induced mouse sarcoma cells selected with actinomycin D, vincristine, or ethidium bromide developed high levels of resistance associated with reduced drug accumulation and suppression of malignancy . To determine whether human tumor cells would undergo similar changes and whether reverse transformation reflected an altered state of differentiation, nine multidrug-resistant sublines were selected with four agents from human neuroblastoma cells with well defined pathways of differentiation . Those five with resistance levels above about 125-fold showed a reduced tumor frequency as compared to control cells . All resistant sublines showed altered differentiation . The changes in transformation phenotype appear to be intrinsic and not the result of altered immunogenicity . Two additional consequences of high level multidrug resistance have been observed: change in ganglioside composition in the Chinese hamster cells, manifested as a block in higher ganglioside biosynthesis and/or a relative increase in GM3, and increase in epidermal growth factor receptor in all three cell systems . A tentative hypothesis links ganglioside and growth factor receptor changes to the change in transformation phenotype . The basis of the reverse transformation phenomenon is not known, but the major alterations in expression of P-glycoprotein, gangliosides, and the epidermal growth factor receptor implicate, in some way, the plasma membrane.

Leuk Lymphoma, 1994 Jun, 14(1-2), 157 - 61
Expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells of patients with granular lymphocyte-proliferative disorders; Kobayashi Y et al.; The immunological findings and clinical course of 33 patients with granular lymphocyte-proliferative disorders (GLPD) are presented . Based on the surface phenotypes of peripheral blood granular lymphocytes (GL), the GLPD were divided into two groups, namely CD3+ T cell-lineage GLPD (T-GLPD) and CD3- CD16+ NK cell-lineage GLPD (NK-GLPD) . Twenty-one patients had T-GLPD, and 12 had NK-GLPD . One patient with T-GLPD and two patients with NK-GLPD had progressive clinical courses and died of the disease despite receiving combination chemotherapy . Among eleven patients analysed for the expression of multidrug resistance P-glycoprotein, six of eight patients with T-GLPD and all three patients with NK-GLPD clearly expressed P-glycoprotein . Since patients with immature NK-GLPD appear to undergo a progressive clinical course, we suggest that therapeutic trials using P-glycoprotein blockers in addition to chemotherapy might be beneficial for such patients.

Leuk Lymphoma, 1994 Jun, 14(1-2), 129 - 35
Analysis of mdr-1 gene expression in human leukemic cells by quantitative competitive PCR; Kato S et al.; The ability to recognize the acquisition of multidrug resistance (MDR) in leukemia patients would improve our ability to predict the responsiveness of patients to chemotherapy . To quantitate the degree of MDR acquisition, we determined the amount of mdr-1 mRNA in leukemic cells from patients by competitive polymerase chain reaction (PCR) analysis . Twenty-one patients including 12 patients prior to treatment and nine relapsed patients with acute myelogenous or lymphoblastic leukemia were examined . The amount of mdr-1 gene expression in K562, K562/ADR500 cells and their mixtures showed a proportional correlation between the ratio of resistant to non-resistant cells and the amount of the mdr-1 gene . Mean mdr-1 gene expression in relapsed patients was greater than that in pretreatment patients . Patients refractory to chemotherapy (NR) showed higher levels of mdr-1 gene expression than the patients who achieved complete remission (CR) . Because of the wide variations in values, no statistical differences were observed between pretreatment and relapsed patients, or CR and NR patients . These results suggest that the competitive PCR technique is a reliable method to quantitatively determine mdr-1 gene expression, but it may be difficult to predict responsiveness to chemotherapy by using this technique alone.

Anticancer Drugs, 1994 Jun, 5(3), 329 - 35
Flow cytometric monitoring of anthracycline accumulation after anti-neoplastic ether phospholipid treatment; Principe P et al.; Ether phospholipids are new anti-neoplastic drugs that have been found active against a variety of tumor cell lines, including drug-resistant sublines . We have characterized the antiproliferative activity of three ether phospholipids, i.e . ET-18-OCH3 (Edelfosine), BM 41.440 (limofosine) and a new aza-derivative (BN 52205), on three leukemic cell lines, i.e . K562 (chronic myeloid leukemia, blast crisis), HL60 (promyelocytic acute leukemia) and CEM (T cell leukemia), and their respective drug-resistant sublines, i.e . K562-ADR (adryamicin resistant), HL60-DNR {daunorubicin (DNR) resistant} and CEM-VLB (vinblastin resistant) . These resistant sublines have been found to express the multidrug-resistant phenotype, revealed by the presence of the P-glycoprotein (PgP) using different monoclonal antibodies . Increased cellular accumulation of the fluorescent anthracycline has been found in both sensitive and resistant cell lines after different ether phospholipid treatment times . In resistant cells, the ether phospholipid effect on DNR accumulation has also been found after blocking the PgP function by verapamil and cyclosporin A . These results confirm that the ether phospholipid action is closely linked with the membrane biochemical composition and that these new anti-tumor drugs are able to change the dynamic structural organization of the tumor cell membrane.

Anticancer Drugs, 1994 Jun, 5(3), 313 - 20
SDZ 280-125: a cyclopeptolide endowed with an in vitro cyclosporin A-like profile of activity for the reversion of the P-glycoprotein-mediated multidrug resistance of tumor cells; Jachez B et al.; Tumor cells whose multidrug resistance is caused by the P-glycoprotein (Pgp) mediated anti-cancer drug (ACD) efflux can be chemosensitized by cyclosporins, whose derivatives were found to display a whole range of resistance-modulating activities . Similarly, derivatives of the non-immunosuppressive natural fungus cyclic peptolide SDZ 90-215 were recently shown to display a broad range of chemosensitizing activities . With highly resistant cells expressing high levels of Pgp, one such compound (SDZ 280-125) was shown here to restore both a normal sensitivity to the growth-inhibitory effects of ACD and a normal retention of an anthracycline antibiotic . With both read-outs, SDZ 280-125 activity was about 3-fold that of cyclosporin A (CsA) . SDZ 280-125 also displayed the same profile of chemosensitization as CsA for different ACD classes.

Tuber Lung Dis, 1994 Jun, 75(3), 163 - 7
Surveillance of resistance to antituberculosis drugs in developing countries; Nunn P et al.; Resistance to antituberculosis drugs is caused by poor management of tuberculosis control . It gives rise to treatment failure, relapse, further transmission of resistant tuberculosis, and multidrug-resistant tuberculosis . Widespread occurrence of multidrug-resistant tuberculosis would constitute a major threat to tuberculosis control in resource-poor countries . Although the impact of HIV on drug resistance is not yet fully understood, it is likely to exacerbate problems caused by drug resistance . In particular, HIV-related adverse effects of thiacetazone, together with the risks of transmission of HIV by parenteral administration of streptomycin, reduce the armamentarium available to tuberculosis control programmes in high HIV prevalence countries, and could encourage the development of resistance to the remaining drugs . While the prime need is to ensure, by good management and supervision, that resistance does not occur in the first place, surveillance of drug resistance is essential to determine the current scale and nature of the drug resistance problem, as well as to define the correct solutionsPIP: Poor management of tuberculosis (TB) control is responsible for resistance to antituberculosis drugs . It leads to treatment failure, relapse, transmission of resistant TB, and multi-drug resistant TB . In developing countries, where resources are already limited, an epidemic of multi-drug resistant TB would jeopardize TB control . The effect of HIV infection is likely to worsen drug resistance-related problems . Specifically, streptomycin injections pose a risk of HIV transmission . It appears that withdrawal of thiacetazone from HIV infected TB patients causes resistance to more powerful drugs . If these 2 antibiotics cannot be used to treat TB patients, the armamentarium available to control TB in high HIV prevalence countries is reduced, which could foster resistance to the fewer remaining antibiotics . Good management and supervision is needed to prevent resistance to antituberculosis drugs . Surveillance of drug resistance is also needed to monitor the current level and characteristics of the drug resistance problem and to identify effective solutions . Specifically, at the national level, a TB surveillance system can assess the TB control program's performance and assess the need to modify the current treatment policy . It can identify districts or health centers with high levels of drug resistance and determine the risk factors for resistance . WHO will assist developing countries in developing their own surveillance systems . WHO and the International Union Against Tuberculosis and Lung Disease plan on setting up a network of supranational reference laboratories to determine the quality control and standardization of susceptibility testing needed for international comparison . WHO also plans on supporting national reference laboratories in developing countries .

J Clin Microbiol, 1994 Jun, 32(6), 1542 - 6
Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis; Plikaytis BB et al.; In 1991, a multidrug-resistant strain of Mycobacterium tuberculosis was isolated from eight people with tuberculosis at a state correctional facility in New York . This strain, which is designated strain W (IS6110 restriction fragment length polymorphism type 212072), was resistant to isoniazid, rifampin, ethambutol, streptomycin, kanamycin, ethionamide, and rifabutin . Since that outbreak, the W strain has been associated with outbreaks in five hospitals in the New York City area and is a continuing public health problem in the area . To be able to identify this strain rapidly, we developed a multiplex PCR assay which targets a direct repeat of IS6110 with a 556-bp intervening sequence (NTF-1) . The amplification generates two amplicons from strain W, which indicate the presence and orientation of the NTF-1 sequence between the direct repeat of IS6110, and a third amplicon, which serves as an internal PCR control . The assay was evaluated with 193 isolates of M . tuberculosis, and all 48 strain W isolates among those 193 isolates were correctly identified.

Cell Biol Int, 1994 Jun, 18(6), 605 - 15
The function of heat-shock proteins in stress tolerance; Venetianer A et al.; We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid-resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived . We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance . Our experiments show that clone 2 cells were not protected from the killing effect of heat by pre-treatment with sodium arsenite, whereas Faza 967 cells were . These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells . Stable heat-resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat-inducibility of hsp68 . These results suggest that several hsps are needed to protect the critical biological processes at high temperature . The heat-resistant hepatoma cells also became resistant to several anticancer drugs . The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P-glycoprotein . Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P-gp may participate in protection against environmental stress such as heat.

Jpn J Cancer Res, 1994 Jun, 85(6), 659 - 64
Circumvention of daunorubicin resistance by a new tamoxifen derivative, toremifene, in multidrug-resistant cell line; Urasaki Y et al.; The reversing effect of toremifene, a new tamoxifen derivative, on multidrug resistance in a K562 subline and its mechanism were studied . K562 cells were cultured in serially increasing concentrations up to 1.0 microM daunorubicin (DNR), and were found to be 28 times more resistant to DNR in comparison to the parent cells . In the resistant cell line (K562/D1-9), intracellular accumulation of DNR was less than that of the parent cell line, and P-glycoprotein was overexpressed . The resistance was reversed by addition of toremifene in a dose-dependent manner in K562/D1-9, while toremifene had no effect in K562 . DNR accumulation was also reversed by toremifene in K562/D1-9, but not in K562 . However, there was no significant difference of toremifene retention between K562/D1-9 and K562, and neither verapamil nor DNR increased toremifene accumulation in K562/D1-9 . Moreover, toremifene and verapamil did not show an additive effect on intracellular DNR accumulation . These results suggested that the reversing mechanism of toremifene is different from that of verapamil, and this compound could be a good candidate for overcoming multidrug resistance.

Clin Sci (Lond), 1994 Jun, 86(6), 749 - 51
Epidemiology of an outbreak of drug-resistant tuberculosis in the U.K . using restriction fragment length polymorphism; Goyal M et al.; 1 . Drug-resistant tuberculosis is a growing health care problem . When a series of cases occur, it is essential to know if patients with multidrug-resistant disease represent one or a number of separate outbreaks . 2 . The epidemiology of an outbreak of isoniazid- and streptomycin-resistant tuberculosis in Blackburn was studied by restriction fragment length polymorphism using a probe for the IS6110 DNA sequence . 3 . Mycobacterium tuberculosis from four cases of isoniazid- and streptomycin-resistant disease had an identical restriction fragment length polymorphism pattern . This pattern was not shared by drug-sensitive isolates of M . tuberculosis obtained from Blackburn (n = 8) or London (n = 13) or a M . tuberculosis isolate from a fifth Blackburn case which was resistant to isoniazid alone . 4 . This methodology confirmed that all four cases of isoniazid- and streptomycin-resistant disease were part of a single epidemiologically related outbreak of drug-resistant disease . This study demonstrates how the epidemiology of an outbreak of multidrug-resistant tuberculosis in the U.K . can be confirmed by restriction fragment length polymorphism.

Anticancer Drug Des, 1994 Jun, 9(3), 251 - 61
Effect of newly synthesized indole derivatives on multidrug resistance in human cancer cells; Matsumoto T et al.; Twenty indole derivatives were investigated for their ability to reverse multidrug resistance (MDR), for their ability to compete with {3H}azidopine in binding to P-glycoprotein (P-gp) and for their hydrophobicity . Six derivatives almost completely reversed the resistance to vincristine (VCR) in multidrug-resistant KB-C2 cells, and other derivatives partially overcame the resistance . The ability of the derivatives to enhance vincristine cytotoxicity did not significantly correlate with the inhibition of {3H}azidopine binding to P-gp or with their hydrophobicity . However, all the derivatives that inhibited > 50% of the photolabeling completely reversed VCR resistance . The 2-pyridyl group with a basic nitrogen atom attached at position 3 of indole in an appropriate spatial orientation seems to be an important feature for the interaction of the indole derivatives with P-gp . One of the derivatives, 1, which has low cytotoxicity and hydrophobicity, completely reversed the resistance of KB-C2 cells to Adriamycin, actinomycin D and VCR . Our data indicate that MDR-reversing indole derivatives with low cytotoxicity and hydrophobicity exist . These characteristics will surely be profitable for clinical use.

DNA Cell Biol, 1994 Jun, 13(6), 641 - 9
Identification of two nuclear protein binding sites and their role in the regulation of the murine multidrug resistance mdr1a promoter; Cohen D et al.; Multidrug resistance genes (mdr) that encode P-glycoproteins (P-gp) are transcriptionally regulated in normal tissues and in some multidrug-resistant (MDR) cells . Several lines of evidence suggest that regulation of P-gp overexpression at the transcriptional level is also important in human tumors . In murine MDR cells, mdr1a and/or mdr1b genes are overexpressed and P-gp isoforms are overproduced . To identify the mdr1a promoter regions that are required for transcription, the promoter has been linked to the chloramphenicol acetyltransferase (CAT) gene in transient expression vectors . 5'-Deletions of the promoter sequences have demonstrated that the region between -155 to +89 bp is crucial for basal activity of the mdr1a gene . DNase I footprinting, methylation interference, and gel retardation assays identified two nuclear protein binding sites within these sequences . One of the nuclear protein binding sites contains an 11-bp DNA sequence that interacts with nuclear protein(s) and is conserved in the promoters of the murine mdr1a and mdr1b, hamster pgp1, and human MDR1 genes . The conserved SP1 site (5'-GGGCGGG-3') that is present further downstream was shown to interact with its nuclear factor . These observations suggest that at least part of mdr gene transcriptional regulation is mediated by conserved mdr cis-regulatory elements and common nuclear factors.

Mol Pharmacol, 1994 Jun, 45(6), 1145 - 52
Characterization of rhodamine 123 binding to P-glycoprotein in human multidrug-resistant cells; Nare B et al.; The overexpression of P-glycoprotein is currently believed to be responsible for the enhanced efflux or decreased influx of cytotoxic drugs across the cell membrane in drug-resistant cells . P-glycoprotein has been proposed to mediate the efflux of a large number of structurally and functionally unrelated drugs . Although it has been suggested that P-glycoprotein binds directly to many lipophilic cations, it remains unclear whether one or more sites in P-glycoprotein mediate its broad substrate specificity . In this report, a photoactive derivative of rhodamine 123 (Rh123) {125I-azidosalicylic acid (ASA)-Rh123} was synthesized and used in a photoaffinity labeling assay to demonstrate, for the first time, direct and specific binding to P-glycoprotein . The photoaffinity labeling of P-glycoprotein by ASA-Rh123 was specifically inhibited in the presence of vinblastine and verapamil but not in the presence of colchicine . Surprisingly, ASA-Rh123 photoaffinity labeled a 6-kDa V8 peptide in P-glycoprotein that was previously shown to be photoaffinity labeled by another multidrug resistance-associated drug, {125I}iodoarylazidoprazosin . Photoaffinity labeling of mitochondria from drug-sensitive or -resistant cells with 125I-ASA-Rh123 did not reveal significant differences in the mitochondrial proteins from sensitive or resistant cells . Interestingly, however, 125I-ASA-Rh123 did photolabel a 66-kDa protein in mitochondria that was not detected in plasma membrane preparations with this assay . Taken together, our results demonstrate for the first time that Rh123 binds specifically to P-glycoprotein and that its binding site may be shared by other multidrug resistance-associated drugs.

J Pharmacol Exp Ther, 1994 Jun, 269(3), 1254 - 60
P-glycoprotein and organic cation secretion by the mammalian kidney; Dutt A et al.; On the basis of physiological localization, broad substrate specificity and energy dependence, the role of the kidney P-glycoprotein was tested in the energy-dependent renal secretion of organic cations . P-glycoprotein-enriched vesicles from Cl 1D/VCR {a multidrug-resistant (MDR) cell line} displayed enhanced transport of the MDR drug vinblastine and the organic cation cimetidine but not of the organic cation tetraethylammonium (TEA) over that shown by vesicles prepared from the drug-sensitive parental line Cl 1D . An outwardly directed proton gradient stimulated TEA and cimetidine uptake by renal brush border membrane vesicles (BBMV) but this gradient did not enhance the uptake of these organic cations into Cl 1D/VCR vesicles . Vinblastine uptake was unaffected by the proton gradient in either vesicle preparation . An outwardly directed gradient of TEA enhanced the uptake of TEA into renal BBMV but did not do so in the case of Cl 1D/VCR vesicles . These data indicate that P-glycoprotein, which is normally energized by ATP hydrolysis, is incapable of catalyzing organic cation/proton exchange or organic cation/organic cation exchange, properties of the organic cation carrier of renal proximal tubule BBMV . The MDR substrates and modulators inhibited the uptake of vinblastine and cimetidine by Cl 1D/VCR vesicles and the uptake of cimetidine and TEA by renal BBMV . Several organic cations studied inhibited TEA and cimetidine uptake by renal BBMV but did not inhibit the uptake of vinblastine and cimetidine by Cl 1D/VCR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia, 1994 Jun, 8(6), 990 - 7
Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemias; Burger H et al.; We determined the expression of the multidrug resistance-associated protein (MRP), a new putative transmembrane drug transporter, in peripheral blood cells from healthy volunteers as well as from 60 patients with acute or chronic leukemia, using an RNase protection assay . MRP appeared to be ubiquitously expressed at low levels in all nonmalignant hemopoietic cell types, reflecting its basal constitutive expression . In acute myelocytic leukemia (AML) (n = 16), one of nine untreated patients and two of seven patients with prior chemotherapy showed significant hyperexpression of MRP . In chronic lymphocytic leukemia (CLL) (n = 21), either treated (n = 8) or untreated (n = 13), a high percentage (15 of 21: 71% had relatively high expression levels of the MRP gene . In contrast, low MRP expression levels were detected in acute lymphocytic leukemia (n = 14), and in chronic myelocytic leukemia (n = 9) . DNA analysis by Southern blotting did not reveal amplification of the MRP gene in the leukemia samples, including those with elevated MRP mRNA levels . We conclude that relatively high expression of MRP is occasionally observed in AML and at high frequency in CLL, irrespective of treatment, probably due to transcriptional activation and/or increased mRNA stability.

Urology, 1994 Jun, 43(6), 787 - 91
DNA ploidy and P-glycoprotein expression as predictive factors of response to neoadjuvant chemotherapy for invasive bladder cancer; Sandlow J et al.; OBJECTIVE . To identify a factor or factors that could predict response of muscle invasive transitional cell carcinoma of the bladder to neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy . METHODS . DNA ploidy analysis and immunohistochemical staining for p-glycoprotein (the product of the multidrug resistance gene, MDR-1) were performed on bladder biopsies obtained prior to chemotherapy . Radical cystectomy specimens were utilized for complete pathologic staging . RESULTS . Tissue was available for DNA ploidy analysis in 25 patients . Ten patients had complete responses to neoadjuvant chemotherapy and 15 patients did not respond . Nineteen patients had aneuploid tumors, of which 7 (37%) were complete responders . Six patients had diploid tumors, of which 3 (50%) were complete responders . This difference was not statistically significant . Tissue was available for immunostaining in 15 patients . Seven of 9 patients (78%) who did not respond to chemotherapy stained positively for p-glycoprotein, whereas 5 of 6 patients (83%) who had complete responses stained positively . This difference was not statistically significant . CONCLUSIONS . Neither DNA ploidy nor p-glycoprotein expression appears to predict successfully those patients likely to respond to neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy in the treatment of invasive bladder cancer.

Int J Cancer, 1994 Jun 1, 57(5), 719 - 26
Establishment and in vivo characterization of multidrug-resistant dunning R3327 rat prostate-carcinoma cell-lines; Bashir I et al.; We describe the selection of 3 new multidrug-resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma . Cell lines of weak (AT2) and strong (AT3 and MAT-LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations . Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay) . Expression of the multidrug-resistance glycoprotein (P-170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219 . The behavior of the parental and resultant drug-resistant cells was assessed by their growth in syngeneic rats . Doxorubicin challenge of the initially drug-sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P-glycoprotein . While lung and lymph-node metastases developed in host animals inoculated with parental AT3 and MAT-LyLu cells, no metastases developed in the multidrug-resistant progeny of these cell lines . This study has shown that Dunning rat prostate-carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug-resistant and express P-glycoprotein following exposure to doxorubicin . Furthermore, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential . Nevertheless, it is unlikely that the non-metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P-glycoprotein . These new multidrug-resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable model with which to analyze the relationship between expression of P-glycoprotein and the metastatic phenotype of prostatic carcinoma cells.

Cancer Res, 1994 Jun 1, 54(11), 2952 - 8
Effect of alpha-interferon on P-glycoprotein expression and function and on verapamil modulation of doxorubicin resistance; Kang Y et al.; Human alpha-interferon (IFN-alpha) is one of the three major classes of interferons which possess antiviral, antiproliferative, and immunomodulatory activities . We recently reported synergism between IFN-alpha and tamoxifen in the modulation of doxorubicin (DOX) cytotoxicity and accumulation in multidrug resistant Chinese hamster ovary ChR C5 cells (Y . Kang and R . R . Perry, Cancer Res., 53: 3040-3045, 1993), but the mechanism was uncertain . In the present study, IFN-alpha (500 units/ml) pretreatment of ChR C5 cells similarly increased verapamil (VPL) modulation of DOX cytotoxicity in a time-dependent manner . Measurement of intracellular DOX fluorescence showed a parallel increase in DOX accumulation . Western blot analysis revealed that IFN-alpha induced a time-dependent increase in p-glycoprotein (p-gp) expression, accompanied by an increase of mdr-1 mRNA as measured using Northern analysis . MDR-1 gene copy number measured using Southern analysis remained unchanged . Assay of specific photoaffinity labeling of p-gp by {3H}-azidopine showed that, although IFN-alpha is not a substrate of p-gp, it significantly increased the ability of VPL to bind to p-gp . These data suggest that IFN-alpha may effect the expression of p-gp in multidrug-resistant ChR C5 cells by transcriptional or posttranscriptional control of mdr-1 gene expression . IFN-alpha enhances the ability of VPL to modulate DOX cytotoxicity and accumulation, possibly by altering the accessibility of p-gp binding site(s) to VPL . By using relatively low concentrations of IFN-alpha and VPL it may be possible to develop less toxic regimens to reverse multidrug resistance.

Cent Eur J Public Health, 1994 Jun, 2(1), 58 - 9
DNA fingerprint analysis of drug resistant Mycobacterium tuberculosis strains isolated in the Czech Republic; Rigouts L et al.; DNA fingerprints were established in 12 drug resistant and 12 susceptible M . tuberculosis strains collected from patients residing in Prague, Czech Republic . The Restriction Fragment Length Polymorphism (RFLP) technique was based on the detection of the insertion sequence IS6110 in PvuII digested chromosomal DNA . All investigated strains possessed at least 5 copies of IS6110 ranging from 5 to 15 copies in the drug resistant isolates and 5 to 11 copies in susceptible isolates . Three multidrug resistant strains displayed identical fingerprints (5 bands), and two strains resistant to pyrazinamid had the same banding pattern (12 bands) . The remaining isolates differed either in number or location of the IS6110 copies . Thorough epidemiological analysis did not furnish proof of direct contact among these patients.

Leukemia, 1994 Jun, 8(6), 998 - 1004
Expression of the multidrug resistance P-glycoprotein and its relationship to hematological characteristics and response to treatment in myelodysplastic syndromes; Lepelley P et al.; Expression of P-glycoprotein (PGP), the product of the multi-drug resistance mdr1 gene was studied by immunocytochemistry on bone marrow slides using JSB1 monoclonal antibody and the alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin-peroxidase (ABC) techniques in 82 cases of untreated myelodysplastic syndromes (MDS), of whom ten had evolved to AML (MDS-AML) . The relationship between PGP expression, myeloperoxidase activity and immunophenotype of blast cells, karyotype and outcome was also analyzed . PGP expression was found in the blasts of 34 of the 82 patients (41%), the majority of blasts being stained in positive cases . PGP positivity was rare in 'low risk' MDS (RA and RARS: 2/12 cases) as opposed to 'high risk' MDS (RAEB, RAEB-T, CMML: 25/60 cases) and MDS-AML (7/10 cases) (p = 0.04) . PGP expression was positively correlated to the presence of myeloperoxidase activity in less than 3% of blasts (p = 0.025), and CD34 antigen expression (p = 0.04), whereas CD33 antigen expression had borderline significance (p = 0.07), demonstrating that PGP expression predominated in blasts with an immature phenotype . An abnormal karyotype, and especially the presence of monosomy 7, was not correlated to a higher incidence of PGP expression, however . There was a trend for more frequent progression to AML and for shorter survival in PGP-positive cases, but differences with PGP-negative cases were not significant . Twenty patients received intensive anthracycline-Ara-C chemotherapy and ten (50%) achieved complete response, including 9/13 (69%) PGP-negative cases and 1/7 (14%) PGP-positive cases (p = 0.03) . Twenty other patients were treated with low-dose Ara-C and ten (50%) responded (complete or partial response) . PGP-positivity did not negatively affect response to low-dose Ara-C: 4/11 responses in PGP-negative, and 6/9 responses in PGP-positive patients (p = 0.18) . Because the treatment choice in advanced MDS (especially between anthracycline-Ara-C or low-dose Ara-C, chemotherapy) is difficult, our preliminary therapeutic results suggest that the analysis of PGP expression could have practical importance in MDS . These findings however, will have to be confirmed on larger numbers of patients . Clinical trials using drugs potentially reverting mdr, activity could also be warranted in MDS.

Leukemia, 1994 Jun, 8(6), 968 - 73
Significantly lower P-glycoprotein expression in acute promyelocytic leukemia than in other types of acute myeloid leukemia: immunological, molecular and functional analyses; Paietta E et al.; Expression of P-glycoprotein (Pgp), the product of the multidrug resistance (MDR1) gene, detected by flow cytometric analysis of the binding of antibody 4E3.16, was found on significantly fewer leukemic cells in 35 adult patients with de novo acute promyelocytic leukemia (APL) (mean 14.8%, median 7%) than in 184 patients with non-APL acute myeloid leukemia (AML) at diagnosis (mean 28.3%, median 18%) (p = 0.0038) . APL was diagnosed based on morphology, the detection of t(15;17) and of the chimeric fusion transcript PML/RAR alpha by PCR . To further substantiate low MDR1 expression in APL, we studied cells from 11 APL patients at the molecular and functional level in comparison to 48 non-APL cases . The diagnosis of APL was associated with the absence of Pgp function by the rhodamine efflux assay (p = 0.0001) . Furthermore, MDR1-specific transcript levels, determined by quantitative PCR with two distinct sets of primers, were significantly lower in mononuclear cells from the APL than the other AML cases (p = 0.013) . The frequency of leukemic cells positive for CD34, an antigen presumably associated with Pgp expression in AML, was significantly lower in APL than other AMLs (p = 0.0001) . In contrast to non-APL leukemias, those few cases of CD34 strongly positive APL neither expressed Pgp nor contained significant MDR1 transcript levels . Low expression of Pgp by APL cells may provide the biologic basis for the high sensitivity of this leukemia subtype to chemotherapeutic agents in vivo.

Ann N Y Acad Sci, 1994 May 31, 716, 126 - 38; discussion 138-43
Gene transfer of drug resistance genes . Implications for cancer therapy; Gottesman MM et al.; Two general approaches to the gene therapy of cancer have been proposed: (1) strategies that use exogenous genes to modify cancer cells so that they are less malignant or more susceptible to host defenses or to killing by exogenous agents; and (2) approaches that modify host cells so that they are more effective in eliminating cancer cells or more resistant to agents that are used to treat cancer . In both cases, the development of vectors that encode in vivo selectable phenotypes, such as drug resistance, would be extremely valuable because of the inherent inefficiency of gene transfer and the potential of such vectors to protect normal tissues against toxic agents . To allow the selection of cells in vivo that have been transduced with vectors for gene therapy, we have utilized the human multidrug resistance (MDR1) gene . The product of this gene is a 170,000-dalton glycoprotein known as P-glycoprotein, which acts as an energy-dependent efflux pump for a great many cytotoxic anticancer drugs, including doxorubicin, daunorubicin, etoposide, teniposide, actinomycin D, and taxol . Vectors encoding an MDR1 cDNA are able to transduce many cell types, including bone marrow cells, with high efficiency to allow selection of drug resistance in vitro and in vivo in mouse models . Thus, it should be possible to protect the bone marrow of patients undergoing intensive chemotherapy by transduction of their bone marrow with MDR1 vectors . Furthermore, the ability to select for the presence of the MDR1 cDNA in vivo means that it can be used to introduce otherwise nonselectable genes into the bone marrow for therapy of cancer and other diseases.

MMWR Morb Mortal Wkly Rep, 1994 May 27, 43(20), 361 - 6
Expanded tuberculosis surveillance and tuberculosis morbidity--United States, 1993; Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane; Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs . It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis) . A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane . Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp . We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition) . The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors . The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux . From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.

FEBS Lett, 1994 May 16, 344(2-3), 221 - 4
Tamoxifen decreases drug efflux from liposomes: relevance to its ability to reverse multidrug resistance in cancer cells?
Kayyali R, Marriott C, Wiseman H.
Tamoxifen decreased the efflux of the fluorescent marker drug, chloroquine, from phosphatidylcholine liposomes . Tamoxifen is a known structural-mimic of cholesterol, which were both found to be similarly effective in preventing drug release from liposomes . This ability of tamoxifen and cholesterol to decrease drug efflux in a concentration-dependent manner is likely to arise from their known ability to decrease membrane fluidity both in liposomes and also in cancer cells . The possible importance of the ability of tamoxifen to inhibit drug efflux from liposomes in relation to its ability to reverse multidrug resistance in cancer patients caused by the efflux of cytotoxic therapeutic agents, is discussed.

Hosp Pract (Off Ed), 1994 May 15, 29(5), 85 - 8, 91, 95-6
Multidrug-resistant tuberculosis: meeting the challenge; Reichman LB; Both public and private institutions are mobilizing to combat drug-resistant TB--indeed, to eliminate all TB . Meeting this goal will require a three-pronged approach: 1) prophylactic isoniazid treatment of latent TB, 2) multidrug (at least six agents in some cases) treatment of active TB to eradicate resistant strains, and 3) correction of the social conditions that have contributed to the current epidemic.

Int J Cancer, 1994 May 15, 57(4), 522 - 8
Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line; Zalcberg JR et al.; Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM . The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml . In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml . P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line . Amplification of the mdrI gene was not observed in the CEM/A7 cell line . Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids . Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line . Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines . The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line . CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2 . Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.

J Med Chem, 1994 May 13, 37(10), 1486 - 94
Synthesis and in vitro evaluation of 9-anilino-3,6-diaminoacridines active against a multidrug-resistant strain of the malaria parasite Plasmodium falciparum; Gamage SA et al.; A series of 9-anilinoacridines have been prepared and evaluated for their activity against a multidrug-resistant K1 strain of the malaria parasite Plasmodium falciparum in erythrocyte suspensions . 3,6-Diamino substitution on the acridine ring resulted in lower mammalian cell cytotoxicity and higher antiparasitic activity than other substitution patterns, providing compounds with the highest in vitro therapeutic indices . A new synthesis of 3,6-diamino-9-anilinoacridines, via reduction of the corresponding diazides, gives much higher yields than traditional methods . Within the subset of 3,6-diamino-9-anilinoacridines, there was considerable tolerance to substitution at the 1'-anilino position . In a sharp divergence with structure-activity relationships for high mammalian cell toxicity and anticancer effects, derivatives bearing electron-withdrawing 1'-substituents (e.g., SO2-NHR and CONHR) showed the most potent antimalarial activity (IC50 values of 10-20 nM) . Representative compounds were shown to be potent inhibitors of the DNA strand-passing activity of human topoisomerase II and of the DNA decatenation activity of the corresponding parasite enzyme . The 1'-SO2NH2derivative 7n completely inhibited strand passage by Jurkat topoisomerase II at 20 microM, and an increase in linear DNA (indicative of inhibition of religation) was seen at or above 1 microM . It also inhibited the decatenating activity of the parasite topoisomerase II at 6 microM and above . In contrast, the analogous compound without the 3,6-diamino substituent was inactive in both assays up to 100 microM . Overall, there was a positive relationship between the ability of the drugs to inhibit parasite growth in culture and their ability to inhibit parasite topoisomerase II activity in an isolated enzyme assay . The 1'-SO2NH2 derivative 7n showed a high IVTI (1000) and was a potent inhibitor of both P . falciparum in vitro (IC50 20 nM) and P . falciparum-derived topoisomerase II . However, the compound was inactive against Plasmodium berghei in mice; reasons may include rapid metabolic inactivation (possibly by N-acetylation) and/or poor distribution.

J Natl Cancer Inst, 1994 May 4, 86(9), 700 - 5
MDR1 gene expression: its effect on drug resistance to doxorubicin in human hepatocellular carcinoma cell lines; Park JG et al.; BACKGROUND: Hepatic tumors are resistant to many chemotherapeutic agents . Although elevated MDR1 (also known as PGY1) gene expression has been shown in such tumors, no direct association has been established between the gene expression and multidrug resistance . PURPOSE: To evaluate the role of the MDR1 gene in the drug resistance of hepatoma, we tested nine human hepatoma cell lines for their expression of the MDR1 gene . METHODS: We measured the MDR1 messenger RNA (mRNA) expression by RNA slot-blot analysis and by immunocytochemical staining with a P-glycoprotein-specific monoclonal antibody, MRK16 . The in vitro chemosensitivity of these cell lines to fluorouracil, doxorubicin, mitomycin C, cisplatin, and etoposide (VP-16) was determined using the MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) colorimetric assay . For doxorubicin cytotoxicity, we also tested the potentiating effect of several multidrug resistance-reversing agents . RESULTS: Slot-blot analysis and immunocytochemistry showed that two cell lines expressed high levels of MDR1 mRNA, one expressed an intermediate level, and all others were low expressors . The MTT assay results showed that all cell lines tested were generally resistant to chemotherapeutic agents . The assay area under the curve (AUC) was within a clinically achievable range only for VP-16 in one of nine cell lines . When the IC50 values were compared among the cell lines, the results revealed a close association with the MDR1 gene expression only for doxorubicin resistance . Verapamil and quinidine lowered the IC50 values of doxorubicin for MDR1-positive cell lines . The lowered assay AUC levels for both reversing agents, however, were still higher than the clinically achievable range . CONCLUSION: These results indicate that the MDR1 gene probably has a role in doxorubicin resistance in hepatocellular carcinoma and that the resistance can be overcome by some multidrug resistance-reversing agents . IMPLICATIONS: Some widely used anticancer agents might be ineffective for treating hepatocellular carcinoma in clinical situations even when combined with reversing agents.

J Natl Cancer Inst, 1994 May 4, 86(9), 688 - 94
P-glycoprotein binding and modulation of the multidrug-resistant phenotype by estramustine; Speicher LA et al.; BACKGROUND: Previous preclinical studies of combinations of estramustine and vinblastine or paclitaxel (Taxol) have shown that it is possible to achieve a greater than additive cytotoxicity with these antimicrotubule drug combinations . Phase II studies in hormone-refractory prostate cancer have demonstrated clinical antitumor activity of sufficient magnitude to stimulate further laboratory and clinical studies of these drugs combinations . PURPOSE: Our purpose was to characterize the interactions of estramustine with P-glycoprotein and to determine its effects . METHODS: Standard laboratory techniques were used to study the effects of estramustine on intracellular drug concentrations, cytotoxicity, and induction of messenger RNA (mRNA) for the MDR1 (also known as PGY1) gene . Using a photoaffinity analogue of estramustine 17-0-{{2-{3-(4-azido-3-{125I}-iodophenyl) propionamido}ethyl}-carbamyl}estradiol-3-N-bis(2-chloroethyl)ca rba mate ({125I}AIPP-estramustine), binding to the membrane proteins of human ovarian (SKOV3) and their multidrug-resistant counterpart SKVLB1 cells was studied . Southern-blot analysis was performed on DNA extracted from human prostate carcinoma wild-type DU145, estramustine-resistant cell line (E4), and SKVLB1 cells . RESULTS: Membrane fractions from SKOV3 and SKVLB1 cells were analyzed for proteins that could be photoaffinity labeled with {125I}AIPP-estramustine . Competitive inhibition of this binding was achieved with excess concentrations of (in order of efficacy) estramustine, vinblastine, verapamil, progesterone, and to a lesser degree, by paclitaxel but not with estramustine phosphate, estradiol, and estriol . SKVLB1 cells accumulated much less {3H}vinblastine and {3H}paclitaxel than did SKOV3 cells . Estramustine caused a concentration-dependent enhancement of drug accumulation in the SKVLB1 cells to a maximum of approximately 12-fold . No effect of estramustine was apparent for the wild-type SKOV3 cells . In comparison with verapamil, estramustine was less effective as a modulator; however estramustine demonstrated good chemosensitizing activity in combination with actinomycin D and vinblastine . Neither short-term, low-dose no longer-term, higher concentration were found to produce measurable transcript (mRNA for the MDR1 gene levels . Such data suggest that, at least levels . Such data suggest that, at least for two distinct human cell line (SKOV3 and DU145), estramustine does not induce the overexpression of the MDR1 gene . CONCLUSION: It is apparent from the P-glycoprotein data that estramustine interacts with this efflux pump, altering intracellular drug accumulation . Overall, the nonempiric basis for including estramustine in clinical protocols that contain other multidrug-resistant drugs is strengthened by the present data.

Am J Med, 1994 May, 96(5), 439 - 50
Tuberculosis and acquired immunodeficiency syndrome: a historical perspective on recent developments; Haas DW et al.; The incidence of tuberculosis has increased in recent years, at least in part as a result of the ongoing worldwide epidemic of acquired immunodeficiency syndrome (AIDS) . In addition, the occurrence of outbreaks caused by multidrug-resistant Mycobacterium tuberculosis organisms has greatly heightened concern . In retrospect, a number of seminal studies that have appeared during the past decade have helped to define changing concepts concerning the epidemiology, pathogenesis, approaches to preventive care, diagnosis, and treatment of tuberculosis in HIV-infected persons . Such reports have shown that the variable clinical manifestations of tuberculosis in patients with AIDS are greatly influenced by the degree of HIV-induced immunosuppression . Explosive outbreaks of tuberculosis occurring in closed environments have emphasized that patients with AIDS and pulmonary tuberculosis may be highly contagious, especially when diagnosis and implementation of appropriate infection control measures are delayed . The extent to which homelessness and illicit drug use complicate management of tuberculosis have been examined, and the high risk of persons who are both tuberculin-positive and HIV-positive ultimately developing active tuberculosis, unless chemoprophylaxis is completed, has been clearly shown . The utility of sputum smears, bronchoscopy, and newer technologies such as polymerase chain reaction for diagnosis has been examined . The risk of relapse appears to be low when patients with AIDS with drug-sensitive tuberculosis complete appropriate multiple-drug therapy . Recent reports have addressed important hospital infection control, tuberculin testing, and chemoprophylaxis issues . This paper describes this evolution of understanding, focusing on reports that we believe have been conceptually important.

Mol Pharmacol, 1994 May, 45(5), 908 - 15
Influence of structural modifications at the 3' and 4' positions of doxorubicin on the drug ability to trap topoisomerase II and to overcome multidrug resistance; Capranico G et al.; To better define the role of the amino sugar in the pharmacological and biochemical properties of anthracyclines related to doxorubicin and daunorubicin, we have investigated the effects of various substituents at the 3'- and 4'-positions of the drug on cytotoxic activity and ability to stimulate DNA cleavage mediated by DNA topoisomerase II . The study shows that the nature of the substituent at the 3'-position but not the 4'-position is critical for drug ability to form cleavable complexes . The amino group at the 3'-position is not essential for cytotoxic and topoisomerase II-targeting activities, because it can be replaced by a hydroxyl group without reduction of activity . However, the presence of bulky substituents at this position (i.e., morpholinyl derivatives) totally inhibited the effects on the enzyme, thus supporting previous observations indicating that the cytotoxic potencies of these particular derivatives are not related to topoisomerase II inhibition . This conclusion is also supported by the observation that 3'-morpholinyl and 3'-methoxymorpholinyl derivatives are able to overcome atypical (i.e., topoisomerase II-mediated) multidrug resistance . Because a bulky substituent at the 4'-position did not reduce the ability to stimulate DNA cleavage, these results support a critical role of the 3'-position in the drug interaction with topoisomerase II in the ternary complex . An analysis of patterns of cross-resistance to the studied derivatives in resistant human tumor cell lines expressing different resistance mechanisms indicated that chemical modifications at the 3'-position of the sugar may have a relevant influence on the ability of the drugs to overcome specific mechanisms of resistance.

Br J Cancer, 1994 May, 69(5), 868 - 74
Multidrug resistance circumvention by a new triazinoaminopiperidine derivative S9788 in vitro: definition of the optimal schedule and comparison with verapamil; Julia AM et al.; The current work was undertaken to investigate the importance of exposure sequence and duration in achieving the maximum reversal action of S9788 on doxorubicin (DOX) cytotoxicity against cells that exhibit the (MDR) multidrug resistance phenotype: the MCF7/DOX cell line . Accumulation and release of DOX were examined in this cell line . The reversal effect was compared with that obtained with verapamil . S9788 activity was schedule dependent: when comparing incubation with S9788 before or after treatment with DOX, the best reversal factor was obtained in the case of a post-treatment incubation (65.6 +/- 7.7 vs 20.8 +/- 7.0) . S9788 was a more potent modulating agent than verapamil, whatever the schedule of exposure of the cells to the reversal agent . The reversal of resistance after short-term DOX exposures was caused not only by prolonged cellular accumulation of DOX, but also by its prolonged retention after transfer of cells to DOX-free medium . A relationship was noted between cellular exposure to DOX and the cytotoxic effect, and so the reversal of resistance induced by S9788 appears to be directly linked to the level of cell exposure to DOX . This work provided a rationale for improving the schedule of administration of S9788 in clinical trials.

J Histochem Cytochem, 1994 May, 42(5), 607 - 11
Immunohistochemical detection of ornithine decarboxylase in individual cells: potential application for in vitro chemosensitivity assays; Shayovits A et al.; Ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in the biosynthesis of polyamines, can serve as a marker of proliferation . The presence of ODC protein in individual cells was quantitatively detected by an immunofluorescence assay using an ACAS 570 computerized fluorescence microscope . ODC was detected in KB-3-1 human epithelial carcinoma cells grown in the absence of any drug . Vinblastine, which inhibits cell growth, caused the disappearance of ODC . On the other hand, ODC was detected in multidrug-resistant cells grown in the absence or in the presence of vinblastine . NIH 3T3 fibroblasts transformed by the c-Ha-ras oncogene also contained ODC protein . This protein disappeared when the cells were treated with cycloheximide, which inhibits cell proliferation . These findings suggest that ODC can be detected in individual cells by immunofluorescence . Whether this method can be used for in vitro chemosensitivity tests remains to be studied.

Clin Infect Dis, 1994 May, 18(5), 755 - 9
Trends in the susceptibility of tuberculosis in New York City, 1987-1991 . New York City Area Tuberculosis Working Group; Sepkowitz KA et al.; The annual number of cases of tuberculosis in New York City has increased since 1978 . In addition, in 1991 a 1-month survey of cases of tuberculosis in New York City found that 33% of all cases were resistant to at least one drug . To determine susceptibility trends from 1987 to 1991, a period during which an unprecedented rise in resistant tuberculosis occurred in New York City, we reviewed the microbiology records of 44 New York City hospitals (comprising > 14,000 cases) . The percentage of cases resistant to at least one drug rose from 19% in 1987 to 28% in 1991, and the percentage of cases resistant to isoniazid rose from 13% in 1987 to 23% in 1991, while resistance to at least both isoniazid and rifampin rose from 6% to 14% . The rise of multidrug-resistant tuberculosis occurred in all four surveyed boroughs (counties) of New York City . These data demonstrate how rapidly multidrug-resistant tuberculosis can appear, and they suggest that initial empirical regimens should be broadened at certain hospitals.

Anticancer Res, 1994 May-Jun, 14(3A), 1221 - 6
Antiproliferative effect of genistein and adriamycin against estrogen-dependent and -independent human breast carcinoma cell lines; Monti E et al.; The effects of the combination of genistein, a tyrosine kinase inhibitor, and adriamycin, an anthracycline anticancer drug, were studied in three human breast carcinoma cell lines (MCF-7/WT, MCF-7/ADR(R) and MDA-231) differing in estrogen receptor status and adriamycin sensitivity . Genistein inhibited cell proliferation in all three cell lines (IC50 between 7.0 and 37.0 microM) . The combination produced additive to synergistic effects; epidermal growth factor receptor modulation by adriamycin does not seem to be involved in this interaction . The possible therapeutic advantage of this drug combination, especially on hormone-independent and multidrug resistant tumor cells, deserves further investigation.

Zhongguo Yao Li Xue Bao, 1994 May, 15(3), 275 - 9
{Intracellular accumulation, retention, and distribution of anthracyclines in a multidrug-resistant variant K562r}; Hu X et al.; A multidrug resistant variant (K562r) of the human K562 erythroleukemia cell line was obtained in vitro by repeated exposure of these cells to vincristine . The K562r cells were resistant to vincristine, harringtonine, mitomycin C, doxorubicin, epirubicin, and daunorubicin but retained the sensitivity to methotrexate . The resistances to vincristine and anthracyclines were reversed in the presence of verapamil . Despite being 60-or over 60-fold resistances to doxorubicin, epirubicin and daunorubicin, the net intracellular uptakes of these drugs at 2 h were reduced by only 15%, 20%, and 11%, respectively in K562r cells compared to their parental line . Verapamil did not enhance drug accumulation but inhibited drug efflux in K562r cells . Though there was no significant difference of drug content in cytoplasm between K562 and K562r cells, the drug content in the nucleus of K562r cells reduced significantly compared to that in K562 cells . The lower concentrations of anthracyclines in the nucleus of K562r cells might contribute to their acquisition of drug resistance.

Haematologica, 1994 May-Jun, 79(3), 200 - 4
Overexpression of MDR-related p170 glycoprotein in chronic myeloid leukemia; Michelutti A et al.; BACKGROUND AND METHODS . Philadelphia (Ph) positive chronic myeloid leukemia (CML) cannot be induced into a true remission with conventional chemotherapy . Blast cells and precursors obtained from 51 Ph+ CML cases were assayed for expression of the multidrug resistance (MDR)-associated glycoprotein (p170) by immunocytochemistry (APAAP) with the MRK-16 monoclonal antibody . RESULTS AND CONCLUSIONS . Positive cells were found in 11/17 cases in chronic phase (65%), in 4/8 cases in accelerated phase, and in 23/26 cases in blastic phase (89%) . The proportion of positive cells, which ranged between less than 1% and 95%, was higher in blastic phase (mean 32 +/- 29.9) than in chronic phase (mean 3 +/- 5.3) (p = 0.006) . These findings show that p170 overexpression is common in Ph+ CML, especially after progression to blastic phase, and suggest that p170-related MDR may contribute significantly to treatment failure.

Anticancer Res, 1994 May-Jun, 14(3A), 869 - 74
Chemoresistance to doxorubicin and cisplatin in a murine cell line . Analysis of P-glycoprotein, topoisomerase II activity, glutathione and related enzymes; Mestdagh N et al.; Resistance to antineoplastic drugs has often been associated with P-glycoprotein overexpression, this certainly being not the sole mechanism . In order to characterize resistance to doxorubicin and cisplatin, we have analysed P-glycoprotein expression, topoisomerase II activity, glutathione and related enzymes in murine leukemic cells (doxorubicin or cisplatin-resistant) . The doxorubicin-resistant cells contained P-glycoprotein, showed lower activities of glutathione S-transferase well as of glutathione reductase and topoisomerase II . The modifications observed in the most cisplatin-resistant cell line were a higher activity of glutathione S-transferase isoenzyme pi and topoisomerase II . These results suggest that drug uptake, glutathione metabolism as well as topoisomerase II activity are all characteristic of multidrug resistance.

Anticancer Res, 1994 May-Jun, 14(3A), 1009 - 16
P-glycoprotein expression and activity of resistance modifying agents in primary cultures of human renal and adrenocortical carcinoma cells; Fridborg H et al.; Tumor cells from patients with renal or adrenocortical carcinomas were tested in vitro for sensitivity to doxorubicin (Dox) and vincristine (Vcr), as well as for the modulation of this sensitivity by resistance modifiers and the immunohistochemical expression of the multidrug resistance associated P-glycoprotein (Pgp) . Normal adrenocortical cells from one patient and Pgp-expressing cells of Dox-resistant myeloma RPMI 8226 sublines were included for comparison . The normal adrenocortical cells and cells from one adrenocortical carcinoma showed high Pgp expression, comparable to the most Dox-resistant myeloma cell line, whereas the other tumor samples showed variable but lower expression . The normal adrenocortical as well as the adrenocortical and renal tumor cells were highly resistant to Dox and Vcr . Whereas the cytotoxic effect of Dox was considerably increased by verapamil, cyclosporin A and its non-immunosuppressive analogue SDZ PSC 833 in the Pgp-expressing Dox-resistant sublines, comparatively small effects on the Dox and Vcr sensitivity were observed in the patient samples, irrespective of their Pgp expression . The results indicate that the Dox and Vcr resistance in human adrenocortical and renal carcinomas is mediated by mechanisms other than Pgp and that resistance modulating agents targeting Pgp may be much less efficient in the clinic than in Pgp-expressing cell lines, at least for the tumor types described in the present study.

Anticancer Res, 1994 May-Jun, 14(3B), 1293 - 5
MDR1 gene expression in myelodysplastic syndrome and in acute myeloid leukemia evolving from myelodysplastic syndrome; Zochbauer S et al.; The expression of the MDR1 gene, a multidrug resistance gene, was determined in patients (N = 24) with myelodysplastic syndrome or acute myeloid leukemia evolving from it . MDR1 RNA expression of the mononuclear cells was detected in 14 (58%) patients . Among patients who had progressed to acute myeloid leukemia (sAML) (N = 14), MDR1 RNA expression was seen in 8 (57%) patients . Expression was observed in the 2 patients who had been pretreated with MDR drugs . These findings indicate that the MDR1 gene is frequently expressed in MDS or sAML and suggest that multidrug resistance might be involved in the clinical drug resistance of these diseases.

Histochem J, 1994 May, 26(5), 417 - 23
Stage-specific distribution of P-glycoprotein in first-trimester and full-term human placenta; MacFarland A et al.; The distribution of P-glycoprotein in human placenta has been examined by immunohistochemistry using a battery of monoclonal antibodies (MRK-16, C219 and JSB-1) . P-glycoprotein was located on the syncytiotrophoblast microvillus border in first-trimester placentas and some of the placental macrophages (Hofbauer cells) showed weak cytoplasmic staining . In term placentas, however, staining was not observed in the trophoblast but most of the Hofbauer cells displayed strong cytoplasmic staining . In situ hybridization with specific gene probes suggested that both human multidrug resistance genes were expressed in the placenta, although only the multidrug resistance-1 gene product would have been detected by the MRK and JSB-1 antibodies . These results point to distinct functions for P-glycoprotein during the different stages of placental development and indicate that its expression may be under developmental control.

Jpn J Cancer Res, 1994 May, 85(5), 536 - 41
Expression of the multidrug resistance gene (MDR1) in non-small cell lung cancer; Abe Y et al.; To ezamine the clinical relevance of P-glycoprotein, encoded by the human multidrug resistance gene (MDR1), to multidrug resistance in lung cancer, we examined the expression of MDR1 in 107 non-small cell lung cancer (NSCLC) specimens and 20 corresponding specimens of normal lung tissues . We also evaluated the relationship between MDR1 expression and the histopathology and pathological staging of NSCLC . The tumors consisted of 60 adenocarcinomas, 38 squamous cell carcinomas, 8 large cell carcinomas, and 1 adenosquamous carcinoma . MDR1 expression was semi-quantified by use of the reverse transcriptase-polymerase chain reaction method . We subclassified the NSCLC into 3 grades according to the MDR1 expression level (-, +, ++) . Sixty-one of the 107 tumor specimens (57%) and 18 of the normal lung tissue specimens (90%) expressed various levels of the MDR1 gene . Only one tumor specimen showed higher MDR1 expression than the corresponding normal lung tissue . The relationship between pathological stage and MDR1 expression levels was not significant . These results suggest that the level of MDR1 expression in lung cells is decreased as cells progress from the normal to the transformed state.

Mol Pharmacol, 1994 May, 45(5), 962 - 70
Cross-resistance to antitumor diarylsulfonylureas and collateral sensitivity to mitochondrial toxins in a human cell line selected for resistance to the antitumor agent N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea; Sosinski J et al.; Diarylsulfonylurea (DSU) antitumor agents represent a new class of oncolytic compounds with an unknown, potentially novel, mechanism of action . At high concentrations of several of these agents, cytotoxicity appears to be a consequence of uncoupling of mitochondria . However, the mechanism of action at pharmacologically achievable concentrations is unknown . To further study these agents a subline of human colon carcinoma, GC3/c1, was selected for resistance to N-(5-indanylsulfonyl)-N'-(4-chlorophenyl)urea (ISCU) (Sulofenur) . This clone (designated LYC5) was stably resistant for 2 years in the absence of selection pressure and was characterized for cross-resistance to other antitumor DSU and therapeutically used oncolytic agents . LYC5 was cross-resistant to six of seven DSU analogues examined when cells were exposed to drugs for 7 days . However, the degree of resistance was inversely related to the potency of the individual DSU against the parental GC3/c1 clone . Consequently, against LYC5 cells there was a relatively narrow range for concentrations inhibiting colony formation by 50% (4-fold), compared with that in GC3/c1 cells (12-fold range) . With a single exception, each DSU examined caused uncoupling of oxidative phosphorylation in isolated mitochondria at 50 microM, and data suggest that cytotoxicity in LYC5 cells may be a consequence of mitochondrial impairment . In contrast, LYC5 cells were collaterally sensitive to the mitochondrial toxins rotenone, antimycin, and oligomycin, by 11.4-, 7.2-, and 36.9-fold respectively . LYC5 cells were also collaterally sensitive to vincristine (7.7-fold), Actinomycin D (5.9-fold), and rhodamine-123 (10.5-fold), agents associated with P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) . LYC5 cells were slightly more sensitive to Melphalan and doxorubicin (2.8- and 2.3-fold, respectively) but not to cisplatin or dideazatetrahydrofolic acid . Collateral sensitivity to vincristine and Actinomycin D was consistent with decreased Pgp levels in LYC5 cells . Immunohistochemical staining and Western blotting with anti-Pgp antibodies indicated an 8-fold reduction in Pgp levels in LYC5 cells, relative to expression in parental GC3/c1 cells . Consequently, association of mitochondrial toxins with resistance in MDR KB8-5 cells was examined in the presence or absence of the MDR-reversing agent verapamil . KB8-5 cells had equal or greater sensitivity, compared with parental KB3-1 cells, to rotenone, antimycin, and oligomycin and also to each DSU analogue examined . In addition, verapamil tended to have a protective effect against these mitochondrial toxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Leukemia, 1994 May, 8(5), 881 - 4
Coexpression of anionic glutathione-S-transferase (GST pi) and multidrug resistance (mdr1) genes in acute myeloid and lymphoid leukemias; Russo D et al.; By using RNA slot-blot technique the frequency and degree of GST pi and mdr1 gene coexpression was investigated in 23 patients with acute myeloid leukemia (AML), and nine patients with acute lymphoid leukemia (ALL) . With respect to the negative controls, MCF7 and HL-60 cell lines, increased GST pi and mdr1 mRNA levels, expressed as arbitrary units (U), were respectively detected both in AML and in ALL patients . A positive and significant correlation between GST pi and mdr1 gene expression was found in the group of AML patients, while in the smaller group of ALL patients only a trend could be shown . These data show that two different mechanisms of drug resistance can be coexpressed at the same time in patients with acute myeloid and lymphoid leukemia . From this evidence many important clinical and therapeutic questions arise.

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