Multidrug resistant

Cancer, 1993 Feb 1, 71(3), 667 - 71
MDR1 gene expression and its clinical relevance in primary gastric carcinomas; Wallner J et al.; BACKGROUND . Drug resistance remains a major problem in gastric carcinomas . To evaluate the mechanisms involved in this resistance, the authors determined the expression of the MDR1 gene, a multidrug resistance gene, in primary gastric carcinomas . METHODS . MDR1 RNA levels of gastric carcinoma specimens (n = 22) were determined by slot blot analysis . An MDR1 cDNA (probe 5A) was used for the hybridization . RESULTS . MDR1 RNA was detected in 41% of the gastric carcinomas, with high levels in 18% of the specimens . No expression of the MDR3 gene was observed in these tumors . MDR1 gene expression was independent of patient age, tumor localization, and lymph node involvement . However, MDR1 RNA expression was less frequent in locally advanced tumors and was absent in the primary tumors of all six patients who had distant metastases . CONCLUSIONS . The data indicate that multidrug-resistant cells are present in primary gastric carcinomas and suggest that multidrug resistance might contribute to the clinical drug resistance of these tumors.

Br J Cancer, 1993 Feb, 67(2), 274 - 8
Chemotherapeutic efficacy of the protein-doxorubicin conjugates on multidrug resistant rat hepatoma cell line in vitro; Ohkawa K et al.; In vitro studies were initiated to study the antitumour effect of protein-doxorubicin (DXR) conjugate on the growth of the multidrug resistant rat ascites hepatoma cell line, AH66DR . The 50% inhibitory concentration (IC50) for DXR in AH66DR cell line was 16 mumol l-1 (AH66 parental cell line, AH66P, IC50 was 0.08 mumol l-1) . Treatment of AH66P and AH66DR cells with various concentrations of DXR or conjugates at equivalent concentrations of DXR was performed . The two types of conjugates used were bovine serum albumin (BSA)-DXR conjugate and immunoglobulin G (IgG)-DXR conjugate . Both of these conjugates showed potent dose-dependent inhibition of cell growth against AH66DR cells as compared with the cells treated with DXR or other controls . The IC50 for BSA-DXR and IgG-DXR conjugates in AH66DR cell line was 0.05 (equivalent DXR) mumol l-1 and 0.07 (equivalent DXR) mumol l-1, respectively . These values were similar to that of the AH66P treated with DXR . Cellular uptake and accumulation of DXR or BSA-DXR conjugate was also quantitated in both cell lines . The cellular concentration of DXR in AH66DR cells was 2-fold lower than that of AH66P cells throughout the experiment . In contrast, by the treatment of AH66DR cells with BSA-DXR conjugate, the intracellular drug concentration increased as a function of time up to 24 h (639.1 +/- 41.8, equivalent DXR, ng 10(-5) cells) and reached the same drug level as AH66P cells treated with DXR (617.9 +/- 17.3 ng-5 cells) . Ammonium chloride treatment inhibited the effects of the conjugates but did not inhibit the free drugs . Intracellular DXR was effluxed rapidly from AH66DR cells, but BSA-DXR conjugate remained in the cells at relatively high concentration for a long time . These results indicate that by chemically modifying DXR, such as by conjugation of the drug with proteins, it may be possible to overcome multidrug resistance.

Gynecol Oncol, 1993 Feb, 48(2), 214 - 20
Sensitivity of drug-resistant human ovarian tumor cell lines to combined effects of tumor necrosis factor (TNF-alpha) and doxorubicin: failure of the combination to modulate the MDR phenotype; Safrit JT et al.; We have examined four human ovarian tumor lines (A2780, AD10, OVC-8, and SKOV-3) selected for their sensitivity and/or resistance to the recombinant human tumor necrosis factor alpha (TNF-alpha) and the chemotherapeutic drug doxorubicin (DOXO) . The tumor lines were either sensitive to both agents, resistant to one or the other, or resistant to both . Of the four lines examined only the DOXO-resistant line AD10 exhibited the multidrug-resistance (MDR) phenotype . Enhanced cytotoxicity was seen with the combination of TNF-alpha and DOXO in each line regardless of their sensitivity or resistance patterns and, thus, demonstrates that drug resistance due to the expression of the MDR phenotype or its absence can be overcome by TNF-alpha and DOXO . We then examined whether TNF-alpha or TNF-alpha and DOXO modulated the MDR phenotype in AD10 as a possible mechanism of overcoming drug resistance . TNF-alpha had no effect on either DOXO intake or efflux as measured by flow cytometry . Further, TNF-alpha treatment showed no effect on the level of MDR-1 mRNA . These results suggest that the enhanced cytotoxicity seen with the combination of TNF-alpha and DOXO is not the result of any modulation of drug influx or efflux levels by TNF-alpha . Overall, these findings suggest that combination treatment with TNF-alpha and DOXO can overcome resistance inflicted by different mechanisms.

J Nat Prod, 1993 Feb, 56(2), 233 - 9
1H- and 13C-nmr assignments of phyllanthin and hypophyllanthin: lignans that enhance cytotoxic responses with cultured multidrug-resistant cells; Somanabandhu A et al.; Complete 1H-nmr data and unambiguous assignments of the 13C-nmr spectra of phyllanthin {1} and hypophyllanthin {2} were obtained through extensive nmr studies, including homonuclear COSY, homonuclear decoupling, APT, HETCOR, nOe difference, selective INEPT, and COLOC experiments . The absolute configuration of hypophyllanthin {2} was determined by cd . Neither of these lignans demonstrated significant cytotoxic activity when evaluated with a battery of cultured mammalian cells, but both were found to enhance the cytotoxic response mediated by vinblastine with multidrug-resistant KB cells . In addition, 1 was found to displace the binding of vinblastine with membrane vesicles derived from this cell line, suggesting an interaction with the P-glycoprotein.

Invest New Drugs, 1993 Feb, 11(1), 75 - 9
Phase II trial of doxorubicin and trifluoperazine in metastatic breast cancer; Budd GT et al.; Pre-clinical and clinical studies have shown that trifluoperazine (TFP) can modulate multidrug resistance . We have performed a Phase II trial of TFP and doxorubicin in doxorubicin-naive patients with metastatic breast cancer . We hypothesized that TFP would inhibit the development of doxorubicin resistance, resulting in an increased rate of complete response or a prolongation in response duration . Twenty patients with metastatic breast cancer were treated every 3 weeks with TFP 5 mg by mouth every 6 hours on days 0-5 and doxorubicin 60 mg/m2/96 hr on days 1-4 by continuous intravenous infusion . The first 5 patients were treated with TFP 15 mg by mouth every 6 hours, but the dose was reduced to 5 mg every 6 hours when grade 3-4 extrapyramidal toxicity was noted in 3 of the first 5 patients . Thereafter, neurologic toxicity was grade 0-2 . No complete and 9 partial responses were produced in 20 patients (45%) . The median response duration was 17 weeks (range 7-112) . The combination of trifluoperazine and doxorubicin did not seem to produce a response rate or duration markedly different than that expected for doxorubicin alone in patients with metastatic breast cancer . Alternative trial designs may be necessary in future clinical trials investigating the inhibition of acquisition of drug resistance.

Leuk Lymphoma, 1993 Feb, 9(3), 255 - 64
A comparative analysis of the sensitivity of multidrug resistant (MDR) and non-MDR cells to different anthracycline derivatives; Michieli M et al.; Because of the fact that tumor cell sensitivity to cytotoxic agents may play a major role in cancer treatment, and several anthracyclines are widely used for first-line treatment of leukemia, lymphoma and other tumors, and since the overexpression of the mdr-1 gene-coded 170 Kd glycoprotein (P170) decreases cell sensitivity to anthracyclines, we investigated the relationship between P170 overexpression and the cytotoxicity of two classic anthracyclines (Daunorubicin or DNR and Doxorubicin or DX) and two lipophilic anthracycline derivatives (Idarubicin or IDA and Iododoxorubicin or IDX) . For these purposes, we used multidrug resistant (MDR) and non-MDR tumor and leukemia cell lines and the MTT-microcultured tetrazolium colorimetric assay . We showed that mdr-1 gene overexpression was strongly associated with the development of a high level of resistance to DNR and DX, but not to the derivatives IDA and IDX . These data suggest that more lipophilic anthracycline derivatives may also be active in MDR cell systems.

Semin Cell Biol, 1993 Feb, 4(1), 63 - 76
P-glycoproteins: mediators of multidrug resistance; Germann UA et al.; Multidrug resistance represents a major obstacle to successful chemotherapy of metastatic disease . Elevated levels in cancer cells of the product of the multidrug resistance gene, P-glycoprotein or the multidrug transporter, have been associated with the development of simultaneous resistance to a great variety of amphiphilic cytotoxic drugs . P-glycoprotein is an integral plasma membrane protein which contains 12 putative transmembrane regions and two ATP binding sites . It confers multidrug resistance by functioning as an energy-dependent drug efflux pump . Here we describe recent studies on the biosynthesis, structure, function, and mechanism of action of P-glycoprotein which have provided insights into the complexity of this multifunctional transport system and revealed an additional chloride channel activity . The physiological role of P-glycoprotein, however, still remains to be elucidated.

Br J Cancer, 1993 Feb, 67(2), 311 - 20
Differential cytotoxicity of 19 anticancer agents in wild type and etoposide resistant small cell lung cancer cell lines; Jensen PB et al.; A panel of six 'wild type' and three VP-16 resistant small cell lung cancer (SCLC) cell lines is used to evaluate to what extent in vitro sensitivity testing using a clonogenic assay can contribute to combine cytotoxic drugs to regimens with improved efficacy against SCLC . The resistant lines include (a) H69/DAU4, which is classical multidrug resistant (MDR) with a P-glycoprotein efflux pump (b) NYH/VM, which exhibits an altered topoisomerase II (topo II) activity and (c) H69/VP, which is cross-resistant to vincristine, exhibits a reduced drug accumulation as H69/DAU4 but is without P-glycoprotein . 19 anticancer agents were compared in the panel . The MDR lines demonstrated, as expected, cross-resistance to all topo II drugs, but also different patterns of collateral sensitivity to BCNU, cisplatin, ara-C, hydroxyurea, and to the topo I inhibitor camptothecin . The complete panel of nine cell lines clearly demonstrated diverse sensitivity patterns to drugs with different modes of action . Correlation analysis showed high correlation coefficients (CC) among drug analogues (e.g . VP-16/VM-26 0.99, vincristine/vindesine 0.89), and between drugs with similar mechanisms of action (e.g . BCNU/Cisplatin 0.89, VP-16/Doxorubicin 0.92), whereas different drug classes demonstrated low or even negative CC (e.g . BCNU/VP-16 -0.21) . When the CC of the 19 drug patterns to VP-16 were plotted against the CC to BCNU, clustering was observed between drugs acting on microtubules, on topo II, alkylating agents, and antimetabolites . In this plot, camptothecin and ara-C patterns were promising by virtue of their lack of cross-resistance to alkylating agents and topo II drugs . Thus, the differential cytotoxicity patterns on this panel of cells can (1) give information about drug mechanism of action, (2) enable the selection and combination of non-cross-resistant drugs, and (3) show where new drugs 'fit in' among established agents.

Br J Cancer, 1993 Feb, 67(2), 284 - 9
Cortisol is transported by the multidrug resistance gene product P-glycoprotein; van Kalken CK et al.; The physiology of the multidrug transporter P-glycoprotein (Pgp) is still poorly understood . We now show evidence that cell lines with a high expression of Pgp display a reduced accumulation of cortisol and an ATP-dependent outward transport of the hormone . Cortisol efflux from Pgp negative cells does not have such an active component . Further we show that the steroid hormones cortisol, testosterone, and progesterone cause an immediate, dose-dependent increase of daunorubicin accumulation in Pgp overexpressing cells . These effects are particularly apparent for the more lipophilic steroids . These results demonstrate that Pgp may function as a transporter for cortisol and suggest a physiological role of the protein in steroid handling by organs such as the adrenal.

Br J Cancer, 1993 Feb, 67(2), 226 - 31
Effect of anthracycline analogs on photolabelling of p-glycoprotein by {125I}iodomycin and {3H}azidopine: relation to lipophilicity and inhibition of daunorubicin transport in multidrug resistant cells; Friche E et al.; Eight anthracycline analogs that have been shown to modulate multidrug resistance (Friche et al., Biochem . Pharmacol., 39, 1721-1726; 1990) were tested for their inhibitory effect on the photolabelling of P-glycoprotein . We photoaffinity labelled P-glycoprotein in daunorubicin (DNR) resistant Ehrlich ascites tumour cells (EHR2/DNR +) with a {125I}iodinated Bolton-Hunter derivative of daunorubicin ({125I}iodomycin) and with {3H}azidopine . The photolabelling of P-glycoprotein by {125I}iodomycin was inhibited more than 50% by 10 microM (1000-fold molar excess) of DNR (52%), N,N-dibenzyl-DNR (52%), and N-benzyladriamycin-14-valerate (AD-198) (85%) . Vincristine at 10 microM inhibited {125I}iodomycin labelling of P-glycoprotein by 95% . Thus vincristine was more potent than any of the eight anthracyclines tested, despite its relatively low lipophilicity . Increasing the concentration of DNR, AD-198 and N,N-dibenzyl-DNR to 40 microM resulted in 90, 99.5 and 99.5% inhibition of P-glycoprotein labelling by {125I}iodomycin, respectively . In comparison with the other anthracycline analogs, N,N-dibenzyl-DNR and Ad-198 were also found to exert the greatest inhibition of {3H}azidopine labelling of P-glycoprotein (about 90% at 100-fold molar excess) . The solvents Cremophor EL and Tween 80 (30 micrograms ml-1; 0.003% v/v), which are modulators of multidrug resistance in EHR2/DNR + cells, also inhibited {125I}iodomycin labelling > 90% . We showed earlier that there is a correlation between the lipid solubility within the anthracycline group of MDR-associated drugs and their ability to enhance DNR accumulation in EHR2/DNR + cells but a corresponding correlation to lipophilicity when it comes to the inhibitory effect on the specific photolabelling of Pgp ligand binding sites could not be demonstrated . Neither could a correlation between the modulating effect of the analogs on DNR accumulation and inhibition on the labelling of Pgp be demonstrated . With increasing lipophilicity of the analogs it seems that the chemical structure plays a lesser role, and the degree of lipophilicity becomes a more important feature.

Leuk Res, 1993 Feb, 17(2), 149 - 56
Expression of P-glycoprotein and anionic glutathione S-transferase genes in non-Hodgkin's lymphoma; Rodriguez C et al.; Non-Hodgkin's lymphomas (NHL) are usually sensitive to chemotherapy . A certain percentage of patients are primarily or subsequently resistant to chemotherapeutic agents . Several biological mechanisms are implicated in this phenomenon, including multidrug resistance (mdr1) and glutathione S-transferase (GST pi) . We investigated these two systems, using dot blot analysis, in 41 patients who presented NHL with advanced disease . There were 15 patients with low grade, 22 with intermediate grade, and 4 patients with high grade using the Working Formulation for Clinical Usage . Twenty-five patients had not been previously treated and 16 had been treated, including 13 with refractory disease . Eleven out of 25 (44%) patients overexpressed mdr1 mRNA at diagnosis as compared to 6/16 (38%) in relapse, corresponding to 6/13 (46%) refractory patients . Nine out of 25 (36%) patients overexpressed GST pi mRNA at the time of diagnosis, and 6/16 (50%) in relapse . These data indicate that overexpression of these two messengers is not acquired after treatment in NHL . Furthermore, there is no relationship between the stage or histological grade and the overexpression of these two markers . This study shows that mdr1 and GST pi gene expressions are independent of one another . With regard to the clinical response, our results also demonstrated a higher level of treatment failure in the group co-expressing the two transcripts, 6/8 (75%) patients died in progressive disease as compared to 9/15 (60%) patients without overexpression, and 2/8 (25%) vs 6/15 (40%) responded to treatment . On the other hand, overexpression of only one of the two mRNAs did not allow us to observe a difference in the clinical response . Since it seems that coexpression of the mechanisms of resistance present a better clinical impact, it would be of interest to analyse simultaneously different mechanisms involved in the resistance phenomenon in NHL.

Cancer, 1993 Feb 1, 71(3 Suppl), 1098 - 109
Cytotoxic chemotherapy for advanced hormone-resistant prostate cancer; Yagoda A et al.; BACKGROUND . Advanced adenocarcinoma of the prostate after hormonal manipulation has been noted to be a relatively chemotherapeutic nonresponsive tumor . Earlier reviews have reported objective responses, that is, complete and partial remissions in 6.5% of 3184 patients, and the current review examines the efficacy of new agents . METHODS . The current review consists of 26 new drug trials culled from papers and abstracts published between 1987-1991 . RESULTS . Results of these 26 drug trials found a similar trend, 8.7% (95% confidence interval, 6.4-9.0%), indicating that hormone-resistant adenocarcinoma of the prostate still fails to respond to most cytotoxic agents . The most interesting of the new therapeutic agents is the combination of vinblastine plus estramustine . Only six agents had an objective response rate greater than 10%, such as vinblastine by continuous infusion, trimetrexate, mitoguazone, and estramustine . The recent introduction of radioactive-labeled monoclonal antibodies is intriguing and these will undoubtably be used as carriers for radiotherapeutic and cytotoxic compounds . CONCLUSIONS . Although multidrug resistance may explain the marginal efficacy of cytotoxic drugs, methods to overcome such resistance and, more importantly, new classes of agents must be developed . In addition, reliable disease markers must be found for osseous and visceral metastases to avoid the prevailing confusion in evaluating more precisely the destruction of prostate cancer cells.

MMWR Morb Mortal Wkly Rep, 1993 Jan 29, 42(3), 48 - 51
Probable transmission of multidrug-resistant tuberculosis in a correctional facility--California; Terfenadine (Seldane): a new drug for restoring sensitivity to multidrug resistant cancer cells; Department of Medicine, Yale University School of Medicine and Comprehensive Cancer Center, New Haven, CT 06510In our efforts to identify clinically effective drugs for reversing multidrug resistance (MDR) mediated by P-glycoprotein, we tested terfenadine for anti-MDR activity because it appeared to sensitize a patient to doxorubicin and because it met structural requirements defined for this activity . Terfenadine sensitized MCF-7/ADR human breast cancer cells and L1210/VMDRC.06 murine leukemia cells to doxorubicin . At concentrations < or = 10 microM, terfenadine decreased the IC50 to doxorubicin by up to 25-fold against MCF-7/ADR cells and completely restored sensitivity to L1210/VMDRC.06 cells . The drug had no effect on the sensitive, parental cell lines and enhanced activity of other drugs affected by the MDR phenotype . Terfenadine was as potent as trans-flupenthixol, one of the most active modulators of MDR . The mechanism of action of terfenadine appeared to be due to inhibition of the function of P-glycoprotein since it augmented the accumulation of doxorubicin and inhibited the efflux of rhodamine 123 from MDR lines but had no effect on drug accumulation or efflux in sensitive cells . Terfenadine displaced azidopine from P-glycoprotein, but at concentrations higher than expected based on its overall potency . Since terfenadine is clinically available, has numerous structural derivatives available for study, and has a relatively low toxicity profile, this drug and drugs of its class should be evaluated for future clinical trials.

J Biol Chem, 1993 Jan 25, 268(3), 1792 - 8
Topology of P-glycoprotein as determined by epitope mapping of MRK-16 monoclonal antibody; Georges E et al.; There is growing evidence for the direct role of P-glycoprotein mediating multidrug resistance in tumor cells . P-glycoprotein is thought to function as an energy-dependent drug efflux pump . The monoclonal antibody MRK-16 binds to an external domain of P-glycoprotein and partially inhibits drug efflux in multidrug-resistant cells . As an approach toward elucidating the mechanism by which MRK-16 affects drug transport, we undertook the definition of the precise binding site of this antibody . In this study we have mapped the epitope of MRK-16 monoclonal antibody to a resolution of a single amino acid using a series of overlapping synthetic peptides . We demonstrate that MRK-16 recognizes only the class I isoform (MDR1) of human P-glycoprotein and that its epitope encompasses at least two (first and fourth) of the six predicted extracellular peptide loops . These results suggest that the epitope of MRK-16 is discontinuous and that the sequences involved which are separated by about 625 amino acids in the linear sequence must be spatially situated in close proximity in the native protein . Based on these results, we present a model for transmembrane alpha-helical packing of P-glycoprotein in the lipid bilayer . This may have implications for understanding the function of P-glycoprotein in drug transport.

Int J Cancer, 1993 Jan 21, 53(2), 323 - 7
Multidrug-resistant human KB carcinoma cells are highly resistant to the protein phosphatase inhibitors okadaic acid and calyculin A . Analysis of potential mechanisms involved in toxin resistance; Chambers TC et al.; In this study we show that multidrug-resistant (MDR) human KB-V1 cells are highly resistant to the cytotoxicity of okadaic acid and calyculin A, 2 toxins from marine sponges that are potent inhibitors of type-1 and type-2A protein phosphatases (PP1 and PP2A) . Cytotoxicity and colony-forming assays indicated that, relative to parental drug-sensitive KB-3 cells, KB-V1 cells are 35-fold more resistant to okadaic acid and 70-fold more resistant to calyculin A . Cytotoxicity of the toxins was associated with mitotic arrest characterized by chromosome scattering and over-condensation, with KB-3 cells being more sensitive than KB-V1 cells and calyculin A being more potent than okadaic acid . The resistance of KB-V1 cells to both okadaic acid and calyculin A was completely reversed by verapamil, suggesting that the toxins may be transported by P-glycoprotein (P-gp) . To further assess the possibility of an interaction with P-gp, the toxins were employed as potential modulators of the photoaffinity labeling of P-gp by {3H}azidopine . Relative to vinblastine, which effectively competed with {3H}azidopine for P-gp photolabeling, calyculin A was 100-fold less potent and okadaic acid did not inhibit photolabeling at concentrations up to 50 microM . To determine whether the resistance mechanism involved differences in toxin-sensitive phosphatase activity, the activity was assayed in extracts from both cell lines and found to be slightly higher (1.6-fold) in KB-V1 than in KB-3 cells . Our results demonstrate a novel, marked resistance of MDR KB-V1 cells to these phosphatase inhibitors and suggest that a major mechanism of resistance may involve toxin transport by P-gp at sites apparently different from those which bind azidopine.

Cancer Lett, 1993 Jan 15, 68(1), 7 - 14
Staurosporine reduces P-glycoprotein expression and modulates multidrug resistance; Sampson KE et al.; We have investigated the effect of staurosporine and other kinase inhibitors on the mRNA and protein levels of the P-glycoprotein (P-gp) in multidrug resistant (MDR) cells . Treatment of human MDR KB-V1 cells with staurosporine for 24 h caused up to a 50% decrease in the amount of P-gp mRNA and protein present . Co-treatment of KB-V1 cells with verapamil, a known reversal agent, plus staurosporine, H-9, or K252a resulted in an enhanced sensitization of cells to vinblastine than with verapamil alone . These findings support a role for protein kinases in the control of multidrug resistance through effects on P-gp levels.

Blood, 1993 Jan 15, 81(2), 490 - 5
P-glycoprotein expression in human plasma cell myeloma: correlation with prior chemotherapy; Grogan TM et al.; Multidrug-resistant (MDR) myeloma patients failing chemotherapy may express P-glycoprotein (PGP), which serves as an efflux pump protecting the neoplastic cells . Unknown is whether PGP expression might relate to prior cytotoxic drug exposure . To address this question, we studied 106 consecutive bone marrow samples from 104 myeloma patients with samples studied either before or after therapy and at the time of relapse . We performed an established immunocytochemical assay of PGP using an MDR-1-specific monoclonal antibody and correlated PGP status with prior chemotherapy dosage . Myeloma patients with no prior therapy had a low incidence of PGP expression (6%, 3/47), whereas those receiving chemotherapy had a significantly higher incidence (43%, 21/49) (P < .0001) . A substantially higher incidence of PGP expression (50%, 83%, respectively) occurred when the total vincristine dose exceeded 20 mg and when doxorubicin exceeded 340 mg . In the 11 patients who received both high vincristine and doxorubicin dosages (> 20 mg, > 340 mg total dose) there was 100% incidence of PGP expression in the tumor cells . These data provided the basis for a predictive mathematical model from which dose-related PGP expression normograms were generated . Time with myeloma for PGP-negative patients (mean 33 months) had overlapping confidence limits with PGP-positive patients (mean 42 months), suggesting that disease duration was not a significant variable . PGP expression did not correlate with other clinical factors or immunophenotypic factors . Our findings indicate a strong correlation between PGP expression in myeloma and past chemotherapy in myeloma, in particular, related to prior exposure to the natural product agents vincristine and doxorubicin . Additionally, the proportion of PGP-positive plasma cells was significantly higher in the doxorubicin-treated patients than the nondoxorubicin-treated patients (87.7% v 65.17%; P = .013) . Combined high vincristine and doxorubicin total dosage appear highly predictive of PGP expression.

Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 79 - 85
Resistance to tetracycline, a hydrophilic antibiotic, is mediated by P-glycoprotein in human multidrug-resistant cells; Kavallaris M et al.; Two multidrug-resistant human leukemic CCRF-CEM sublines (CEM/VCR R and CEM/VLB100) were significantly more resistant to tetracycline, a hydrophilic antibiotic, than parental cells (P < 0.001) . Verapamil and cyclosporin A completely reversed tetracycline resistance in CEM/VCR R cells, which also accumulated and retained significantly less {3H}tetracycline than CCRF-CEM cells . Like verapamil, addition of tetracycline to CEM/VCR R cells which had achieved steady-state vincristine levels resulted in augmented vincristine accumulation . {3H}Azidopine photoaffinity labelling of CEM/VCR R membrane proteins was inhibited by tetracycline in a dose-dependent manner . Although drugs associated with the multidrug-resistance phenotype are typically hydrophobic compounds, these data suggest that resistance to tetracycline, despite its hydrophilic nature, is mediated by P-glycoprotein in these cell lines.

Cancer Res, 1993 Jan 15, 53(2), 310 - 7
Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein; Arceci RJ et al.; A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents . P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these cytotoxic agents from the resistant cell . In order to study the phenomenon of multidrug resistance in both normal and neoplastic cells, we have generated a mouse monoclonal antibody directed to an external epitope of the human P-glycoprotein . This monoclonal antibody, 4E3, is an IgG2a class antibody which specifically recognizes the human mdr1 P-glycoprotein but not the mdr3 gene product . The 4E3 monoclonal antibody immunoprecipitates both the glycosylated and nonglycosylated forms of P-glycoprotein under mild denaturation conditions . In addition, 4E3 can detect P-glycoprotein in immunocytochemical analysis of fixed tissue-cultured cells and in analysis of frozen sections of human tissue . Binding of the monoclonal antibody to multidrug-resistant cells does not significantly affect the intracellular accumulation or potentiate the cytotoxicity of daunomycin in multidrug-resistant cells . However, at high concentrations of antibody, 4E3 produces a mild potentiation of vinblastine and actinomycin cytotoxicity in multidrug-resistant cells . This monoclonal antibody will be useful both for analyzing P-glycoprotein expression in normal and neoplastic cells and for isolating live cells expressing the P-glycoprotein without significantly affecting the efflux functions of the transporter.

J Biol Chem, 1993 Jan 5, 268(1), 658 - 64
Selective regulation of expression of protein kinase C (PKC) isoenzymes in multidrug-resistant MCF-7 cells . Functional significance of enhanced expression of PKC alpha; Blobe GC et al.; The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells . Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype . In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined . The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography . This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis . The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon . The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate . Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate . These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.

Eur J Cancer, 1993, 29A(8), 1152 - 7
Selective reversal of vinblastine resistance in multidrug-resistant cell lines by tamoxifen, toremifene and their metabolites; Kirk J et al.; In this study we describe the effects of tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites on the toxicity of a range of drugs to human breast and lung cancer and to Chinese hamster ovary cell lines, determined using a tetrazolium-based semi-automated colorimetric assay . Vinblastine resistance was completely abolished in an mdr1-transfected lung cancer cell line (S1/1.1), indicating that P-glycoprotein-mediated multidrug resistance can be fully reversed by anti-oestrogens . A substantial (14- to 39-fold) enhancement of vinblastine toxicity to highly multidrug-resistant (MCF-7Adr) cells expressing P-glycoprotein was also observed in the presence of tamoxifen, toremifene and their metabolites, while m-amsacrine, cisplatin and melphalan toxicity was unaffected.

In Vivo, 1993 Jan-Feb, 7(1), 73 - 9
In vivo characterization of immunogenicity of a mitoxantrone-resistant murine P388 leukemia; Fichtner I et al.; The Mitoxantrone-resistant murine leukemia P388/Mitox, expressing the multidrug-resistant phenotype, has a higher immunogenicity than the parent sensitive P388 . This could be shown in vivo by immunization with lethally-irradiated tumor cells . If the P388/Mitox was used for immunization before subsequent challenge with viable tumor cells of the same line, this resulted in a partial rejection of tumors and production of a substantial number of tumor-free survivors . For an effective immunization at least two primings s.c., i.v . or i.p . with at least 10(6) irradiated cells were necessary . This protected the recipient mice from a challenge of up to 10(8) viable cells over a period of at least 75 days . Treatment of BDF1 mice with the T-cell suppressor Cyclosporin A prevents immunization . In nude mice no immunization effect could be obtained . It was possible to transfer immunity adoptively with spleen cells from mice, which were treated with irradiated tumor cells of the P388/Mitox line . Treatment of tumor-bearing mice with IL-2 resulted in a prolongation of survival both when it was administered prophylactically before transplantation of P388/Mitox and at an advanced stage (day 7-11) . Also the alkyl-phosphocholine hexadecylphosphocholine was significantly effective in the resistant but not in the parent P388 leukemia . The data presented demonstrate that by development of a multidrug-resistance, concomitantly a xenogenization must have taken place which leads to a recognition of cells by immune mechanisms . In our model, T-lymphocytes and NK-/LAK-cells probably play a role in the immunologically conditioned rejection of tumor cells of the P388/Mitox leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Chemother Pharmacol, 1993, 32(3), 173 - 8
Antitumor activity and cytotoxicity of a new ankinomycin derivative, 3'-,11-dibutyryl ankinomycin; Ishii S et al.; Ankinomycin is a new antitumor antibiotic found in the culture broth of Streptomyces sp . SF2587 . Ankinomycin showed marked cytotoxicity and antitumor activity against some murine leukemias, but the activity against murine solid tumors was rather weak because of its strong acute toxicity . We synthesized ankinomycin acyl derivatives and examined their antitumor activity . Among the derivatives, 3',11-dibutyryl ankinomycin (AN1006) exhibited the highest antitumor activity . The antitumor activity of AN1006 was dependent on the administration schedule, and on the most effective schedule, AN1006 showed activity comparable with that of Adriamycin (ADM) against murine solid tumors and leukemias . AN1006 showed a cytotoxic spectrum different from that of ADM, exhibiting cytotoxicity stronger than that of ADM against colon carcinoma, stomach carcinoma, and some leukemia cell lines . According to these in vitro effects, AN1006 showed antitumor activity superior to and equal to that of ADM against human colon xenografts and stomach carcinoma xenografts in athymic nude mice, respectively . AN1006 was effective against multidrug-resistant tumors in vitro and in vivo . AN1006 is an interesting candidate for further evaluation.

Eur J Cancer, 1993, 29A(7), 1000 - 2
Titanocendichloride activity in cisplatin and doxorubicin-resistant human ovarian carcinoma cell lines; Harstrick A et al.; The activity of a new organometallic compound, titanocendichloride, was evaluated in doxorubicin- and cisplatin-resistant human ovarian carcinoma cell lines in vitro . Titanocendichloride showed no cross resistance to doxorubicin in two multidrug resistant sublines of A2780 . Furthermore, the cell line A2780 CP3, which is about 20-fold resistant to cisplatin was only 2.5-fold resistant to titanocendichloride, indicating a lack of cross resistance between the two metal compounds . These results were confirmed in vivo where titanocendichloride showed a much stronger inhibitory effect in cisplatin-resistant human ovarian carcinoma xenografts than cisplatin.

Int J Hematol, 1993 Jan, 57(1), 31 - 7
MDR1 (multidrug resistance) gene expression in adult acute leukemia: correlations with blast phenotype; Miyachi H et al.; Resistance to multiple chemotherapeutic agents is related to the production of P-glycoprotein, a transmembrane drug efflux pump that is encoded by the multidrug resistance gene (MDR1) . To detect low-level or heterogenous expression of the MDR1 gene in acute leukemia, we have developed sensitive, specific and semi-quantitative protocols for measuring levels of MDR1 mRNA, based on the polymerase chain reaction . Using this assay, we screened blasts from 20 patients with untreated adult acute leukemia for evidence of MDR1 gene expression . The level of MDR1 mRNA was normalized to beta 2-microglobulin mRNA and was defined by reference to the highly resistant trimetrexate-selected leukemia cells MOLT-3/TMQ200 (1.80) . MDR1 mRNA was observed in 14 out of 20 patients . Higher MDR1 mRNAs were observed in three patients with phenotypes of undifferentiated or minimally differentiated nonlymphocytic acute leukemia, as compared with other types of acute leukemia (0.98 vs . 0.25) . In contrast, lower MDR1 mRNAs were found in five patients with acute promyelocytic leukemia, as compared with other types of acute leukemia (0.08 vs . 0.45) . These findings suggest that MDR1 gene expression is correlated with the leucocyte differentiation stage of leukemia . MDR1 gene expression may, in part, explain the responsiveness to chemotherapy in these distinct subtypes of acute leukemia.

Anticancer Res, 1993 Jan-Feb, 13(1), 249 - 55
An analysis of vincristine-resistance in BHK cells pretreated with 1-beta-D-arabinofuranosylcytosine; Chen Y et al.; As with SV40-transformed BHK cells (1), pretreatment of BHK cells with 1-beta-D-arabinofuranosylcytosine (araC) very markedly enhanced the resistance frequency to N-phosphonoacetyl-L-aspartate (PALA) but only modestly increased the resistance frequency to vincristine (VCR) . AraC pretreatment of cells that already had moderate VCR-resistance again only modestly enhanced the resistance frequency at a higher VCR concentration . By contrast, delaying the addition of VCR to BHK cells by 7-24 hours after pretreatment with araC increased the enhancement of VCR-resistance frequency to a level comparable to the enhancement of PALA-resistance frequency . VCR-resistant clones isolated without or after araC pretreatment were analysed for amplification of the multidrug resistance (mdr) gene . One of four clones isolated by single-step selection with no araC pretreatment and four of six clones isolated after araC pretreatment showed amplification of the mdr gene . All VCR-resistant clones tested were cross-resistant to actinomycin D and VCR-resistance was inhibited by treatment with verapamil.

Int Rev Neurobiol, 1993, 35, 279 - 390
Acetylcholine transport, storage, and release; Parsons SM et al.; ACh is released from cholinergic nerve terminals under both resting and stimulated conditions . Stimulated release is mediated by exocytosis of synaptic vesicle contents . The structure and function of cholinergic vesicles are becoming known . The concentration of ACh in vesicles is about 100-fold greater than the concentration in the cytoplasm . The AChT exhibits the lowest binding specificity among known ACh-binding proteins . It is driven by efflux of protons pumped into the vesicle by the V-type ATPase . A potent pharmacology of the AChT based on the allosteric VR has been developed . It has promise for clinical applications that include in vivo evaluation of the density of cholinergic innervation in organs based on PET and SPECT . The microscopic kinetics model that has been developed and the very low transport specificity of the vesicular AChT-VR suggest that the transporter has a channel-like or multidrug resistance protein-like structure . The AChT-VR has been shown to be tightly associated with proteoglycan, which is an unexpected macromolecular relationship . Vesamicol and its analogs block evoked release of ACh from cholinergic nerve terminals after a lag period that depends on the rate of release . Recycling quanta of ACh that are sensitive to vesamicol have been identified electrophysiologically, and they constitute a functional correlate of the biochemically identified VP2 synaptic vesicles . The concept of transmitter mobilization, including the observation that the most recently synthesized ACh is the first to be released, has been greatly clarified because of the availability of vesamicol . Differences among different cholinergic nerve terminal types in the sensitivity to vesamicol, the relative amounts of readily and less releasable ACh, and other aspects of the intracellular metabolism of ACh probably are more apparent than real . They easily could arise from differences in the relative rates of competing or sequential steps in the complicated intraterminal metabolism of ACh rather than from fundamental differences among the terminals . Nonquantal release of ACh from motor nerve terminals arises at least in part from the movement of cytoplasmic ACh through the AChT located in the cytoplasmic membrane, and it is blocked by vesamicol . Possibly, the proteoglycan component of the AChT-VR produces long-term residence of the macromolecular complex in the cytoplasmic membrane through interaction with the synaptic matrix . The preponderance of evidence suggests that a significant fraction of what previously, heretofore, had been considered to be nonquantal release from the motor neuron actually is quantal release from the neuron at sites not detected electrophysiologically.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer Chemother Pharmacol, 1993, 32(1), 25 - 30
Reversal of multidrug resistance in Friend leukemia cells by dexniguldipine-HCl; Reymann A et al.; Dexniguldipine-HCl (DNIG)--a prospective clinical modulator of p170-glycoprotein (pgp170)-mediated multidrug resistance (MDR)--was evaluated in a drug-accumulation assay in MDR murine leukemia cell strain F4-6RADR expressing pgp170 . The compound elevated low accumulation of either doxorubicin (DOX), daunorubicin (DNR), or mitoxantrone (MITO) in resistant F4-6RADR cells to the very levels observed in drug-sensitive F4-6 precursor cells . In parallel with the increase in DNR content (F4-6RADR, solvent: 303 +/- 27 pmol/mg protein; DNIG (3.3 mumol/l): 1,067 +/- 174 pmol/mg protein; F4-6P, solvent: 948 +/- 110 pmol/mg protein; n = 8-9, SEM), the amount of DNR tightly bound to the acid precipitate pellet obtained from F4-6RADR (i.e., protein, DNA, RNA) increased 3.9-times to the levels observed in sensitive F4-6 cells . The main pyridine metabolite of DNIG displayed similar activity . Concentration-response analysis revealed that DNIG and R,S-verapamil (VER) induced 100% reversal of the DNR accumulation shortage associated with the MDR phenotype but DNIG was 8 times more potent than VER (50% inhibitory concentration (IC50), 0.73 vs 5.4 mumol/l) . In keeping with the accumulation assay, DNIG was about 10 times more potent than VER in sensitizing F4-6RADR cells to the cytostatic and cytotoxic effects of DNR in proliferation assays . In conclusion, DNIG is a potent in vitro modulator, improving (a) the accumulation of anthracycline-like cytostatics, (b) drug access to cellular binding sites, and (c) the cytostatic action of DNR in F4-6RADR leukemia cells of the MDR phenotype.

Eur J Cancer, 1993, 29A(4), 559 - 61
Effect of D,L-verapamil, verapamil enantiomers and verapamil metabolites on the binding of vincristine to alpha 1-acid glycoprotein; Woodcock BG et al.; Vincristine binding to solutions of alpha 1-acid glycoprotein (AGP, 2 mg/ml) and the effect of D,L-verapamil, verapamil enantiomers and the verapamil metabolites norverapamil and D617 were investigated in vitro using equilibrium dialysis and 3H-labelled vincristine . Vincristine binding to AGP (52.3 +/- 3.6%) was concentration independent over the range 0.002-2.0 micrograms/ml . The displacement of vincristine from AGP varied between 25.1 and 81.3% with D,L-verapamil and verapamil enantiomers added at concentrations in the range 5-50 micrograms/ml . In contrast, the displacement by D617 (5-100 micrograms/ml) was weaker and varied between 0 and 47% . The displacement at 20 micrograms/ml produced by D,L-verapamil, R-verapamil, S-verapamil and norverapamil was 53.1%, 56.8%, 58.9% and 53.9%, respectively, was more than double that for D617 (25%; P = 0.002) . It is concluded that vincristine, D,L-verapamil and verapamil isomers and metabolites interact at binding sites on AGP . These interactions may be clinically important in multidrug resistance, for example in cancer patients with elevated levels of AGP undergoing treatment with verapamil and vinca alkaloids.

Cancer Chemother Pharmacol, 1993, 31(5), 412 - 4
Targeting chemosensitizing doses of toremifene based on protein binding; Wurz GT et al.; Toremifene is currently being evaluated as a chemosensitizing agent in doxorubicin-resistant patients . Although concentrations of > 2 microM reverse resistance in vitro, target concentrations required to reverse multidrug resistance (MDR) in vivo may be highly influenced by variables such as protein binding in serum . We examined the effects of high serum concentrations on the cellular accumulation of toremifene in an MDR MDA-MB-A-1 human breast-cancer cell line . We then examined the cellular accumulation of doxorubicin at various toremifene concentrations in 5% - 100% serum . We also measured the concentrations of toremifene and its major metabolites in plasma specimens obtained from two patients receiving 360 mg/day for 5 days in a phase I study . Our results show that (1) high serum concentrations decrease toremifene accumulation, (2) toremifene concentrations of < or = 2.5 microM enhance doxorubicin accumulation, and (3) patients achieve plasma toremifene concentrations of 10-15 microM following doses of 360 mg/day x 5 days . Our findings suggest that in vivo toremifene concentrations well above those used to reverse resistance in vitro are required to overcome the effect of high serum-protein binding.

Cancer Chemother Pharmacol, 1993, 31(5), 369 - 75
Effects of verapamil on the pharmacokinetics and metabolism of epirubicin; Mross K et al.; Experimental data suggest that multidrug resistance in cancer may be overcome by using an increased dose of anticancer agent(s) in combination with a resistance-modifying agent (RMA) . We studied the pharmacokinetics and metabolism of both epirubicin (EPI) and verapamil (VPL) to explore the possible pharmacokinetic interactions between these two drugs . Ten patients with advanced breast cancer were given EPI (40 mg/m2 in a daily i.v . bolus for 3 consecutive days), and five of them also received VPL (4 x 120 mg/daily p.o . for 4 consecutive days) . The data indicated a significant interaction between these two drugs that affected their metabolism . The areas under the concentration-time curves (AUC) obtained for epirubicin glucuronide, epirubicinol glucuronide, and both of the 7-deoxy-aglycones were higher in the EPI + VPL group as compared with the EPI group . The AUC, terminal half-life, mean residence time, volume of distribution at steady state, and plasma clearance of EPI alone as compared with EPI + VPL did not differ significantly . These results suggest either an induction of enzymes necessary for drug metabolism or an increase in the liver blood flow, resulting in an enhanced generation of metabolites with time or in an inhibition of excretion processes . Comparisons of the AUC values obtained for EPI and its metabolites after the first, second, and third injections of EPI revealed a cumulative effect for the metabolites that was more pronounced in the EPI + VPL group, being significant (P < 0.05) for epirubicin glucuronide in both treatment groups and for epirubicinol glucuronide in the EPI + VPL group . Maximal concentrations of VPL and nor-VPL reached 705 +/- 473 and 308 +/- 122 ng/ml, respectively, with the steady-state concentrations being 265 +/- 42 ng/ml for VPL and 180 +/- 12 ng/ml for nor-VPL.

Cancer Chemother Pharmacol, 1993, 31(5), 343 - 9
Modulation of drug cytotoxicity in wild-type and multidrug-resistant tumor cells by stereoisomeric series of C-20'-vinblastine congeners that lack antimicrotubule activity; Borman LS et al.; Seven binary vinca alkaloid congeners were newly synthesized as the C14' or C16'(20') or C14'16'(20') stereoisomers of C20'-modified VBL . These congeners lacked detectable antimicrotubule activity in assays of polymerization of purified microtubule protein and of mitotic arrest induction . The compounds modulated the cytotoxicity of VBL, VCR, and DOX in sarcoma and colon-tumor cell lines . In wild-type cell lines, each congener elicited a concentration-dependent enhancement of cytotoxicity that was drug- and cell-type-selective . For example, C20'-deoxy C14'16'20'-epi VBL sensitized sarcoma S180 cells 19-fold to DOX and 11-fold to VCR but had no effect on VBL cytotoxicity . In the rat colon-cancer cell lines there was preferential enhancement of VCR cytotoxicity by most congeners . In two MDR cell strains of S180, the modulation potency of each congener was independent of specific drug or of resistance level . As a result, the amount of modulator (concentration) required for reversal was proportional to the drug-resistance level . Such properties were not displayed by the monomeric vinca alkaloid modulator vindoline . The potency of drug modulation in both wild-type and MDR cells strains was dependent on the stereoisomeric form of the congener and its C20'-substituents.

Cell Growth Differ, 1993 Jan, 4(1), 41 - 7
Regulation by bcl-2, c-myc, and p53 of susceptibility to induction of apoptosis by heat shock and cancer chemotherapy compounds in differentiation-competent and -defective myeloid leukemic cells; Lotem J et al.; Myeloid leukemias that differ in their competence for induction of differentiation were analyzed for expression of bcl-2 and c-myc and for their sensitivity to induction of apoptosis by heat shock and cancer chemotherapy compounds . The M1 leukemia expressed a high level of bcl-2 and showed a much lower susceptibility to induction of apoptosis by heat shock, Adriamycin, 1-beta-D-arabinofuranosylcytosine, methotrexate, and cycloheximide, compared to five other leukemias which expressed a low level of bcl-2 . There was no association between susceptibility to induction of apoptosis and competence for induction of differentiation . The difference in susceptibility to methotrexate, which is not regulated by the multidrug resistance (MDR) genes, and treatment with verapamil, which blocks MDR activity, have indicated that the higher resistance of the M1 leukemia to these agents was not due to MDR activity . The results indicate that the level of regulated bcl-2 expression in these myeloid leukemias was associated with cell susceptibility to induction of apoptosis by different apoptosis-inducing agents . Screening for expression of bcl-2 may thus be useful to characterize leukemias regarding susceptibility to induction of apoptosis by different agents . The level of regulated c-myc expressed in these leukemias was not associated with susceptibility to induction of apoptosis . Transfection with a deregulated mutant p53 into the M1 leukemia did not change susceptibility to apoptosis induction, but transfection with deregulated c-myc increased susceptibility to apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Growth Differ, 1993 Jan, 4(1), 31 - 40
Extracellular matrix regulation of multidrug resistance in primary monolayer cultures of adult rat hepatocytes; Schuetz JD et al.; Previous studies reported that, in the absence of drug exposure, multidrug resistance, including resistance to Adriamycin (ADR), could develop in primary rat hepatocyte cultures (B . Carr, Proc . Am . Assoc . Cancer Res., 29:1158, 1988) . However, the hepatocytes in that report were cultured on plastic without the benefit of an extracellular matrix (ECM) . Because the ECM regulates hepatic gene expression, we have critically evaluated in primary cultures of rat hepatocytes how the ECM affects hepatic ADR resistance, the level of the drug efflux transporter associated with MDR, P-glycoprotein (pgp), and transport of a prototypical pgp substrate, vincristine . Hepatocytes cultured on type I collagen (Vitrogen) had greater resistance to ADR toxicity accompanied by parallel increases in the level of pgp mRNA, decreased drug accumulation, and enhanced drug efflux when compared with the hepatocytes maintained on the basement membrane matrix Matrigel . The development of ADR resistance coincided with the time course of increased pgp mRNA but was not coincident with the time course of expression of either the placental isozyme of glutathione S-transferase or P-450 reductase, proteins associated with MDR in some resistance models . Southern blot analysis revealed neither gross changes in pgp gene structure or gene copy number to account for the increase in pgp RNA levels for hepatocytes cultured on Vitrogen . ECM also regulated xenobiotic-inducible expression of hepatic pgp, since chemotherapeutic agents, including vincristine and colchicine, induced pgp mRNA exclusively in hepatocytes cultured on Vitrogen . The critical matrix proteins in Matrigel responsible for regulation of pgp were determined by the selective addition of its components to the culture environment . The presentation of the individual matrix elements as a rigid substratum to the hepatocyte did not decrease pgp mRNA . In contrast, the presentation to the same hepatocytes of either laminin or type IV collagen in a nonrigid state (solubly in the medium) selectively decreased hepatocellular pgp mRNA . We conclude that primary rat hepatocytes develop ADR resistance with time in culture due to increased expression of pgp and that ECM proteins represent endogenous physiological modulators of both basal and chemotherapeutically inducible expression of hepatic P-glycoprotein.

Mol Pharmacol, 1993 Jan, 43(1), 51 - 6
Estradiol induction of rhodamine 123 efflux and the multidrug resistance pump in rat pituitary tumor cells; Jancis EM et al.; Rhodamine 123 is a fluorescent dye that localizes in mitochondria, is a substrate for the multidrug resistance pump, and is retained for long periods of time by carcinoma cells . 17 beta-Estradiol causes GH4C1 cells (rat pituitary tumor cells) to lose rhodamine 123 fluorescence faster than untreated cells . We found that estradiol induces accumulation of the mRNA for the multidrug resistance pump 3-5-fold, with maximum induction occurring within 1 day at 10(-9) M estradiol . Immunoblot analysis demonstrated that estradiol induces a protein of 150 kDa that reacts with an antibody to P-glycoprotein, the multidrug resistance pump . The reduced retention of rhodamine 123 caused by estradiol is prevented by verapamil and cyclosporin, inhibitors of the pump . A clone resistant to the effects of estradiol on rhodamine 123 has greatly reduced levels of mRNA for the pump . The effect of estradiol is more marked on rhodamine 123 retention than it is on that of rhodamine 110 or tetramethylrhodamine methyl ester . We conclude that estradiol enhances rhodamine 123 efflux by inducing the multidrug resistance gene . The specificity for rhodamine 123, compared with other analogs, may be caused by differences in accessibility to the pump.

Cancer Chemother Pharmacol, 1993, 31(4), 301 - 7
Decreased resistance to N,N-dimethylated anthracyclines in multidrug-resistant Friend erythroleukemia cells; Schaefer A et al.; Doxorubicin-resistant Friend erythroleukemia cells, line F4-6 ADM2R, were selected by exposure of wild-type F4-6 cells to doxorubicin concentrations of up to 1 microgram/ml . In these cells, increased expression of multidrug resistance (MDR) genes was demonstrated by Northern blot analysis . The growth-inhibitory effect of doxorubicin, daunorubicin, N,N-dimethyldoxorubicin, N,N-dimethyldaunorubicin, morpholinodoxorubicin, and pyrromycin was comparatively investigated in resistant and wild-type cells . The doxorubicin-resistant F4-6 cells showed approx . 200-fold resistance to doxorubicin and about 100-fold resistance to daunorubicin with respect to the drug-sensitive counterpart . A dramatic decrease in resistance was observed for the N,N-dimethylated derivatives of doxorubicin and daunorubicin as well as for the N,N-dimethylated natural anthracycline pyrromycin and for morpholinodoxorubicin . Uptake studies using {14C}-daunorubicin and {14C}-N,N-dimethyldaunorubicin in resistant F4-6 cells showed a decreased accumulation of daunorubicin but no significant reduction in N,N-dimethyldaunorubicin accumulation as compared with the wild-type cells . Treatment with verapamil led to increased intracellular levels of daunorubicin in resistant cells, whereas an excess of N,N-dimethyldaunorubicin did not have this effect . Thus, the decreased resistance of the doxorubicin-resistant F4-6 cells to the N-alkylated anthracyclines may at least in part be due to a reduced affinity of these compounds for the efflux pump . The results indicate that the dimethylation of the amino group of the anthracycline sugar moiety and its incorporation within a morpholinyl ring may overcome MDR by similar mechanisms.

Eur J Cancer, 1993, 29A(2), 245 - 7
In vitro effect of suramin on lung tumour cells; Morocz IA et al.; In the search for new therapeutic concepts in lung cancer chemotherapy, suramin, a potential anticancer drug which evades multidrug resistance, was tested in vitro on 25 lung-derived cell lines, either non-tumorigenic cells, or established cell lines from five different tumour types . Suramin treatment resulted in a time- and dose-dependent decrease in {3H}thymidine incorporation, except in one adenocarcinoma cell line where DNA synthesis was highly stimulated . {3H}Leucine incorporation was less affected, indicating that suramin acted cytostatically rather than cytotoxically . Our results show that suramin affected DNA synthesis of the different types of lung derived cells, including non-tumorigenic and tumour cell lines, to a similar extent.

Adv Cancer Res, 1993, 60, 157 - 80
Function and regulation of the human multidrug resistance gene; Chin KV et al.; The discovery of an energy-dependent pump system for natural product anticancer drugs has important implications for the biology of related energy-dependent transport systems as well as for the treatment of human cancer . To fully realize the therapeutic potential associated with manipulation of the multidrug transporter, it will be necessary to understand the mechanisms of action of the transporter and its mode of regulation . This review has summarized recent developments in these areas which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies.

J Urol, 1993 Jan, 149(1), 174 - 8
Pseudomonas exotoxin conjugated to monoclonal antibody MRK16 specifically kills multidrug resistant cells in cultured renal carcinomas and in MDR-transgenic mice; Mickisch GH et al.; Using renal carcinoma and prostate carcinoma cell lines, we investigated the concept of targeting and killing multidrug resistant cells in urogenital cancers . Renal carcinoma lines HTB44, 45, 46, and 47 expressed a relatively low, but detectable level of multidrug resistance (MDR)1 mRNA as indicated by Northern blot analysis, whereas prostate lines LNCaP and DU145 were found to be MDR1-negative . Anti-P-glycoprotein monoclonal antibody MRK16 was conjugated to Pseudomonas exotoxin (PE) by a stable thioether bond . Treatment with MRK16-PE resulted in a dose-dependent killing of multidrug resistant renal carcinoma cells, while non-MDR expressing prostate carcinoma cells were not affected . Addition of excess MRK16 blocked the effect of MRK16-PE . Furthermore, MOPC-PE, a non-MDR associated monoclonal antibody control conjugate, did not target and kill multidrug resistant renal carcinoma cells . Having established that MRK16-PE was active against and specific for multidrug resistant cells in culture, we also tested bioactivity in MDR-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers . Again, MRK16-PE killed multidrug resistant bone marrow cells with high efficiency in an intact animal, and killing was blocked by unconjugated MRK16.

Indian Pediatr, 1993 Jan, 30(1), 47 - 50
Clinical profile and outcome in enteric fever; Sharma A et al.; Sixty five blood culture positive cases of S . typhi were studied for clinical profile . A total of 64.6% were multidrug resistant and 35.4% were chloramphenicol sensitive . In patients with multidrug resistant S . typhi the age was higher (p < 0.01), and incidence of complications such as shock (35.7%), encephalopathy (42.9%), myocarditis (14.3%) and gastric hemorrhage (4.7%) were more frequent, compared to chloramphenicol sensitive group . Cases with multidrug resistant S . typhi (MDRST) were treated with oral ciprofloxacin; the period of defervescence of fever was significantly less (p < 0.05) compared to the chloramphenicol group . Our study suggests the use of ciprofloxacin in the treatment of MDRST without any side effects.

Cancer Detect Prev, 1993, 17(3), 425 - 32
Doxorubicin, vincristine, and actinomycin-D, but not teniposide, require long-lasting uninterrupted verapamil pressure to overcome drug resistance in multidrug-resistant cells; Toffoli G et al.; The cytotoxic efficacy of doxorubicin (DOX), vincristine (VCR), actinomycin-D (ACT-D), and teniposide (VM-26) toward the LoVo and SW948 multidrug-resistant (MDR) cell sublines was significantly enhanced by the concomitant presence of verapamil (VER) during pharmacological treatment (1-h exposure) without, however, achieving a complete reversion of the MDR phenotype . Long-lasting VER in the culture medium, at the end of the combined drug+VER treatment, conferred to DOX, VCR, and ACT-D cytotoxic efficacy roughly similar to that exerted on the drug-sensitive parent cell lines . On the contrary, no enhancement of VM-26 cytotoxic efficacy was derived from continuous VER treatment . Enhancement of DOX, VCR, and ACT-D cytotoxic efficacy requires that VER be present in an uninterrupted manner in the culture medium since brief interruptions (15 to 60 min) in the VER pressure completely counteract any effect . These findings offer new insight into the modalities of pharmacological treatment that MDR cells require to obtain a complete reversion of cellular resistance to the various drug families included in the MDR spectrum.

Breast Cancer Res Treat, 1993, 26(1), 23 - 39
Differential expression of steroid receptors, hsp27, and pS2 in a series of drug resistant human breast tumor cell lines derived following exposure to antitumor drugs or to fractionated X-irradiation; Whelan RD et al.; This study examined whether levels of estrogen receptor (ER), progesterone receptor (PR), and expression of estrogen regulated pS2 and/or heat shock protein (hsp) 27 were associated with drug resistance in a series of MCF-7 sublines expressing modest (i.e . 3- to 14-fold), yet clinically relevant, levels of resistance to vincristine (VCR) . These sublines were variously derived following pulsed exposures to VCR, to fractionated X-irradiation, or to alternating drug and X-ray treatments . This selection procedure more closely reflects the clinical treatment of breast tumors than the use of continuous drug exposures . The drug-selected sublines exhibited the classical multidrug resistance phenotype (MDR) characterized by cross-resistance to vinblastine (VLB), etoposide (VP-16), and Adriamycin (ADR), overexpression of P-glycoprotein (Pgp), impaired accumulation of {3H}-VCR and of Rhodamine-123 (Rh 123), and altered activities of certain drug detoxification enzymes . This classic MDR phenotype was associated with a lack of mitogenic response to estrogen or antiestrogen, related to loss of detectable ER and PR; consistent with these data, neither pS2 nor hsp27 expression was detectable . In contrast, X-ray-pretreated VCR-resistant cells (MCF/DXR-10) cells exhibited a distinctive resistance phenotype proving cross-resistant to VLB and VP-16 but not to ADR, and Pgp overexpression was not detectable . Furthermore, these VCR-resistant DXR-10 cells retained parental levels of ER and PR, exhibited sensitivity to estrogen and 4-hydroxytamoxifen, and expressed detectable levels of pS2 and hsp27 . Comparable characteristics to these MCF-7/DXR-10 cells were also identified in a similarly-derived X-ray-pretreated VCR-resistant subline of the ZR-75-1 human breast tumor cell line . These data therefore indicate that functional ER are frequently, but not invariably, modified in tumor cells which express resistance to multiple drugs.

Eur J Cancer, 1993, 29A(3), 408 - 15
Chemosensitisation and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein; Barrand MA et al.; The doxorubicin-selected multidrug resistant (MDR) human large cell lung cancer line COR-L23/R, lacks P-glycoprotein but shows a drug accumulation deficit . It does however overexpress a 190k membrane protein which shares an epitope with, but is otherwise distinct from, P-glycoprotein . The resistant cells show only a small sensitisation to vincristine and daunorubicin on treatment with cyclosporin A and its more potent analogue, PSC-833 despite an increase in drug accumulation . Verapamil, another effective resistance modifier in P-glycoprotein MDR cells, is slightly more effective . Fluorescent daunorubicin distributes in the cytoplasm and nucleus of sensitive parent COR-L23 cells but is confined to cytoplasmic perinuclear vesicles in resistant cells . Addition of cyclosporin A or PSC-833 slightly increases cytoplasmic fluorescence whereas verapamil also increases nuclear fluorescence . Resistance in this non-P-glycoprotein MDR line, COR-L23/R where these resistance modifiers have little effect may be associated with expression of the 190k protein.

Eur J Cancer, 1993, 29A(12), 1776 - 8
Increased chemosensitivity to doxorubicin of intrinsically multidrug-resistant human colon carcinoma cells by prolonged exposure to verapamil; Toffoli G et al.; Resistance modifying agents (RMA) such as verapamil (VER) have proved effective in reversing multidrug resistance (MDR) in many in vitro experimental models, but clinical results with RMA have been disappointing . To clarify this apparent discrepancy we have evaluated the cytotoxic effects of doxorubicin (DOX) plus VER in four human colon carcinoma (HCOC) cell lines (LoVo, DLD-1, SW948, SW1116) . These lines were selected on the basis of their levels of mdr1 mRNA being similar to those expressed by HCC obtained from non-drug-treated patients . In all cell lines the sensitising effect of VER on DOX cytotoxicity was schedule-dependent and maximal potentiation of DOX cytotoxicity was obtained by exposure to VER for a time > or = the cells' population doubling time.

Eur J Cancer, 1993, 29A(10), 1377 - 83
Reversal of multidrug resistance by a new lipophilic cationic molecule, S9788 . Comparison with 11 other MDR-modulating agents in a model of doxorubicin-resistant rat glioblastoma cells; Huet S et al.; We have compared the properties of the novel multidrug resistance modulator, S9788, to a panel of 11 well-known modulators in a model of rat glioblastoma cells resistant to doxorubicin and displaying a P-glycoprotein-mediated multidrug-resistance phenotype complemented by a mechanism of intracellular drug tolerance not yet identified (Br J Cancer 1992, 65, 538-544) . S9788, like most modulators, was able to completely restore drug accumulation in the resistant line to the level obtained in the sensitive cells . This was obtained with 10 mumol/l of modulator, which is slightly higher than required for cyclosporine A (3 mumol/l) verapamil and nicardipine (6 mumol/l), but lower than for amiodarone, trifluoperazine and dipyridamole (20 mumol/l), tamoxifen and diltiazem (40 mumol/l), quinine, quinidine and nifedipine (> 100 mumol/l) . Complete restoration of drug cytotoxicity was, however, obtained only with amiodarone, and a residual resistance factor of 4 could not be overcome by cyclosporine A or S9788, while other modulators gave residual resistance factors of 5-20 (trifluoperazine, tamoxifen, verapamil, quinine, nicardipine, dipyridamole) or even higher (diltiazem, quinidine, nifedipine) . When studying doxorubicin accumulation obtained for an exposure to the IC50 of this drug, it appeared that some modulators were able to decrease this "intracellular IC50" independently of their efficiency in resistance reversal (cyclosporine A, S9788, amiodarone, trifluoperazine, quinine, tamoxifen), thus reversing intracellular drug tolerance, whereas other modulators could not reduce this parameter (verapamil, nicardipine, dipyridamole, diltiazem, quinidine) . It is suggested that drugs of the first group could be able to segregate doxorubicin in subcellular compartments from which it could not reach its nuclear targets.

Eur J Cancer, 1993, 29A(5), 753 - 9
A study of the expression of four chemoresistance-related genes in human primary and metastatic brain tumours; Mousseau M et al.; We investigated four mechanisms of intrinsic chemoresistance in a series of 67 human brain tumours including 31 gliomas (one grade I ganglioglioma, nine grade II and 10 grade III astrocytomas, 11 glioblastomas), 13 cerebral metastases, one medulloblastoma, one malignant teratoma, three ependymomas and 18 meningiomas . We studied four genes by northern blotting: multidrug-resistance (MDR 1), glutathione-s transferase (GST pi), dihydrofolate reductase (DHFR), and topoisomerase II (Topo II) . The Topo II gene was absent in the normal adult brain (100%) and in 64% of the tumour samples tested . A second gene, GST pi, was found to be overexpressed in 38% of brain tumours . The two other chemoresistance-related genes were occasionally overexpressed in brain tumours (2% for MDR1, 9% for DHFR) . Our results provide evidence that chemoresistance is intrinsic to the brain tissue and seems likely to be a multifactorial process.

Cancer Chemother Pharmacol, 1993, 31(5), 401 - 6
In vitro assessment of N-{2-(dimethylamino)ethyl}acridine-4-carboxamide, a DNA-intercalating antitumour drug with reduced sensitivity to multidrug resistance; Finlay GJ et al.; The successful treatment of cancer requires the identification of new drugs with novel actions . N-{2-(Dimethylamino)ethyl}acridine-4-carboxamide dihydrochloride (DACA) is a topoisomerase II-targeted antitumour drug with curative activity against murine Lewis lung carcinoma . DACA was assessed for novel patterns of growth inhibition using normal and multidrug-resistant human cell lines . Cells were cultured in 96-well microtitre trays and tested against DACA and related topoisomerase-directed drugs, including amsacrine, etoposide and doxorubicin, and drug concentrations for 50% growth inhibition (IC50 or GI50 values) were determined . In a series of Jurkat leukaemia lines characterised as exhibiting atypical multidrug resistance, DACA was to a large extent capable of overcoming multidrug resistance exhibited towards the other topoisomerase-directed agents . DACA was also tested against the National Cancer Institute 60-tumour-specific cell-line panel (GI50 values ranging from 420 to 5,400 nM; mean, 2,100 nM) and against a series of primary cultures of surgically excised melanomas (IC50 values ranging from 60 to 1,600 nM; mean, 590 nM) . DELTA values (deviations of logarithmic IC50 or GI50 values from the mean) were calculated and compared by correlation analysis . The standard deviation of DELTA values was found to be lower for DACA than for the other topoisomerase II-directed drugs amsacrine, etoposide, doxorubicin and mitozantrone in both the cell lines and the primary cultures . These lower standard deviations appear to have resulted from the reduced susceptibility of DACA to both P-glycoprotein- and topoisomerase II-mediated multidrug-resistance mechanisms occurring naturally in cell lines and primary cultures.

Oncol Res, 1993, 5(2), 75 - 82
Cytoplasmic membrane cholesterol and doxorubicin cytotoxicity in drug-sensitive and multidrug-resistant human ovarian cancer cells; Mazzoni A et al.; The possible involvement of cholesterol (CHOL) in the cellular transformations leading to the acquisition of the multidrug-resistance (MDR) phenotype has been evaluated in human ovarian cancer cells . To this end, the A2780 cell line and the 52-fold doxorubicin (DX)-resistant counterpart A2780-DX3 were analyzed under two different growth conditions: standard culture medium (FCS medium), or medium deprived of CHOL (LPDS medium) . The following variables were investigated: free and esterified cytoplasmic membrane CHOL, cell growth, DX uptake and cytotoxicity, and low-density lipoprotein uptake and degradation (as indirect variables of CHOL homeostasis) . The impact of the calcium antagonist verapamil (VER) on these variables was assessed . The results obtained indicate that under standard growth conditions, A2780 and A2780-DX3 cells are different not only with respect to DX uptake and sensitivity, but also with respect to membrane CHOL content and the ratio of free-to-esterified CHOL . The deprivation of lipoproteins in the culture medium, apart from slowing cell growth, induced a decrease in the cytoplasmic membrane CHOL content (mainly of the esterified form) that was particularly evident in A2780 sensitive cells . In LPDS medium, a reduced DX uptake occurred in both cell lines, but to a greater extent in A2780 cells, in which DX cytotoxicity decreased to values comparable to that of A2780-DX3 resistant cells . Restoration of DX sensitivity was achieved with the addition of 10 microM VER.(ABSTRACT TRUNCATED AT 250 WORDS)

Trop Gastroenterol, 1993 Jan-Mar, 14(1), 21 - 7
The anatomy of an epidemic (the final report on an epidemic of multidrug resistant enteric fever in eastern India); Anand AC; Two hundred and forty two patients of enteric fever were treated at Command Hospital (EC) Calcutta, during the period 1989-90 . The mean age of the patients was 28.4 (range 2-60) years and 119 (49.2%) of them were males . Fever (100%), headache (55%), diarrhoea (25.2%), intestinal bleeding (2.9%) and icterus (3.7%) were the main presenting features . Blood cultures were positive in 216 (%) patients . A majority of the isolates were found to be resistant to all four commonly used drugs for enteric fever i.e . chloramphenicol, ampicillin, cotrimoxazole and furazolidone . The commonest phage type found in the resistant strains was 51 (biotype-1) . Two plasmids of 14 and 120 kd size were detected in the resistant strains but not in the sensitive strains . Clinical response to gentamicin was not satisfactory in spite of all strains showing in vitro sensitivity . Ciprofloxacin proved to be a safe and effective drug for the treatment of multidrug resistant enteric fever.

Cancer Chemother Pharmacol, 1993, 32(5), 396 - 8
Tamoxifen stimulates in vivo growth of drug-resistant estrogen receptor-negative breast cancer; Maenpaa J et al.; An estrogen receptor-negative, multidrug-resistant MDA-MB-A1 human breast cancer cell line was grown in culture with and without a noninhibitory concentration (0.5 microM) of tamoxifen for 122 days . Tamoxifen-treated and control cells were inoculated into opposite flanks of nine nude mice, where they produced measurable tumors in every case . Six of the animals were treated with tamoxifen at 500 micrograms/day for 22 days . Although no inhibitory nor stimulatory effect of tamoxifen was seen in vitro, tamoxifen had a clear tumor-growth-stimulating effect in mice . The most pronounced stimulatory effects were observed in the cells that had been cultured with tamoxifen . Within 3 weeks of the start of tamoxifen therapy, the cells grown in the presence of tamoxifen produced tumors with a mean size of 380 mm2, whereas the cells not pretreated with tamoxifen had tumors of 220 mm2 . In contrast, in mice not receiving tamoxifen, the sizes of the tumors were 190 and 140 mm2, respectively . These preliminary results suggest that prolonged in vitro tamoxifen exposure induces cellular changes that result in tumors that are stimulated to grow faster in mice following tamoxifen treatment.

Ann Oncol, 1993, 4 Suppl 4, 57 - 62
Advanced ovarian cancer . Drug resistance, supportive care and dose intensity; de Vries EG et al.; BACKGROUND: Both intrinsic and acquired drug resistance occur in ovarian cancer . Much work on in vivo or in vitro, obtained drug resistance has been done and this knowledge is presently being converted into clinical studies . MATERIALS AND METHODS: The review focuses on the detoxifying system, MDR (multidrug resistance), and supportive care in relation to dose intensity . RESULTS: In vitro models suggest that the amount of glutathione, glutathione S-transferase activity or metallothioneins could play a role in the outcome of chemotherapy treatment . The results of human tumour samples studies however do not support this idea . Expression of the cell membrane P-glycoprotein in tumour cells appears in vitro to be an important adverse prognostic factor concerning the effect of natural products . Again the results in human tumour samples vary . The supportive agent sodium thiosulphate protects against cisplatin induced nephrotoxicity, but the exact role of sulphur compounds in ameliorating the neurotoxicity is not yet established . The neuropeptide Org 2766 may be a possible neuroprotector . Hemopoietic growth factors such as GM-CSF, G-CSF and interleukin-3, protect the bone marrow to varying degrees . Autologous bone marrow transplantation induces high, but often short lasting response rates in chemotherapy resistant patients . CONCLUSIONS: More clinical studies on intervention of drug resistance and on high dose chemotherapy are needed to define the role of these strategies in ovarian cancer.

Microbios, 1993, 76(309), 251 - 61
Use of beta-lactam/beta-lactamase-inhibitor combinations as antimycobacterial agents; Prabhakaran K et al.; Mycobacterium tuberculosis and Mycobacterium leprae develop resistance against the drugs used to treat tuberculosis and leprosy, respectively . Now multidrug-resistant tuberculosis is spreading in many countries, especially with the emergence of AIDS . Multidrug treatment is being promoted at present to eradicate leprosy . Since M . leprae may also become multidrug-resistant, new approaches have to be adopted for controlling mycobacterial diseases . Mycobacteria usually synthesize beta-lactamase and are insensitive to beta-lactam antibiotics . M . tuberculosis contains a constitutive beta-lactamase; de-repression of beta-lactamase has been reported in M . leprae . Three different beta-lactam/beta-lactamase-inhibitor combinations (ampicillin/sulbactam, amoxicillin/clavulanate and piperacillin/tazobactam) were used to suppress the growth of several strains of mycobacteria (including M . tuberculosis H37Rv) in vitro . Ampicillin/sulbactam is a potent bactericidal agent against M . leprae multiplying in mouse foot pads . In the present work, ampicillin/sulbactam showed higher activity than the other drug combinations . The beta-lactam/beta-lactamase inhibitors are likely to be effective as rational therapeutic agents against mycobacterial infections.

J Cancer Res Clin Oncol, 1993, 120(1-2), 27 - 34
Effects of interferon gamma on the proliferation and modulation of cell-surface structures of human ovarian carcinoma cell lines; Mobus VJ et al.; Platinum-containing regimens are very effective in the primary treatment of ovarian cancer . However, upon subsequent treatment most tumors develop multidrug resistance . The clinical application of biological response modifiers like interferon gamma (IFN gamma) in advanced ovarian cancer is therefore of increasing interest . Permanent ovarian cancer cell lines are suitable for investigating the mode of action and the potential clinical effectiveness of such response modifiers . IFN gamma is known to modulate many cellular functions . In this study it was compared for its antiproliferative and antigen-modulatory activity on the expression of tumor-associated (CA-125, HMFG, CEA) and major histocompatibility complex (MHC) class I and II antigens as well as of the epidermal growth factor (EGF) receptor on 20 newly established human ovarian carcinoma cell lines . IFN gamma in concentrations of 10, 50 and 100 U/ml was used to study its antigen-modulatory effect, and at additional 1 U/ml and 1000 U/ml to assess its antiproliferative effect on the cells . The cells were incubated with IFN for 4 days . Two cell lines showed strong antiproliferative activity even at minimal doses (up to 50 U/ml) . Intermediate growth inhibition between 34% and 84% was observed in 15 cell lines with higher doses . Three lines were resistant to IFN gamma . Independent of the antiproliferative effect, IFN gamma enhanced the expression of MHC class I and MHC class II in nearly all cell lines . Upregulation was also observed for most of the tumor-associated antigens (TAA) and EGF receptor expression . A down-regulation was noticed but rarely . The fact that IFN gamma showed an antiproliferative activity on the majority of the cell lines is of clinical relevance . The in vitro modulation of cell-surface determinants by IFN gamma warrants special attention . The enhanced expression of TAA and MHC antigens can improve immunogenicity of the tumor cells and may explain the therapeutic effects observed under IFN therapy in ovarian cancer . By contrast, enhanced expression of the EGF receptor, often associated with poor patient survival rates, may be an undesirable side-effect of IFN therapy.

Cancer Chemother Pharmacol, 1993, 33(1), 10 - 6
Effects of the methoxymorpholino derivative of doxorubicin and its bioactivated form versus doxorubicin on human leukemia and lymphoma cell lines and normal bone marrow; Kuhl JS et al.; The methoxymorpholino derivative of doxorubicin (MMDX; FCE 23672) has recently entered clinical trials because of its broad spectrum of preclinical antitumor activity and non-cross-resistance in multidrug-resistant (MDR) tumor models . MMDX is activated in the liver to a > 10 times more potent metabolite that cross-links DNA . To assess the potential of this drug in hematologic malignancies, we studied the myelotoxicity in vitro and antitumor effect of MMDX as well as its bioactivated form (MMDX+) in a panel of 14 different human leukemia and lymphoma cell lines . The tumor specificity of MMDX in CEM and K562 cells was similar to that of doxorubicin (DOX), and that of MMDX+ was slightly superior . All of the 14 cell lines were found to be more sensitive to MMDX and MMDX+ than were granulocyte-macrophage progenitors . On a molar basis, MMDX was approximately 3-100 times more active than DOX, and MMDX+ was 10-1,000 times more potent than DOX . The cytotoxic effect of MMDX and MMDX+ in two P-glycoprotein-positive MDR sublines was greatly improved in comparison with that of DOX . Whereas the response to DOX in the different leukemia and lymphoma cell lines was highly heterogeneous, the response to MMDX and MMDX+ was rather homogeneous . The novel anthracycline MMDX and its bioactivated form MMDX+ are highly active against this panel of human leukemia and lymphoma cell lines and demonstrate potentially greater selectivity for tumor cells in vitro as compared with normal bone marrow precursors.

Oncol Res, 1993, 5(3), 119 - 26
31P and 13C NMR spectroscopic study of wild type and multidrug resistant Ehrlich ascites tumor cells; Rasmussen J et al.; Steady-state 31P NMR spectra of wild type EHR2 Ehrlich ascites tumor cells and multidrug resistant EHR2/DNR+ cells, immobilized in agarose threads, and continuously perfused with medium, showed temperature-dependent differences in the levels of intracellular phosphate metabolites . At 37 degrees C, the EHR2/DNR+ cells contained four times more phosphocreatine (PCr) than the EHR2 cells . At 20 degrees C, the EHR2 cells contained 80% more of phosphodiesters (PDE), the levels of PCr being equal . The quantitative metabolite level data are based on T1 relaxation times data and are normalized for the protein content of the cells . Perfusion of the cells with azide, an inhibitor of mitochondrial respiration, had no effect on the ATP level, and caused no changes in glucose consumption and lactate production . Azide perfusion, combined with glucose depletion, caused rapid drop in the ATP content, which was reestablished after renewed perfusion with glucose . Similarly, perfusion with 2,4-dinitrophenol, an uncoupler of the respiration chain, had no effect on the phosphate metabolites . These results demonstrate that aerobic glycolysis is the main route by which glucose is metabolised under the conditions used (glucose concentration in medium 2 g/L) . Rates of uptake and phosphorylation of 2-deoxy-D-glucose were measured by following the formation of intracellular {6-13C}2-deoxy-D-glucose-6-phosphate by 13C NMR; at 37 degrees C the observed rates for EHR2 and EHR2/DNR+ cells were equal, about 10 nmol/(min x mg protein), whereas at 20 degrees C the wild type cells produced the 6-phosphate at an approximately twice the rate found for the resistant cells {about 4 and 2 nmol/(min x mg protein), respectively}.(ABSTRACT TRUNCATED AT 250 WORDS)

Life Sci, 1993, 53(25), PL433 - 8
Gossypol inhibits basal and estrogen-stimulated DNA synthesis in human breast carcinoma cells; Hu YF et al.; Estrogen stimulates the growth of hormone-dependent human breast cancer . Failure of chemotherapy frequently results from the development of multidrug resistance . Gossypol (GP), a naturally occurring toxin, inhibits the growth of various carcinoma cells . Thus, the effects of GP on 17 beta-estradiol (E2)-stimulated DNA synthesis were studied in two hormone-dependent human breast carcinoma cell lines: the wild-type MCF-7 and the multidrug-resistant MCF-7 Adr cells . Cells (5 x 104/well) were cultured for 24 hrs in a chemically-defined, serum-free medium consisting of 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's nutrient mixture F12 (DMEM/F12) supplemented with insulin (5.0 micrograms/ml), transferrin (5.0 micrograms/ml), epidermal growth factor (EGF; 10.0 ng/ml) and antibiotics E2 (0 or 10.0 nM), GP (0, 2.5, 5.0, 10.0 or 20.0 microM) and bovine serum albumin (BSA; 0 or 0.1 mg/ml) were used as treatments in a factorial experimental design . Cells were treated for 24 hrs and finally pulsed with 3H-thymidine (5.0 microCi/ml) for 3 hrs . E2 significantly stimulated 3H-thymidine incorporation in both MCF-7 and MCF-7 Adr cells . GP at 10.0 and 20.0 microM inhibited both basal and E2-stimulated DNA synthesis in human breast cancer cells . The inhibitory effects of GP at 10.0 microM, but not at 20.0 microM, were blocked by BSA treatment . Results from the present study indicate that GP treatment was antiproliferative in both drug-sensitive and multidrug-resistant cancer cells and that the antiproliferative effects of GP on human breast cancer cells were mediated through mechanisms independent of estrogenic responses . Thus, GP could be potentially very useful for treatment of human breast cancer patients, especially those who have developed multidrug resistance.

Tumour Biol, 1993, 14(5), 288 - 94
Cytotoxic effect of the protein-doxorubicin conjugates on the multidrug-resistant human myelogenous leukemia cell line, K562, in vitro; Hatano T et al.; In vitro studies were performed to examine the antitumor effect of protein-doxorubicin (DXR) conjugate on the growth of the multidrug-resistant human chronic myelogenous leukemia cell line, K562/DXR . The 50% inhibitory concentration (IC50) for DXR in the K562/DXR cell line was 20 nM (in the K562 parental cell line, IC50 was 3.2 nM) . Treatment of both types of cells with various concentrations of DXR or conjugates at equivalent concentrations of DXR was carried out . One type of the conjugates used was human serum albumin (HSA)-DXR conjugate and human transferrin (Tf)-DXR conjugate via a glutaraldehyde bridge (HSA-ga-DXR, Tf-ga-DXR, respectively) and another type used was HSA-DXR conjugate with a dextran bridge (HSA-dex-DXR) . All of these conjugates showed potent dose-dependent inhibition of cell growth against the K562/DXR cells as compared with the cells treated with DXR or other controls . IC50 for HSA-ga-DXR, Tf-ga-DXR and HSA-dex-DXR conjugates in the K562/DXR cell line was 2.4, 3.6 and 1.0 (equivalent DXR) nM, respectively, which were approximately similar to the value of the K562 treated with DXR . Through the treatment of K562/DXR cells with HSA-DXR conjugate, the intracellular drug concentration increased as a function of time up to 24 h compared with the cells treated with DXR . Intracellular DXR effluxed rapidly from K562/DXR cells, but HSA-ga-DXR as well as HSA-dex-DXR conjugates remained in the cells at a relatively high concentration for a long time . These results indicate that it may be possible to overcome multidrug resistance by chemically modifying DXR, such as by conjugation of the drug with proteins.

Free Radic Res Commun, 1993, 19(6), 355 - 69
High field proton NMR investigations of the metabolic profiles of multidrug-sensitive and -resistant leukaemic cell lines: evidence for diminished taurine levels in multidrug-resistant cells; Jiang XR et al.; High field proton (1H) nuclear magnetic resonance (NMR) spectroscopy has for the first time been employed to investigate and compare the metabolic profiles of vinblastine-sensitive and -resistant T-lymphoid leukaemic cell lines (CCRF-CEM and CEM/VLB100 respectively) and evidence is presented for a significantly lower taurine content in the CEM/VLB100 resistant subline when expressed relative to that of its drug-sensitive parental counterpart . These data suggest differences in the nature and relative involvements of taurine biosynthetic pathways between the two cell lines, a phenomenon that may be related to their differing sensitivities towards chemotherapeutic agents such as adriamycin which promote the generation of cytotoxic reactive oxygen species (ROS) in vivo . However, the 1H NMR data obtained provided no evidence for an increased metabolic consumption of hypotaurine (a metabolic precursor of taurine with powerful .OH radical scavenging properties) in CCRF-CEM cells since differences observed in the hypotaurine: taurine concentration ratio between the drug-sensitive and -resistant cell lines were not statistically significant . Furthermore, hypotaurine is unlikely to compete with alternative endogenous .OH radical scavengers present such as lactate since its level in either of the two cell lines investigated (ca . 6.0 x 10(-8) mol./10(8) cells) is insufficient for it to act as an antioxidant in this context . The biochemical and therapeutic significance of these results are discussed.

Acta Biochim Pol, 1993, 40(4), 487 - 96
Interactions between the gene products of pma1 encoding plasma membrane H(+)-ATPase, and pdr1 controlling multiple drug resistance in Saccharomyces cerevisiae; Ulaszewski S; In Saccharomyces cerevisiae, the pma1 mutations controlling the vanadate resistance of the H(+)-ATPase activity from the plasma membrane, map on chromosome VII in the vicinity of pdr1 mutations controlling multiple drug resistance . However, the pma1-1 mutants exhibit a genotype and a multidrug resistant phenotype quite different from those obtained for pdr1 mutants . Quantitative modifications of cycloheximide and N,N'-(p-xylylidene)-bis-aminoguanidine-2HCl resistance are observed in diploids containing the pma1 and pdr1 genes in trans configuration . Each of the pdr1 mutations interacts with pma1 as shown by a decrease in the ATPase activity in pdr1/pma1 diploids . The in vitro resistance of ATPase activity to vanadate is totally or partially suppressed in pdr1 mutants in haploid double mutants . These results suggest that the expression of PMA1 might be controlled by the PDR1 gene product.

Oncol Res, 1993, 5(6-7), 207 - 12
Toremifene enhances cell cycle block and growth inhibition by vinblastine in multidrug resistant human breast cancer cells; Baker WJ et al.; The clinical study of compounds that modulate multidrug resistance has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at the level of the tumor cell . Previously, toremifene has been shown to be well tolerated clinically and to sensitize multidrug resistant cells to the effects of cytotoxic chemotherapeutic agents . The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-MB-A1 human breast cancer cells were studied using flow cytometric analysis and growth inhibition assays . Cell cycle kinetics of MDA-MB-A1 cells were not significantly affected by treatment with either toremifene, N-desmethyltoremifene, Toremifene IV or vinblastine alone, as the majority of cells remained in G0/G1 . However, preincubation with toremifene or one of its metabolites for 72 hours followed by treatment for one hour with vinblastine caused a marked shift of cells to G2/M, as cells appeared to be blocked in that phase of the cell cycle . This result was nearly identical to the effect of vinblastine alone on vinblastine-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-MB-A1 cells to vinblastine . This chemosensitizing effect of toremifene was accompanied by an enhanced inhibition of cell growth by vinblastine . The chemosensitizing effects of toremifene or one of its metabolites in combination with cytotoxic chemotherapy can be effectively monitored by flow cytometry, an easily accessible technique.

Eur J Cancer, 1993, 29A(16), 2264 - 8
Antiproliferative activity of thermosensitive liposome-encapsulated doxorubicin combined with 43 degrees C hyperthermia in sensitive and multidrug-resistant MCF-7 cells; Merlin JL et al.; Thermosensitive liposome-encapsulated doxorubicin (TLED) was compared to free doxorubicin, at 37 degrees C or combined with 43 degrees C hyperthermia, in sensitive and multidrug-resistant MCF-7 human tumour cells using clonogenic assays . In the resistant subline, TLED was found to partly circumvent multidrug resistance (MDR) . The reversal was comparable to that obtained when verapamil was added to free doxorubicin . When hyperthermic treatment was applied, no difference in thermosensitivity was found between sensitive and resistant cells . The combination of hyperthermia with free doxorubicin did not reverse MDR . Hyperthermia and TLED yielded additive effects in the resistant cells while potentiation was observed in the sensitive cells . These results confirmed the usefulness of the liposome encapsulation of doxorubicin in reversing MDR . The possibility of obtaining additive cytotoxicity using TLED combined with hyperthermia may represent an alternative way of intensification of doxorubicin cytotoxicity concomitant with the circumvention of MDR without using MDR reversing agents, which often generate limiting toxic side-effects.

Biol Cell, 1993, 78(1-2), 63 - 8
Flow cytometry: a useful technique in the study of multidrug resistance; Leonce S et al.; Flow cytometry has recently become a very important tool in the study of the mechanisms of multidrug resistance (MDR) . This technique allows rapid access to information regarding two characteristics related to the MDR phenotype: i) the level of overexpression of the P-glycoprotein; and ii) its functional aspect in expulsion of the cytotoxic agents to which the cell is exposed . In pharmacology, flow cytometry also allows evaluation of the capacity of modulating agents to reverse this resistance, and contributes a deeper insight into the mechanism of action of new molecules.

Acta Haematol, 1993, 89(4), 184 - 8
Vitamin D3 administration and multidrug resistance in acute nonlymphoblastic leukemia; Petrini M et al.; This article reports preliminary results from a pilot study started in 1986 on patients with acute myeloblastic leukemia treated for several months with low-dose arabinosylcytosine and 1(OH)D3 . During treatment or at the time of relapse, a monoblastic component was frequently found . A high percentage of patients were P-170-positive . In 2 patients it was possible to show that blasts, previously P-170-negative, became positive after treatment . In these 2 patients, failure of clinical response to antileukemic therapy was associated with this phenotype . The addition of the revertant drug nicardipine to the previously inactive treatment induced a partial response . Thus, previously reported in vitro observations on the differentiating activity of vitamin D3 metabolites, possible induction of multidrug chemoresistance by differentiating agents and the revertant activity of the Ca++ antagonist nicardipine appear to be confirmed in vivo in the reported patients.

Eur J Cancer, 1993, 29A(3), 389 - 94
Derivation and characterisation of a mouse tumour cell line with acquired resistance to cyclosporin A; Wright KA et al.; Cyclosporin A (CsA) is an effective modifier of multidrug resistance . We have studied (a) the possibility that cells grown in increasing concentrations of CsA acquire cellular resistance to the agent and, (b) whether such cells have a multidrug resistant phenotype . Sublines of the EMT6 mouse tumour cell line were developed which were able to grow in 75 and 200 micrograms/ml of CsA, respectively . The resistant sublines grew slowly in the presence of CsA but reverted to control growth rates, whilst maintaining resistance, when the drug was removed . P-glycoprotein (Pgp) was not detectable in the resistant sublines by immunocytochemistry . The CsA-resistant cells were not cross-resistant to doxorubicin or vincristine but showed a clear degree of cross-resistance to the calcium transport blocker, verapamil . Cellular accumulation of both {3H}CsA and {3H}daunorubicin was significantly increased in the EMT6/CsA200R subline compared with the parent line . In the EMT6 parent line, which expresses very low levels of Pgp, 10-30-fold sensitisation to doxorubicin may be achieved using 0.1-5 microgram/ml of CsA . Similar sensitisation by CsA was also seen in the CsA-resistant sublines.

Eur J Cancer, 1993, 29A(3), 378 - 83
Verapamil enhances doxorubicin activity in cultured human renal carcinoma cells; Lai T et al.; Cells from 22 renal cell carcinomas (RCC) were established in culture . Sensitivity of the tumour cells to doxorubicin alone and in combination with racemic verapamil, which reverses multidrug resistance, was tested using a {75Se}selenomethionine uptake assay to measure protein synthesis . The effect of verapamil was expressed as a potentiation index: LD50doxorubicin/LD50doxorubicin + verapamil . The potentiation index in 15 of these carcinomas was determined for cells within the first 14 days of culture . At 3.3 mumol/l concentration of verapamil, of the tumours sensitive to doxorubicin alone (LD50 < 0.75 microgram/ml) five of seven showed a potentiation index of > 2 . For the less sensitive tumours the analogous proportion was seven of eight . Tumour cell expression of glycoprotein P-170, associated with multidrug resistance, was estimated using the monoclonal antibody C-219 . Initial expression levels were unrelated to the action of verapamil . In five tumours the proportion of cells expressing P-170 declined as the period of culture increased . This was not associated with any consistent change in the LD50 for doxorubicin or in potentiation of doxorubicin sensitivity by verapamil . Cell cloning associated with prolonged cell growth in vitro could mimic tumour cell cloning which accompanies the formation of metastases . Thus reduced expression of P-170 on prolonged cell growth in vitro may be a pointer to the efficacy of combination therapy in the treatment of patients with metastatic renal cell carcinoma.

Mol Carcinog, 1993, 8(2), 67 - 73
Activation of distinct multidrug-resistance (P-glycoprotein) genes during rat liver regeneration and hepatocarcinogenesis; Teeter LD et al.; The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2 . Only the first two genes are functionally related to multidrug resistance . Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported . In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration . In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols . Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene . Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy . Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice . These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.

Eur Urol, 1993, 24(1), 156 - 60
Expression of P-glycoprotein and multidrug resistance in renal cell carcinoma; Naito S et al.; The expression of the multidrug-resistance gene product, P-glycoprotein was examined immunohistochemically in 31 untreated human renal cell carcinomas . In 17 of these, chemosensitivity to Adriamycin and vinblastine was also assessed by a microtiter succinate dehydrogenase inhibition test and the correlation between the expression of P-glycoprotein and intrinsic multidrug resistance was investigated . P-glycoprotein was detected in 16 (51.6%) of the 31 carcinomas . In the chemosensitivity test, 14 (82.4%) of the 17 carcinomas were estimated to be resistant to both drugs (multidrug resistant; MDR) . Eight (72.7%) of the 11 carcinomas with a positive expression of P-glycoprotein were MDR, and none of them were sensitive of both drugs . On the other hand, MDR carcinomas were not necessarily associated with the expression of P-glycoprotein . Eight (61.5%) of the 13 MDR carcinomas showed a positive expression of P-glycoprotein while the remaining 5 (38.5%) were negative . These results suggest that the expression of P-glycoprotein is an important factor responsible for the intrinsic MDR phenotype of renal cell carcinoma, however, there are probably other factors involved as well which have yet to be fully elucidated.

J Cancer Res Clin Oncol, 1993, 119(10), 609 - 14
In vitro and in vivo chemosensitizing effect of cyclosporin A on an intrinsic multidrug-resistant rat colon tumour; Van de Vrie W et al.; Colon tumours are intrinsically resistant to chemotherapy and most of them express the multidrug transporter P glycoprotein (Pgp) . Whether this Pgp expression determines their resistance to anticancer agents in patients is not known . We report here on the reversibility of intrinsic multidrug resistance in a syngeneic, solid tumour model . CC531 is a rat colon carcinoma that expresses Pgp, as was shown with the monoclonal antibody C-219 . In vitro the sensitivity to doxorubicin, daunorubicin and colchicine was enhanced by the addition of the chemosensitizers verapamil and cyclosporin A (CsA), while the sensitivity to cisplatin was not enhanced . In a daunorubicin accumulation assay verapamil and CsA enhanced the daunorubicin content of CC531 cells . In vivo CsA was injected intramuscularly for 3 consecutive days at a dose of 20 mg kg-1 day-1 . This resulted in whole-blood CsA levels above 2 mumol/l, while intratumoral CsA levels amounted to 3.6 mumol/kg . In a subrenal capsule assay the maximal tolerable dose of doxorubicin (4 mg/kg) significantly reduced tumour growth . Doxorubicin at 3 mg/kg was not effective, but in combination with CsA this dose was as effective as 4 mg/kg doxorubicin . These experiments show that adequate doses of the chemosensitizing drug CsA can be obtained in vivo, resulting in increased antitumoral activity of doxorubicin in vivo . The in vitro and in vivo data together suggest that the chemosensitization by CsA is mediated by Pgp . This finding may have implications for the application of CsA and CsA-like chemosensitizers in the clinical setting.

Hereditas, 1993, 118(2), 121 - 30
Amplification and overexpression of the mouse mdr 1a gene in nine independently derived multidrug-resistant SEWA murine cell lines; Stahl F et al.; Many different drugs may be used in selecting cells for multidrug resistance (MDR) . Enhanced expression and/or gene amplification is known to cause overproduction of membrane-bound 170,000 P-glycoproteins, responsible for the MDR . In rodents, the P-glycoproteins are encoded by a small gene family: mdr 1a, mdr 1b, and mdr 2 . To evaluate the relationship between the pattern of MDR and the selecting drug, nine MDR sublines were independently selected from a sensitive mouse tumor cell line, SEWATC13K, using three different drugs . Each MDR subline displayed amplification of one or more of the three mdr genes, but only one, mdr 1a, was consistently overexpressed . Thus, our results indicate that the pattern of mdr gene amplification and overexpression is independent of the selective agent . Furthermore, in four of the MDR sublines, where all three mdr genes had been originally amplified, pulsed field gel electrophoresis (PFGE) revealed that amplification of mdr 1a, only, was a second step of gene amplification . In addition, the gene for the calcium-binding protein, sorcin, was coamplified in eight of the nine MDR sublines . The sorcin gene was overexpressed in seven of these eight sublines . Finally, hybridizations with a probe homologous with a putative region of RFLP (restriction fragment length polymorphism), indicated that the amplified sequences originate from one or the other of the two homologous chromosomes with no preference.

J Cancer Res Clin Oncol, 1993, 119(9), 527 - 32
Pharmacological and molecular characterization of intrinsic and acquired doxorubicin resistance in murine tumor cell lines; Schott B et al.; We have studied the pharmacological parameters of doxorubicin resistance in three lines of murine cells selected by long-term culture in the presence of this drug or vincristine . A line originating from rat hepatoma spontaneously presented an intrinsic doxorubicin resistance as compared to the other lines, originating from a rat glioblastoma and from simian-virus-40-transformed mouse hepatocytes . This intrinsic resistance, as well as the doxorubicin resistance exhibited by the vincristine-selected glioblastoma variant, could be entirely attribute to decreased drug accumulation due to drug efflux . In contrast, the doxorubicin-selected variants of the three lines exhibited an intracellular tolerance to this drug . Despite a reduction in drug accumulation when exposed to the same amount of doxorubicin, they accumulated 6-12 times more doxorubicin than wild lines when submitted to equitoxic exposures . Verapamil could restore in these lines the doxorubicin accumulation observed in sensitive lines but could not restore doxorubicin cytotoxicity . Quantitative evaluation of P-glycoprotein expression by Western blotting with the C219 antibody indicated that the wild hepatoma line overexpressed P-glycoprotein by a factor of 5 in comparison with the other wild lines, and that the vincristine-selected glioblastoma variant overexpressed this protein almost as much as the doxorubicin-selected variants . These observations favor the existence of P-glycoprotein-independent mechanisms of doxorubicin resistance, which are added to the classical multidrug-resistant phenotype in doxorubicin-selected highly resistant variant cell lines.

Eur J Cancer, 1993, 29A(8), 1078 - 81
Multiple drug resistance in the human ovarian carcinoma cell line OAW42-A; Redmond A et al.; A new multidrug-resistant variant (OAW42-A) of a human ovarian carcinoma line has been selected by exposure to increasing concentrations of doxorubicin . The variant is resistant to doxorubicin, vincristine (but surprisingly not to colchicine), etoposide, tenoposide and also to cisplatin (a drug not usually involved in classical multidrug resistance), but not to 5-fluorouracil . Overexpression of P-glycoprotein in the resistant line was demonstrated by immunofluorescence and western blotting . Direct evidence for P-glycoprotein as a determinant of resistance was provided by transfection with a specific antisense oligonucleotide . Reversal was incomplete and this, along with the pattern of cross-resistance observed, suggests that additional mechanisms of resistance may also be involved . Substantial clonal variation in resistance exists within the cell line.

Eur J Cancer, 1993, 29A(7), 1024 - 7
Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3; Wall DM et al.; A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL) . Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp . Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL . The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively . When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004) . The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Haematologica, 1993 Jan-Feb, 78(1), 12 - 7
Expression of multidrug resistance gene (MDR-1) in human normal leukocytes; Damiani D et al.; BACKGROUND . The mdr-1 gene, which codes for a 170-kd transmembrane glycoprotein (P170), is frequently overexpressed in multidrug resistant (MDR) tumor cell lines and in spontaneous tumors, including leukemia and lymphoma . However, it is also constitutively expressed as a normal gene in normal tissues . METHODS . We used human mdr-1 cDNA and three anti-P170 monoclonal antibodies (MoAbs: MRK-16, C-219 and JSB-1) to investigate the normal peripheral blood leukocyte content of mdr-1 specific mRNA and of P170 through immunocytochemistry and flow cytometry . RESULTS . We did not find any increase in mdr-1-specific mRNA, while small amounts of P170 were easily detectable in about two thirds of the lymphocytes and monocytes and in about one third of the granulocytes . The level of P170 expression in leukocytes was similar to that found in non-MDR tumor cell lines . CONCLUSIONS . mdr-1 is constitutively expressed in human normal leukocytes at levels that cannot significantly affect drug resistance . Accordingly, low-level mdr-1 expression in leukemic cells should not be considered a label of pleiotropic drug resistance.

Cancer Chemother Pharmacol, 1993, 32(2), 151 - 5
Influence of sequential exposure to R-verapamil or B8509-035 on rhodamine 123 accumulation in human lymphoblastoid cell lines; Roller E et al.; Modulators for the reversal of multidrug resistance such as R-verapamil and B8509-035, a dihydropyridine, effectively overcome multidrug resistance in vitro and are currently undergoing clinical trial . One problem with their use is the application protocol; the question as to whether they should be given by continuous administration or in sequential doses in combination with the cytotoxic drugs has to be addressed . Therefore, we examined the influence of the exposure time and the sequence of modulator administration on the active transport of the fluorescent dye rhodamine 123 (R123), a substrate for the P-glycoprotein, in the resistant lymphoblastoid cell line VCR1000 and the parental nonresistant cell line CCRF-CEM . Our results demonstrate the importance of coadministration of R-verapamil and the cytotoxic agent for the modulation of multidrug resistance, whereas the exposure sequence does not seem to be such an essential parameter in the case of B8509-035 . This observation should be considered for the further design of clinical studies.

Cancer Chemother Pharmacol, 1993, 32(2), 116 - 22
Non-glucocorticoid steroid analogues (21-aminosteroids) sensitize multidrug resistant cells to vinblastine; Abraham I et al.; Several members of a group of compounds developed to treat stroke and trauma of the central nervous system are shown also to reverse multidrug resistance in human KB-V1 cells . The most potent reversal agents studied are 21-aminosteroid derivatives (lazaroids), tirilazad mesylate (tirilazad, U-74006F) and U-74389F . Tirilazad sensitizes resistant human cells (KB-V1) to killing by vinblastine by 66-fold, but does not change the sensitivity of the nonresistant parental line, KB-3-1, to vinblastine . KB-V1 cell membranes have high levels of P-glycoprotein, a protein that acts as an efflux pump and is thought to be the major cause of multidrug resistance in these cells . Tirilazad inhibits the photoaffinity labeling of P-glycoprotein with {3H}azidopine in KB-V1 cells more effectively than does verapamil, a standard reversal agent . In addition, tirilazad causes the increased accumulation of {3H}vinblastine in multidrug resistant KB-V1 cells . Studies of the resistance reversal potential of related compounds suggest that the complex amine portion of tirilazad is important for its reversal activity, while the steroid portion is less important.

Cancer Chemother Pharmacol, 1993, 31(6), 431 - 41
MDR-1 gene expression, anthracycline retention and cytotoxicity in human lung-tumor cells from refractory patients; Ramachandran C et al.; Lung-tumor cells from pleural effusion of four refractory patients and in cell lines established from them were analyzed for anthracycline retention, cytotoxicity, and MDR-1 gene and P-glycoprotein expression . Murine leukemic P388 and doxorubicin-resistant P388/R84 lines were used as controls . The 50% growth-inhibitory concentration (IC50) for doxorubicin among lung-tumor lines varied from 0.16 to 0.31 microM in soft agar . Heterogeneity in doxorubicin or daunorubicin retention and response to the efflux-blocking action of 25 microM prochlorperazine was noted in pleural effusion of FCCL-1, -4, and -8 . Among the cell lines established, an efflux-blocking effect in a subpopulation was noticed only in FCCL-1 and -4 . Although the MDR-1 gene was present in all cell lines, including P388, its expression was pronounced only in P388/R84 and FCCL-1 . In situ hybridization of antisense RNA probe to tumor cells showed high heterogeneity for MDR-1 message in the human lung-tumor cells as compared with the murine cells . Northern and slot blot hybridization confirmed in situ hybridization in lines with high levels of MDR-1 expression . The synthesis of MDR-1 mRNA and P-glycoprotein in tumor lines was correlated . The results suggest that because of extensive tumor-cell heterogeneity in human tumors, monitoring of MDR expression by in situ hybridization, quantitation of P-glycoprotein content by laser flow cytometry (and/or immunohistochemical methods), and drug efflux (by laser flow cytometry) may be the best ways to monitor multidrug resistance in human tumors.

J Heart Lung Transplant, 1993 Jan-Feb, 12(1 Pt 1), 20 - 6
Multidrug resistance in lung allograft recipients: possible correlation with the development of acute and chronic rejection; Yousem SA et al.; The expression of drug resistance antigens in mononuclear inflammatory cells was studied in transbronchial lung biopsy specimens of lung allograft recipients who experienced steroid-sensitive and steroid-resistant bouts of acute rejection and bronchiolitis obliterans . Immunostains for C494 and C219 epitopes of p-glycoprotein and human metallothionein revealed that (1) mononuclear cells expressing these antigens are present in the lung allograft during rejection, (2) that steroid-resistant acute rejection is associated with increased percentages of C494 and metallothionein-positive cells as compared to steroid-sensitive cases, (3) that bronchiolitis obliterans was associated with a higher percentage of cells with drug-resistant antigen expression, and (4) that steroid-resistant bronchiolitis obliterans is associated with the highest percentage of C494 and metallothionein-positive cells in the five clinical situations studied . P-glycoprotein and metallothionein expression may be a marker of aggressive or persistent cases of acute rejection and bronchiolitis obliterans.

J Surg Oncol, 1993 Jan, 52(1), 21 - 5
P-glycoprotein expression in hepatocellular carcinoma; Isshiki K et al.; In hepatocellular carcinoma cell lines, the intensity of staining with the monoclonal antibody C-219 to the multidrug-resistant gene (mdr1) product P-glycoprotein and the intensity of the band at a molecular weight of 170 KDa on Western blot were associated closely with resistance to Adriamycin but not with the resistance to cis-dichlorodiamine platinum (CDDP) . In clinical specimens, noncancerous liver tissue was regularly stained with this antibody on the biliary canalicular front of the hepatocyte cell membrane . In liver cancer tissue, however, regular staining as in the noncancerous regions of the liver was observed in only 16% of the patients, irregular staining was seen in only 24%, and no staining was seen at all in 60% . Staining of P-glycoprotein with the C-219 antibody is technically simple and is useful for studying the role of P-glycoprotein in drug-resistant hepatocellular carcinoma.

Mol Microbiol, 1993 Jan, 7(1), 69 - 79
The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP-dependent translocators; Karow M et al.; We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature-sensitive phenotype . The msbA gene maps to 20.5 min on the Escherichia coli genetic map and encodes a protein with an estimated molecular mass of 64,460 Da, with the properties of an integral membrane protein . The amino acid sequence of MsbA is very similar to those of the family of ATP-dependent translocators, which includes the haemolysin B protein of E . coli and the mammalian multidrug resistance (MDR) proteins . Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.

Eur J Cancer, 1993, 29A(4), 554 - 8
High rate of expression of multidrug resistance-associated P-glycoprotein in human endometrial carcinoma and normal endometrial tissue; Schneider J et al.; The overexpression of P-glycoprotein was studied in 10 normal endometrial controls (five from the proliferative and five from the secretory phase of the menstrual cycle) and in 23 endometrial carcinomas of different histological varieties, using the C219 and JSB-1 monoclonal antibodies . Three of the tumours had been previously treated with combination chemotherapy containing doxorubicin . All endometrial carcinomas, whether treated or untreated, as well as the normal endometrial controls from both the proliferative and the secretory phase of the menstrual cycle, overexpressed P-glycoprotein . This puts endometrial carcinoma into the same category as other tumours arising in organs which normally overexpress P-glycoprotein, all of which tend to be intrinsically resistant to chemotherapy.

Genomics, 1993 Jan, 15(1), 182 - 4
Mapping of a rat multidrug resistance gene by fluorescence in situ hybridization; Popescu NC et al.; A cDNA clone encoding the rat mdr1b (Pgy2) gene was recently isolated and characterized . This gene has a high degree of sequence identity with other Pgy genes, particularly the mouse Pgy2 gene . By means of in situ fluorescence hybridization, the rat Pgy gene was localized on chromosome 4 band q12 . This regional mapping will facilitate the identification of synteny groups on rat, mouse, and human genomes and chromosomal rearrangements during mammalian evolution.

Cancer Immunol Immunother, 1993, 36(2), 133 - 9
Diverse multidrug-resistance-modification agents inhibit cytolytic activity of natural killer cells; Chong AS et al.; Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells selected for resistance to one chemotherapeutic agent simultaneously acquire resistance to several apparently unrelated drugs . MDR in tumor cells is associated with the over-expression of P-glycoprotein, an ATP-dependent cell-membrane transport molecule . P-glycoprotein is also expressed in several normal tissues but its physiological role(s) is unknown . We recently observed that a hierarchy of MDR-like activity exists among human peripheral blood lymphocytes in the order CD8 > CD4 > CD20 (cytotoxic/suppressor T cells, helper T cells and B cells respectively) . In this study, we report that natural killer (NK) cells also express MDR-like activity . This activity could be inhibited with verapamil or solutol HS-15, two agents that reverse MDR in tumor cells . These, and four additional reversing agents, were used to investigate the possible role of P-glycoprotein in NK cells . We observed that at 10% of their IC50, five of six reversing agents inhibited NK-cell-mediated cytotoxicity; at higher (but non-toxic) doses, all six agents were inhibitory . These data suggest that NK-cell-mediated cytotoxicity may require the functional expression of an efflux molecule similar or identical to P-glycoprotein.

J Cancer Res Clin Oncol, 1993, 119(4), 185 - 9
Prognostic implication of immunodetection of P glycoprotein in Ewing's sarcoma; Roessner A et al.; Increased expression of P glycoprotein is associated with multidrug resistance in many cell lines . P glycoprotein has been detected in different human tumors . To assess the implication of multidrug resistance in the prognosis of Ewing's sarcoma the expression of P glycoprotein was studied immunohistochemically in pre- and post-therapeutic tumor tissues of 21 cases treated according to the CESS 81 or 86 protocol . The response to chemotherapy was evaluated histologically . Formalin-fixed, paraffin-embedded and fresh frozen sections were immunostained with a monoclonal antibody to P glycoprotein, clone JSB 1, using the double APAAP method . P glycoprotein was detected in 12 cases of 21 (57%) in either pre- or postchemotherapy tumor tissues . From the 21 cases 8 revealed a good morphological response to chemotherapy (33%); 10 of the 13 non-responders were positive for P glycoprotein (77%), but only 2 of the 8 responders (25%) . The difference was statistically significant (P < 0.05) . Comparing P glycoprotein expression with the clinical outcome, we found that 7 of 12 positive cases had died (58%) . From the negative cases only 3 of 9 had died (33%) . However, judged by the Kaplan Meyer life tables, these data were not significant . In conclusion our results suggest that the immunodetection of P glycoprotein indicates a poor response to chemotherapy and probably a bad clinical outcome for Ewing's sarcoma patients.

J Clin Oncol, 1993 Jan, 11(1), 109 - 15
Expression of P-glycoprotein and glutathione-S-transferase in recurrent lymphomas: the possible role of Epstein-Barr virus, immunophenotypes, and other predisposing factors; Cheng AL et al.; PURPOSE: We previously have reported the poor prognoses of recurrent peripheral T-cell lymphoma (PTCL) and Epstein-Barr virus (EBV)-associated PTCL (J Clin Oncol 7:725-731, 1989; Blood 77:799-808, 1991) . To study the role that drug resistance plays in this scenario, we conducted a retrospective study of 23 adult patients . PATIENTS AND METHODS: All patients had recurrent lymphoma tissue available for immunophenotyping and screening for the existence of EBV DNA in tumor cells by Southern blot analysis and in situ hybridization . Expression of a multidrug resistance P-glycoprotein ({P-gp}mdr-1) and a glutathione redox cycle-related glutathione-S-transferase pi (GST-pi) was determined by an immunohistochemistry method . RESULTS: Expression of mdr-1 or GST-pi was found in 11 (48%) and 12 (52%) cases, respectively . Most (11 of 12) of the GST-pi expression occurred simultaneously with mdr-1 . Prechemotherapy tumor tissues were available in 11 cases; only two (18.2%) of these cases expressed mdr-1 . Four (36%) of 11 cases that expressed mdr-1 (mdr-1(+)) and nine (90%) of 10 cases that did not express mdr-1 (mdr-1(-)) responded to second-line chemotherapy (P < .05) . The survival-after-recurrence (SAR) curves significantly favored mdr-1(-) recurrent lymphoma (P < .05) . The mdr-1 expression was further correlated with the immunophenotype and EBV association . All six cases of EBV-associated lymphoma (PTCL, five cases; Hodgkin's disease, one case) had significant simultaneous expression of mdr-1 and GST-pi in their recurrent tumor tissues . CONCLUSION: (1) mdr-1 expression is a significant prognostic factor in recurrent lymphomas; (2) high expression of mdr-1 is observed in recurrent EBV-associated PTCL; and (3) GST-pi usually expresses simultaneously with mdr-1 in recurrent lymphomas . The role of EBV in the development of mdr-1 and the biologic significance of the simultaneous expression of mdr-1 and GST-pi in recurrent lymphomas are well worth further exploration.

Oncol Res, 1993, 5(12), 467 - 74
In vitro and in vivo effects of clinically important camptothecin analogues on multidrug-resistant cells; Mattern MR et al.; The cytotoxic alkaloid camptothecin (CPT) and several of its analogues, including the clinically relevant topotecan (TPT), irinotecan (CPT-11), and 9-aminocamptothecin, were evaluated for differential cytotoxic effect and DNA damage induction in multidrug-sensitive (AuxB1) and multidrug-resistant (MDR) (CHRC5) Chinese hamster ovary cells . CPT, 10-hydroxycamptothecin, and 10,11-methylenedioxycamptothecin produced equivalent amounts of cell growth inhibition and/or DNA single-strand breakage in the two cell lines . TPT, SN-38 (the active metabolite of CPT-11), and 9-aminocamptothecin were 12-, 9-, and 10-fold, respectively, less toxic to the MDR than to the wild-type cells . These findings are consistent with differences in yields of DNA single-strand breaks produced in AuxB1 and CHRC5 cells by 2-hr incubations with the various compounds . In both assays, the resistance ratios of the topoisomerase I inhibitors were approximately one-tenth those of known MDR drugs such as vinblastine or amsacrine . Thus, cultured cells that overexpress P-glycoprotein have the potential to develop some level of cross-resistance to all three topoisomerase I inhibitors currently in the clinic . The chemical basis for cross-resistance of cultured MDR cell lines to certain CPT analogues is not yet understood, but is likely more complex than positive charge alone . TPT had a reasonable therapeutic effect on B6D2F1 female mice implanted with MDR sublines of P388 leukemia, compared with its effect on mice implanted with wild-type P388 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Oncol Res, 1993, 5(9), 373 - 81
Development of a safe and effective adriamycin plus interleukin 2 therapy against both adriamycin-sensitive and -resistant lymphomas; Ho RL et al.; This laboratory has extensively studied Adriamycin (doxorubicin)-induced immunomodulation . Despite demonstration of favorable effects, little therapeutic advantage was seen, and it was decided to test Adriamycin in combination with interleukin 2 (IL2) . Considerable toxicity was seen with either high-dose IL2 or high-dose Adriamycin alone, using the syngeneic C57B1/6-EL4 T cell lymphoma model . When the doses of either agent were reduced to decrease toxicity, little therapeutic effect was seen . In contrast, an effective protocol without apparent toxicity was developed by combining a moderate dose of Adriamycin (4 mg/kg, IV, Days 1 and 8 or only Day 8) with prolonged administration of a moderate dose of IL2 (2 micrograms, b.i.d., i.p., Days 9 to 40) . This protocol resulted in up to 80% long-term survivors among mice inoculated on Day 0 with EL4 lymphoma (5 x 10(4) cells) . It should be noted that under these conditions, neither agent, when administered singly, induced long term survivors, and that following the inoculation of only 10-100 EL4 tumor cells all animals died in the absence of treatment . The survivors developed protective immunity as demonstrated by their ability to resist reimplantation with EL4 tumor . Furthermore, this resistance to tumor reimplantation could be transferred into naive hosts with spleen cells from tumor-bearing mice receiving the combination protocol; exposure of mice to sublethal whole body irradiation prior to tumor implantation completely abrogated the efficacy of this combination treatment . Finally, it was shown that this combination protocol was equally effective against an Adriamycin-resistant subline of EL4 that expresses the multidrug resistance phenotype.

Oncol Res, 1993, 5(8), 311 - 23
Reversal of "atypical"-multidrug resistance by recombinant human tumor necrosis factor in the human ovarian cancer cell line A2780-DX3; Cimoli G et al.; A2780 human ovarian cancer cell line and its multidrug resistant counterpart A2780-DX3 were utilized for this in vitro study . A2780-DX3 is resistant in various degrees to several topoisomerase II inhibitors but sensitive to vinca alkaloids . Simultaneous treatment of the A2780-DX3 line with 1000 U/mL rHuTNF largely reverses resistance to most topoisomerase II inhibitors . By itself, 1000 U/mL rHuTNF is not toxic to the resistant line . Uptake and retention of {3H}-Mitoxantrone are not modified by rHuTNF, whereas rHuTNF is very active in potentiating the effects of Mitoxantrone . After treatment with topoisomerase II inhibitors, Doxorubicin, Mitoxantrone, or VP16, rHuTNF restores DNA-SSB and DNA-protein cross-links in the resistant line to the level of the wild type . The cleavage activity of topoisomerase II in the resistant line is about 40% of the level present in the parental line . Five minutes after the addition of 1000 U/mL of rHuTNF, the cleavage activity in the resistant line is about 85% of the level present in the parental line . The catalytic activity of topoisomerase II is only 15% lower in the resistant line, but it is increased by about 50% 5 min after the addition of rHuTNF to the resistant line . These effects are transient and cannot be observed after 30 min . These transient direct effects of rHuTNF on topoisomerase II could be associated with its ability to restore sensitivity to inhibitors of topoisomerase II in the A2780-DX3 line.

Oncol Res, 1993, 5(8), 301 - 9
Anticancer drug sensitivity profiles of new and established melanoma cell lines; Marshall ES et al.; Metastatic melanoma is notable for its resistance to chemotherapy, and methods for determining resistance in cultures would be advantageous . We investigated the chemosensitivities of seven newly derived low passage lines and eight established melanoma lines . A 96-well microculture system utilising {3H}-thymidine incorporation was used to determine IC50 values (50% inhibitory concentrations) for lomustine, mitomycin C, 4-hydroperoxycyclophosphamide, cisplatinum, 5-fluorouracil, vincristine, doxorubicin, etoposide, amsacrine and the amsacrine analogue CI-921 . Cytokinetic parameters were determined using 5-bromodeoxyuridine and flow cytometry . The presence of "transport" and "atypical" multidrug resistance was investigated with an IC50 ratio method, using pairs of drugs with differing sensitivity to these multidrug resistance mechanisms . A wide range of chemosensitivity for each of the antitumor agents was observed, and a spectrum of activity was observed in each of the assays for multidrug resistance . Unexpectedly, a number of cell lines displayed coordinate sensitivity or resistance to cytotoxic agents with unrelated mechanisms of action . Resistance mechanisms apart from "transport" and "atypical" multidrug resistance may be required to account for the observed 40-fold range in overall chemosensitivity of the melanoma lines.

Yao Xue Xue Bao, 1993, 28(11), 808 - 11
{Studies on effective reversal of adriamycin-resistant Chinese hamster ovary cell line by verapamil}; Jiang B et al.; A noncytotoxic dose of verapamil (Ver) 3 micrograms/ml was found to potentiate 10-fold the growth-inhibitory effects of adriamycin (ADM) in ADM-resistant Chinese hamster ovary cell line (RC1) . Ver 3 micrograms/ml also reduced the IC50 value of ADM from 1.2 micrograms/ml to 0.08 microgram/ml in RC1 in clonogenic assay . The index of reversing resistance was 15-fold . In an attempt to elucidate the mechanism of the reversion of the multidrug resistance by Ver, Ver in combination with ADM was found to enhance intracellular ADM accumulation in RC1, and brought about more toxicity in RC1 than ADM alone; and Ver in combination with ADM was shown to retard RC1 in G2 + M phase by flow cytometry analysis.

Receptors Channels, 1993, 1(4), 305 - 13
Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein; Mintenig GM et al.; Expression of the human multidrug resistance P-glycoprotein is associated with two activities, active drug transport and a volume-regulated chloride channel . In this study we define four classes of compound, based on their differential effects on these two activities . Class I compounds are substrates transported by P-glycoprotein . They also prevent channel activation when added to the cytoplasmic face of the membrane . Class II compounds include reversers of multidrug resistance such as verapamil . These compounds inhibit drug transport and block the chloride channel when added to the outer face of the membrane . Class III compounds include conventional channel blockers which block the chloride channel but do not influence drug transport . Class IV compounds, for example cyclosporin A, appear to inhibit drug transport but do not affect chloride channel activity . These findings have implications for the relationship between the channel and transporter functions associated with P-glycoprotein expression, and for the development of clinical agents which reverse multidrug resistance.

Biotherapy, 1993, 6(4), 291 - 302
Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr1 gene; Boesen JJ et al.; Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients . 'Rescue' of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs . Despite this improvement, patients often relapse with drug resistance disease . The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene . By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy . This review explores the feasibility of mdr1 gene therapy.

J Natl Cancer Inst Monogr, 1993, (15), 55 - 61
Taxol: mechanisms of action and resistance; Horwitz SB et al.; Information on the mechanisms of action and of resistance to Taxol, as well as new data from our laboratory on the promoter regions of the genes that encode P-glycoprotein in a murine Taxol-resistant cell line, is discussed . Taxol induces the formation of stable bundles of microtubules, thereby interfering with the normal function of cellular microtubules . The drug can induce the multidrug-resistance (MDR) phenotype that includes the overproduction of P-glycoprotein, a membrane glycoprotein that acts as a drug efflux pump . In human tumors resistant to Taxol, P-glycoprotein could be responsible for maintaining the drug below cytotoxic levels . Analyses of the MDR promoters that are involved in P-glycoprotein expression and overproduction revealed an interesting recombination event in a Taxol-resistant cell line . As an important new clinical agent for the treatment of malignancies, Taxol requires further mechanistic investigations at the preclinical level.

Rev Med Interne, 1993, 14(8), 772 - 9
{Multidrug resistance induced by surexpression of the mdrl gene}; Guerci AP; Drug resistance of some tumors and hematopoietic diseases is a major problem for clinicians . One of the mechanisms of resistance is called the multidrug resistance . Multidrug resistance is a cross-reactive resistance between molecules different in structure and activities . Multidrug resistance is characterized by an active drug efflux through the cell membrane with a decrease in drug cellular accumulation . This efflux out of the cell is due to a membrane glycoprotein called P-glycoprotein or P-gp 170 encoded by the mdr1 gene . In humans, high levels of mdr1 gene expression are observed in normal tissues as adrenal gland, colon and kidney . Tumors derived from these tissues are usually resistant to chemotherapy . By contrast to this intrinsic drug resistance, acquired drug resistance is defined by the mdr1 gene expression in tumors which relapse and become refractory to chemotherapy as acute lymphoid and myeloid leukemia, breast and ovarian cancers, non-Hodgkin lymphomas and multiple myeloma . When the clinical significance of multidrug resistance will be defined, identification of non-responders patients should lead to the use of therapeutic regimens including mdr reversing agents.

Wien Med Wochenschr, 1993, 143(19-20), 526 - 32
{Calcium antagonists as modulators of multi-drug resistant tumor cells}; Hamilton G et al.; Multidrug-resistance (MDR) is a cellular mechanism, which in certain tumors reduces the chemosensitivity of cells to a characteristic group of structural different cytostatic drugs, as anthracyclines, Vinca alkaloids and others, and correlates with a unfavourable clinical prognosis . In MDR-cells the intracellular concentration of cytostatic drugs is reduced due to the action of the mdr-1-gene-encoded P-glycoprotein (P-gp/gp 170), which functions as drug efflux pump with broad substrate specificity . Many calcium channel blockers of all subclasses (phenylalkylamine, dihydropyridine and benzothiazepine type) and other calcium antagonists inhibit the P-gp-mediated drug efflux and represent modulators of MDR (resistance modifiers, chemosensitizers) . Since the sensitized tumor cells express no voltage-gated calcium channels, the induction of changes in intracellular free calcium showed no effect on MDR and the MDR-activity of antagonists is not correlated with their cardiovascular effects, the chemosensitization by these drugs is independent from their action on calcium channels and Ca(++)-regulation . Photoaffinity labelling with reactive derivatives proved the direct competitive interaction of calcium antagonists with the binding site of P-gp for cytostatic drugs as mechanism of action . The MDR-modulation of the doxorubicin or Vinca alkaloid resistance of tumor cells in vitro by verapamil and other calcium channel blockers was confirmed in vivo as increased survival length in mice with tumor transplants in combination with cytostatic therapy . The clinical application of calcium antagonists is limited by their severe cardiovascular side effects associated with the high concentrations required for successful reversal of MDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Oncol Res, 1993, 5(6-7), 229 - 34
N-benzyladriamycin-14-valerate (AD 198)-resistant cells exhibit highly selective cross-resistance to other anthracyclines that circumvent multidrug resistance; Lothstein L et al.; N-Benzyladriamycin-14-valerate (AD 198)-resistant murine J774.2 macrophage-like cells (A300) exhibited a novel mechanism of resistance in which P-glycoprotein was overexpressed without decreased AD 198 accumulation . Cross-resistance to Adriamycin (ADR), N-benzyladriamycin, and Adriamycin-14-valerate was due, at least in part, to reduced accumulation, suggesting that circumvention of P-glycoprotein-mediated transport was associated with extreme lipophilicity conferred by both substitutions . Thus, unlike multidrug resistance mediated by either P-glycoprotein, the multidrug resistance-associated protein (MRP), or decreased topoisomerase II activity, cross-resistance in A300 cells was highly structure-specific . In order to further characterize the specificity of AD 198 resistance, the cytotoxicity, accumulation, and intracellular localization of a series of 3'-morpholinyl, 3'-deamino and halogenated ADR congeners that have been reported to circumvent MDR was determined in AD 198-resistant J774.2 and P388 AD 198-resistant cells . Cross-resistance correlating with increased AD 198 resistance was observed for 2'-bromo-4'-epi-hydroxy-daunomycin (13-fold), morpholinyl doxorubicin (24-fold), and 4'-iodo-4'-deoxydoxorubicin (2.8-fold), but was attributable to decreased accumulation . Cross-resistance to 3'-hydroxy-14-O-palmitoyl-doxorubicin (6-fold) was not due to reduced accumulation . No cross-resistance was observed for the highly cytotoxic metabolite of WP474, 3'-hydroxyldoxorubicin (hydroxyrubicin; WP159), nor for the much less cytotoxic 3'-O-benzylated congeners, including 3'-O-benzyl-doxorubicin-14-valerate . These findings indicate that AD 198 resistance confers cross-resistance to compounds that, like AD 198, localize in the cytoplasm but are metabolized to highly cytotoxic, nuclear-localizing compounds.

Int J Tissue React, 1993, 15(1), 17 - 23
Activity of different revertant agents on multidrug resistance: in vitro evaluation of their combination; Petrini M et al.; The revertant activity of different compounds has been assayed on a multidrug-resistant human breast-cancer cell line (MCF 7/Dx) . The calcium-channel blocker nicardipine showed the higher revertant ability when compared to cefoperazone or cyclosporin A at concentrations close to the pharmacological range . Interestingly, nicardipine was able to increase the revertant activities of both cefoperazone and cyclosporin A, but these latter were not able to enhance each other over a plateau . However, a limit of about 70% of growth inhibition of the line cultured in the presence of 60 microM doxorubicin seems to be insuperable at the concentrations employed . The combination of the three drugs brings the concentrations of drugs to the point at which the maximum possible inhibition is reached in the pharmacological range, but the complete reversion of chemoresistance is not reached when the doxorubicin is added at the concentration capable of reducing the cell proliferation by 50%.

J Cancer Res Clin Oncol, 1993, 120(1-2), 17 - 23
Response of a multidrug-resistant human small-cell lung cancer xenograft to chemotherapy; Arvelo F et al.; Small-cell lung carcinomas (SCLC) are highly responsive to various chemotherapies . However only a minority of patients benefit from long survival . SCLC patients treated at the Institut Gustave Roussy received a combined chemotherapy (CCAV) including cisplatin, cyclophosphamide (Cpa), Adriamycin (doxorubicin; Adm) and vepeside (VP16) . We report here the intrinsic sensitivity of a small-cell lung carcinoma, designated SCLC-6, grafted in nude mice . This xenografted tumour was derived from an untreated patient . The CCAV regimen given to the patient donor of the tumour sample resulted in a complete response followed by recurrence and death, 8 months after the initial cure . The expression of P-glycoprotein encoded by the MDR1 gene was detected with the C219 antibody on the membrane of SCLC-6 tumour cells . When given to SCLC-6-tumour-bearing nude mice, CCAV induced a strong inhibition of tumour growth (84% of growth inhibition, 20 days after start of the treatment), but no cure . Intensification of CCAV doses did not improve the response . The efficacy of individual agents of the CCAV, given at maximal tolerated doses was analysed . Only cisplatin (10 mg/kg) and Cpa (3 x 50 mg/kg) inhibited SCLC-6 growth (79% and 100% inhibition respectively), VP16 (3 x 24 mg/kg) was poorly efficient (42%) and Adm (10 mg/kg) not at all . Two-drug combinations such as cisplatin plus VP16 or cisplatin plus Cpa inhibited tumour growth (81% and 70%, respectively) . Curiously, the efficacy of Cpa, given in combination with cisplatin was less than that of Cpa alone . Repeated treatments with CCAV administered to mice at each in vivo passage of the tumour induced a loss of chemosensitivity, which was observed until the ninth passage . An improvement of the therapeutic response was obtained by adding a headline reverser of multi-drug resistance, verapamil (25 mg/kg), to CCAV (81% versus 63% inhibition) . MDR1-related resistance appeared to play a role in the failure of SCLC-6 chemotherapy; frequent recurrences after treatment with cisplatin and Cpa, two drugs that are not recognized by the P-glycoprotein, indicated that other modes of resistance were simultaneously active.

Lasers Surg Med, 1993, 13(5), 511 - 6
Effect of multidrug-resistant P-glycoprotein gene expression on chloroaluminum tetrasulfonate phthalocyanine concentration; Frazier DL et al.; The effect of multidrug-resistant P-glycoprotein gene expression (MDR1) in 3T3 cells on cellular concentrations and cytotoxicity induced by the photodynamic agent chloroaluminum tetrasulfonate phthalocyanine (AlSPc) was evaluated . 3T3 cells transfected with a retroviral vector expressing human MDR1 cDNA were resistant to colchicine . Resistant cells incubated with daunomycin accumulated only 40-50% of the quantity of daunomycin accumulated in control cells . Resistant cells incubated with daunomycin in the presence of verapamil had intracellular daunomycin concentrations approximately equal to control cells without verapamil . When these MDR1 3T3 cells were incubated with AlSPc, cellular concentrations of AlSPc did not differ between cells resistant to colchicine and those that were not . Similarly, there was little difference in cytotoxicity demonstrated by 51Cromium release in the two cell lines exposed to AlSPc and light (675 nm; 6 J/cm2) . This study suggests photodynamic therapy using AlSPc may be a useful treatment modality for tumors in which the MDR1 P-glycoprotein confers resistance to cancer chemotherapeutics.

Cancer Chemother Pharmacol, 1993, 33(2), 123 - 9
Differential effects of estrogen, tamoxifen and the pure antiestrogen ICI 182,780 in human drug-resistant leukemia cell lines; Zalcberg JR et al.; ICI 182,780, a potent, new steroidal antiestrogen without apparent agonist activity, appears to be a potent modulator of the classic multidrug resistance (MDR) phenotype in the CEM/A7, CEM/VLB100 and K562/VIN100 MDR cell lines . This reagent had no effect on the respective parental CCRF-CEM and K562 cell lines . The use of 1.25 microM ICI 182,780 resulted in a 6- to 7-fold decrease in doxorubicin resistance in the CEM/A7 and CEM/VLB100 cell lines . A dose-response effect was observed at ICI 182,780 concentrations of up to 5 microM . As compared with tamoxifen (TAM), ICI 182,780 was 2 and 4 times more effective in the K562/VIN100 and CEM/A7 cell lines, respectively . ICI 182,780 at 0.625 microM increased {3H}-daunomycin uptake (P < 0.0001) as effectively as 5 microM TAM in the resistant CEM/A7 line . Drug-efflux studies showed that 5 microM ICI 182,780 significantly decreased drug efflux as compared with 5 microM TAM (P < 0.0001) . Estradiol (EST) at 10 microM increased doxorubicin resistance by 1.2-1.3 times in the CEM/A7 and CEM/VLB100 cell lines and significantly decreased drug accumulation (P = 0.002) and retention (P < 0.001) in the CEM/A7 cell line . However, the addition of 10 microM EST to 1-2 microM ICI 182,780 did not inhibit the ability of ICI 182,780 to modulate doxorubicin resistance in the two resistant cell lines . Using reverse-phase high-performance liquid chromatography (HPLC) to measure lipophilicity, we found no apparent association between the ability of ICI 182,780, TAM or EST to modulate resistance and their relative hydrophobicity.

Cancer Biother, 1993 Spring, 8(1), 77 - 85
Sensitization of P388 murine leukemia cells to epirubicin cytotoxicity by reserpine; Viladkar A et al.; Reserpine, the crystalline active substance isolated from the Rauvolfia plant, produces a characteristic vasodepressor effect in hypertensive patients . Apart from its antihypertensive property, reserpine also possesses transquillising and vasodepressor action, hence it is employed as supportive therapy in the treatment of cardiac disorders . Doxorubicin is a potent anticancer agent, the use of which is limited by its cumulative dose-dependent cardiotoxicity . Epirubicin is a derivative of doxorubicin having more favourable therapeutic index than doxorubicin and possessing less hematologic and cardiac toxicity at comparable doses . The data presented in this paper show the effect of reserpine as a chemosensitizer, when used in combination with epirubicin on P388 murine leukemia cells sensitive (P388/S) and resistant to doxorubicin (P388/DOX) cells . Inhibition of 3H-TdR incorporation into DNA was used as an index of the cytotoxic effects of drug when used alone or in combination . The combination of reserpine (1 microM) and epirubicin (1.7, 8.6 and 17.2 microM) indicated a significant enhancement in the DNA biosynthesis inhibition in P388/S and P388/DOX cell lines . The most prominent feature of the multidrug-resistant cell is the reduced accumulation of the drug intracellularly . P388/DOX cells showed less accumulation of epirubicin in the cell as compared to that of the parental cell line . Further studies demonstrated that reserpine significantly enhanced the intracellular accumulation of epirubicin in both the cell lines . The nature of DNA damage caused by the combination of reserpine and epirubicin was irreversible when studied in P388/DOX cell line . The combination of reserpine (5mg/kg) and epirubicin (1mg/kg) significantly potentiated the antitumor activity of epirubicin in P388/DOX tumor bearing mice . These studies suggest that reserpine can be used as an adjuvant in the cancer chemotherapy to potentiate the antiproliferative activity of anticancer drugs.

Cancer Biother, 1993 Spring, 8(1), 67 - 75
Reversal of C1300 murine neuroblastoma multidrug resistance by cremophorEL, a solvent for cyclosporin A; Chervinsky DS et al.; We previously developed a homoharringtonine resistant C-1300 neuroblastoma cell line with cross-resistance to adriamycin and increased levels of p-glycoprotein, and showed that drug resistance could be reversed in this cell line by cyclosporin A . The present study shows that cremophor EL, a parenteral vehicle for cyclosporin A, can also completely reverse this multidrug resistance in a clonogenic assay system . Cremophor EL incubated with resistant cells for up to six days did not reduce levels of p-glycoprotein . Intracellular homoharringtonine analysis using HPLC revealed increased drug accumulation in resistant cells treated with cremophor EL . The increased drug level was not due to blocking of drug efflux commonly seen in other multidrug resistant models . The data suggest that resistance modulation with cyclosporin A should be interpreted with caution when cremophor EL is a solvent . Our work suggests cremophor EL, a relatively nontoxic lipophylic solvent, may have a direct effect on membrane permeability, although other mechanisms cannot be ruled out.

Cytotechnology, 1993, 12(1-3), 63 - 89
Molecular cytogenetics of multiple drug resistance; Schoenlein PV; The refractory nature of many human cancers to multi-agent chemotherapy is termed multidrug resistance (MDR) . In the past several decades, a major focus of clinical and basic research has been to characterize the genetic and biochemical mechanisms mediating this phenomenon . To provide model systems in which to study mechanisms of multidrug resistance, in vitro studies have established MDR cultured cell lines expressing resistance to a broad spectrum of unrelated drugs . In many of these cell lines, the expression of high levels of multidrug resistance developed in parallel to the appearance of cytogenetically-detectable chromosomal anomalies resulting from gene amplification . This review describes cytogenetic and molecular-based studies that have characterized DNA amplification structures in MDR cell lines and describes the important role gene amplification played in the cloning and characterization of the mammalian multidrug resistance genes (mdr) . In addition, this review discusses the genetic selection generally used to establish the MDR cell lines, and how drug selections performed in transformed cell lines generally favor the genetic process of gene amplification, which is still exploited to identify drug resistance genes that may play an important role in clinical MDR.

Cytotechnology, 1993, 12(1-3), 33 - 62
Molecular analysis of the multidrug transporter; Germann UA; The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold . P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump . The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold . This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds . These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel . With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions . Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established . The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.

Cytotechnology, 1993, 12(1-3), 315 - 24
Mathematical models for multidrug resistance and its reversal; Michelson S; Mathematical models describing drug resistance are briefly reviewed . One model which describes the molecular function of the P-glycoprotein pump in multidrug resistant (MDR) cell lines has been developed and is presented in detail . The pump is modeled as an energy dependent facilitated diffusion process . A partial differential equation linked to a pair of ordinary differential equations forms the core of the model . To describe MDR reversal, the model is extended to add an inhibitor . Equations for competitive, one-site noncompetitive, and two-site noncompetitive inhibition are derived . Numerical simulations have been run to describe P-glycoprotein dynamics both in the presence and absence of these kinds of inhibition . These results are briefly reviewed . The character of the pump and its response to inhibition are discussed within the context of the models . All discussions, descriptions, and conclusions are presented in nonmathematical terms . The paper is aimed at a scientifically sophisticated but mathematically innocent audience.

Cytotechnology, 1993, 12(1-3), 265 - 88
Differing patterns of cross-resistance resulting from exposures to specific antitumour drugs or to radiation in vitro; Hill BT; This article reviews the patterns of cross-resistance identified in various P-glycoprotein-mediated and non-P-glycoprotein-mediated drug resistant mammalian tumour cell lines . The differing patterns of cross-resistance and the variable levels of resistance expressed are summarised and discussed . Although the mechanism by which P-glycoprotein can recognise and transport a large group of structurally-unrelated substrates remains to be defined, the recent evidence indicating that membrane associated domains participate in substrate recognition and binding is summarised, and other possible explanations for these variable cross-resistance patterns are considered . Amongst the non-P-glycoprotein-overexpressing multidrug resistant cell lines, two subsets are clearly identifiable, one lacking and the other expressing cross-resistance to the Vinca alkaloids . Resistance mechanisms implicated in these various sublines and possible explanations for their differing levels and patterns of cross-resistance are summarised . Clinical resistance is identified in patients following treatment not only with antitumour drugs, but also after radiotherapy . Experimental data providing a biological basis for this observation are summarised . A distinctive multiple drug resistance phenotype has been identified in tumour cells following exposure in vitro to fractionated X-irradiation characterised by: the expression of resistance to the Vinca alkaloids and the epipodophyllotoxins but not the anthracyclines and overexpression of P-glycoprotein which is post-translationally regulated, but without any concomitant overexpression of P-glycoprotein mRNA . Finally, the possible clinical relevance of these variable patterns of cross-resistance to the antitumour drugs commonly used in the clinic is considered.

Cytotechnology, 1993, 12(1-3), 231 - 56
Human cell lines as models for multidrug resistance in solid tumours; Clynes M et al.; In spite of our expanding knowledge on the molecular biology of cancer, relatively little progress has been made in improving therapy for the solid tumours which are major killers, e.g., lung, colon, breast . Significant advances over the past 10-15 years in chemotherapy of some tumours such as testicular cancer and some leukaemias indicates that, in spite of the undesirable side-effects, chemotherapy has the potential to effect cure in the majority of patients with certain types of cancer . Multidrug resistance, inherent or acquired, is one important limiting factor in extending this success to most solid tumours . In vitro studies described in this review are now uncovering a diversity of possible mechanisms of cross-resistance to different types of drug . Sensitive methods such as immunocytochemistry, RT-PCR or in situ RNA hybridisation may be necessary to identify corresponding changes in clinical material . Only by classifying individual tumours according to their specific resistance mechanisms will it be possible to define the multidrug resistance problem properly . Such rigorous definition is a prerequisite to design (and choice on an individual basis) of specific therapies suited to individual patients . Since a much larger proportion of cancer biopsies should be susceptible to accurate analysis by the immunochemical and molecular biological techniques described above than to direct assessment of drug response, it seems reasonable to hope that this approach will succeed in improving results for cancer chemotherapy of solid tumours where other approaches such as individualised in vitro chemosensitivity testing have essentially failed . Results from clinical trials using cyclosporin A or verapamil are encouraging, but these agents are far from ideal, and reverse resistance in only a subset of resistant tumours . Proper definition of the other mechanisms of MDR, and how to antagonize them, is an urgent research priority.

Cytotechnology, 1993, 12(1-3), 213 - 30
Multidrug resistance (MDR) genes in haematological malignancies; Nooter K et al.; The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies . Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs . Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR) . Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistant in vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype . P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value . Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas . Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation . Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors . However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.

Cytotechnology, 1993, 12(1-3), 171 - 212
Pharmacologic circumvention of multidrug resistance; Ford JM et al.; The ability of malignant cells to develop resistance to chemotherapeutic drugs is a major obstacle to the successful treatment of clinical tumors . The phenomenon multidrug resistance (MDR) in cancer cells results in cross-resistance to a broad range of structurally diverse antineoplastic agents, due to outward efflux of cytotoxic substrates by the mdr1 gene product, P-glycoprotein (P-gp) . Numerous pharmacologic agents have been identified which inhibit the efflux pump and modulate MDR . The biochemical, cellular and clinical pharmacology of agents used to circumvent MDR is analyzed in terms of their mechanism of action and potential clinical utility . MDR antagonists, termed chemosensitizers, may be grouped into several classes, and include calcium channel blockers, calmodulin antagonists, anthracycline and Vinca alkaloid analogs, cyclosporines, dipyridamole, and other hydrophobic, cationic compounds . Structural features important for chemosensitizer activity have been identified, and a model for the interaction of these drugs with P-gp is proposed . Other possible cellular targets for the reversal of MDR are also discussed, such as protein kinase C . Strategies for the clinical modulation of MDR and trials combining chemosensitizers with chemotherapeutic drugs in humans are reviewed . Several novel approaches for the modulation of MDR are examined.

Cytotechnology, 1993, 12(1-3), 155 - 70
Glutathione-related enzymes, glutathione and multidrug resistance; Moscow JA et al.; This review examines the hypothesis that glutathione and its associated enzymes contribute to the overall drug-resistance seen in multidrug resistant cell lines . Reports of 34 cell lines independently selected for resistance to MDR drugs are compared for evidence of consistent changes in activity of glutathione-related enzymes as well as for changes in glutathione content . The role of glutathione S-transferases in MDR is further analyzed by comparing changes in sensitivity to MDR drugs in cell lines selected for resistance to non-MDR drugs that have resulting increases in glutathione S-transferase activity . In addition, results of studies in which genes for glutathione S-transferase isozymes were transfected into drug-sensitive cells are reviewed . The role of the glutathione redox cycle is examined by comparing changes in elements of this cycle in MDR cell lines as well as by analyzing reports of the effects of glutathione depletion on MDR drug sensitivity . Overall, there is no consistent or compelling evidence that glutathione and its associated enzymes augment resistance in multidrug resistant cell lines.

Cytotechnology, 1993, 12(1-3), 137 - 54
Topoisomerase II in multiple drug resistance; Hofmann GA et al.; Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents . Two types of multiple drug resistance are associated with these enzymes . In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein . In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors . Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance . In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5') or noncovalently (3') to the enzyme while a second double strand is passed through the break in the first . This transition state complex then decays to enzyme and DNA of altered linking number . Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates . Toxicity is related to topoisomerase II content as well as to drug concentration . Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical) . Mixed types, i.e., both typical and atypical, are known . Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.

Cytotechnology, 1993, 12(1-3), 109 - 25
Non-P-glycoprotein multidrug resistance in cell lines which are defective in the cellular accumulation of drug; Center MS; Non-Pgp mdr related to a defect in drug accumulation has now been documented in a number of different cell lines exposed to certain cytotoxic agents . In studies conducted thus far most isolates have been obtained after selection in either adriamycin or mitoxantrone . The work in this area is in its early stages and very little is known about the molecular events which contribute to this mode of drug resistance . At the present time no protein with drug binding properties comparable to Pgp has been identified in non-Pgp mdr isolates . Evidence based on the finding that all isolates do not respond in the same way to reversal agents such as verapamil suggests the possibility that more than one mechanism may exist for non-Pgp mdr . Future studies may thus reveal that cells contain a multiplicity of genes which upon transcriptional activation can function to alter drug transport processes and thus contribute to the development of mdr . Identifying and characterizing these genes will be important since they may function in transport systems of normal cells . The exact identify of proteins which contribute to non-Pgp mdr remains to be determined . One protein designated P190 has been found to be overexpressed in cell lines of human promyelocytic leukemia, lung and adenocarcinoma treated with adriamycin . The protein also is increased in some clinical samples from patients undergoing chemotherapy . P190 which has a minor sequence homology with Pgp can bind ATP and may thus contribute to the energy dependent drug efflux systems found in cells containing this protein . Transfection studies with a P190 cDNA should determine whether this protein actually contributes to drug resistance . Many other protein changes have been detected in non-Pgp mdr cells but the importance of these in resistance also remains to be determined . In some systems a particular protein change can be identified in multiple independent isolates suggesting a correlation between the development of resistance and the presence of this cellular alteration . Experiments conducted thus far on the mechanism of non-Pgp mdr are intriguing . Studies utilizing fluorescence microscopy to follow the fate of daunomycin suggests that the drug passes to the interior of the cell and eventually localizes in the Golgi apparatus . Drug located at this site may move directly into an efflux pathway for rapid extrusion from the cell . Evidence also indicates that as drug leaves the Golgi some may be sequestered into other organelles such as lysosomes or mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotechnology, 1993, 12(1-3), 1 - 32
P-glycoprotein structure and evolutionary homologies; Croop JM; Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family . Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents . P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold . The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes . This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require 12 alpha-helical transmembrane domains and two nucleotide binding folds for substrate transport . P-glycoprotein homologs have been isolated and characterized from a wide range of species . Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein.

Cytotechnology, 1993, 12(1-3), 91 - 107
Antibodies in the study of multiple drug resistance; Heike Y et al.; Multidrug resistance (MDR) is a major problem in cancer chemotherapy . As P-glycoprotein is the key molecule in MDR, many investigators have constructed anti-P-glycoprotein monoclonal antibodies (MAbs) . Those antibodies, including MRK16 and C219, were used for elucidation of the mechanism of MDR and for overcoming of MDR . This article describes the characterization of the antibodies against the P-glycoprotein and other proteins of multidrug-resistant tumor cells, and discusses the therapeutic implication of the antibodies.

Cytotechnology, 1993, 11(2), 115 - 9
Altered DNA topoisomerase II in multidrug resistance; Beck WT et al.; The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR) . We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26 . Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities . Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA . Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells . We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases . We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Neoplasma, 1993, 40(1), 21 - 5
Overcoming of vincristine resistance in L1210/VCR cells by several corticosteroids . Collateral sensitivity of resistant cells; Barancik M et al.; Five corticosteroids were tested to determine whether they are able to overcome the multidrug resistance of vincristine resistant mouse leukemia cells L1210/VCR . The most effective in reversing multidrug resistance were cortisone and dexamethasone, less effective as reversing agents were 11-deoxycorticosterone, 1-dehydrocortisone and hydrocortisone . By testing of collateral sensitivity of vincristine resistant cell line to these corticosteroids it was found that only 11-deoxycorticosterone and dexamethasone were toxic to multidrug resistant cells at doses much lower than required for toxicity to the drug-sensitive L1210/S cells . Using thin layer chromatography the polarity of tested corticosteroids was estimated, and good correlation was found between polarity of corticosteroids and their increased toxicity to vincristine resistant cells.

Virchows Arch A Pathol Anat Histopathol, 1993, 422(3), 233 - 8
Establishment of a transplantable carcinoma arising from the intrahepatic bile duct in Syrian golden hamsters; Fukahori T et al.; A subcutaneously transplantable cancer line from the intrahepatic bile duct (IHBD) induced by N-nitrosobis(2-oxopropyl) amine was established in Syrian golden hamsters . The doubling time of this tumour was 2.6 days when 2 x 10(5) tumour cells were inoculated subcutaneously (take-up rate was 100%) . Growth of the tumour was significantly faster in male hamsters but neither oestrogen nor androgen receptors were detected in the tumour . The primary and all allograft tumours were tubular adenocarcinomas with fibrosis and a scirrhous pattern resembling human IHBD carcinoma of the peripheral type . Transmission electron microscopic findings showed irregular glands covered with numerous microvilli . Blood-group-related antigens including A, B and H were positive . P-Glycoprotein, which is an indicator of multidrug resistance, was also positive . Carcinoembryonic antigen and CA19-9 as general tumour markers of the biliary tract were negative . The deoxyribonucleic acid (DNA) pattern of this transplantable carcinoma was diploid . This newly established animal model of a transplantable IHBD carcinoma can be used to examine the mechanisms of synthesis and secretion of tumour-associated antigens and to study potential therapeutic agents.

Pflugers Arch, 1993 Jan, 422(4), 347 - 53
Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin; Diaz M et al.; The possible role of Cl- currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique . Cells showed very small currents in voltage-clamp experiments performed with Cl(-)-rich, permeant-cation-free (N-methyl-D-glucamine replacement) intracellular and bathing solutions . Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl- current, which decayed at the most depolarised voltages used . Replacement of extracellular Cl- by a series of halide anions, SCN- and gluconate was consistent with an anion selectivity sequence: SCN- > I- > Br- > Cl- > F- > gluconate . The volume-regulated Cl- current was effectively inhibited by 100 microM 5-nitro-2-(3-phenyl-propylamino)-benzoic acid and by 100 microM 4,4'-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl- channels in a variety of cells . Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP{gamma S} and Mg(2+)-free ATP . The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells . The characteristics of this Cl- current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein . Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.

Br J Haematol, 1993 Jan, 83(1), 63 - 7
Analysis of multidrug-resistance (MDR-1) glycoprotein and CD56 expression to separate monoclonal gammopathy from multiple myeloma; Sonneveld P et al.; Monoclonal gammopathy of undetermined significance (MGUS) is different from multiple myeloma (MM) by a low proliferation and by its indolent clinical course . In this study, two biological parameters were investigated which mark the transition from MGUS to MM, i.e . expression of the P-170 glycoprotein associated with the multidrug resistance phenotype (MDR-1) and expression of the natural killer cell antigen . CD56 . Strong MDR-1 expression was found in plasma cells of 32/38 untreated MGUS as compared with 33/105 untreated MM stage I-III (84% v 32%, P < 0.001) and in 0/10 normal plasma cell samples . CD56 expression in high density was present in 43/57 analysed untreated MM but in none of 23 MGUS (78% v 0%, P < 0.0001) . Plasma cells did characteristically show a low Ki-67 proliferation index in 14/15 MGUS patients (mean 0.05%, range 0-0.2%) and a higher index in 25 analysed MM patients (mean 2.31%, range 1-7%, P < 0.03) . These data indicate that MDR-1 expression together with absence of CD56 expression and a low proliferation index can be used to separate MGUS from MM.

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 312 - 6
The multidrug resistance (mdr1) gene product functions as an ATP channel; Abraham EH et al.; The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells . The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane . Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane . These findings suggest an unusual role for the P-glycoprotein.

Mutat Res, 1993 Jan, 285(1), 79 - 90
Multidrug resistance and mutagenesis; Ferguson LR et al.; Multidrug resistance, the phenomenon whereby the development of resistance to one drug is sometimes accompanied by the simultaneous development of resistance to a variety of other, often structurally unrelated, drugs, is frequently associated with the presence of an energy-dependent membrane-transport system which reduces the concentration of a drug or other chemical in the cytoplasm . The latter process (termed here MDR) occurs naturally in a number of normal mammalian tissues, including colon, jejunum, liver, kidney and bone marrow, as well as in other species including bacteria . The presence of MDR can reduce the mutagenic potential of a variety of compounds in mammalian and microbiological assays . MDR can be reversed by a diverse collection of compounds, many of which are hydrophobic cations with other physiological effects . An important consequence of these considerations is that MDR-reversing agents are potentially dangerous because, while having no intrinsic mutagenicity, they may significantly increase the mutagenicity of other compounds by poisoning protective MDR mechanisms in the body.

Cytotechnology, 1993, 12(1-3), 289 - 314
The use of reverse transcriptase-polymerase chain reaction (RT-PCR) to investigate specific gene expression in multidrug-resistant cells; O'Driscoll L et al.; Expression of specific genes at the level of mRNA can be studied using techniques such as Northern blot, slot/dot blot, RNase protection assay, in situ hybridisation and RT-PCR . In this article these methods of analysis are compared; RT-PCR offers higher levels of specificity and sensitivity than traditional methods of RNA analysis and as such has become the method of choice for the study of gene expression . The RT-PCR technique is described in detail with sections dealing with RNA extraction, choice of primers (including the use of cDNA sequence data bases), PCR and RT-PCR protocols in addition to the limitations of the method . The study of one particular mRNA transcript (MDR1) using RT-PCR is discussed in detail . Recently described methods for quantitation of PCR products are discussed . Quantitative PCR would appear to offer a method of studying gene expression in a more extensive way than has been possible to date.

Tumori, 1992 Dec 31, 78(6), 403 - 6
Simultaneous occurrence of monoclonal gammopathy and acute secondary leukemia with overexpression of P-glycoprotein; Stasi R et al.; A 52-year-old woman, previously treated with chemo- and radiotherapy for Hodgkin's disease, developed an acute non-lymphoid leukemia and, contemporarily, an IgG-kappa paraproteinemia . Cytogenetic analysis showed a major clone, representing 90% of observed metaphases, with monosomy of chromosomes 5 and 14 . In addition, leukemic cells exhibited a high expression of the P-glycoprotein, a transmembrane glycoprotein involved in the multidrug-resistance mechanism . Possible explanations for this cluster of findings are provided.

Biochemistry, 1992 Dec 22, 31(50), 12555 - 64
Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrug-resistant cells expressing different levels of P-glycoprotein; Roepe PD; Continuous monitoring of fluorescence (CMF) has been used to examine doxorubicin efflux from intact human myeloma cells . The time resolution of these measurements has enabled detailed comparison of the initial rates of efflux for the drug-sensitive myeloma line RPMI 8226 and a series of sequentially derived multidrug-resistant (MDR) lines expressing different amounts of human MDR protein (P-glycoprotein) . Cells that are 3-, 10-, 60-, or 120-fold resistant to doxorubicin export approximately 10, 20, 30, or 33% more doxorubicin than the parental sensitive cells, respectively, when all are preloaded to the same level of total intracellular drug . Remarkably, however, when cells are loaded to the same level of exchangeable drug the initial rates of efflux are found to be virtually identical . This agreement between rates is apparently not dependent on the drug concentration . Approximately 50% of the increase in the steady-state level of doxorubicin efflux for the resistant cells is abolished upon glucose starvation . However, surprisingly, the apparent initial rates of efflux from the treated and untreated cells are found to be virtually the same . Pretreatment of the resistant cells with verapamil reduces the steady-state level of efflux but increases the apparent initial rate at some concentrations . Conversely, vincristine does not alter steady state but slows the initial rate of efflux from both sensitive and resistant cells by approximately the same extent . Finally, quite interestingly, a nearly linear relationship between pHi and relative steady state of efflux is found for the series of cell lines . These data are interpreted in terms of existing models for MDR.

J Natl Cancer Inst, 1992 Dec 16, 84(24), 1909 - 15
Liposome-mediated modulation of multidrug resistance in human HL-60 leukemia cells; Rahman A et al.; BACKGROUND: Multidrug resistance (MDR) is a major obstacle in cancer treatment . Resistance of cultured tumor cells to major classes of cytotoxic drugs is frequently due to expression of a plasma membrane P-glycoprotein encoded by MDR genes . We have demonstrated that liposome-encapsulated doxorubicin is more toxic than the free drug and that it modulates MDR in Chinese hamster LZ cells and human colon cancer cells . PURPOSE: To investigate further the association between expression of P-glycoprotein and modulation of MDR by liposome-encapsulated doxorubicin, we studied vincristine-resistant HL-60/VCR leukemia cells, which express P-glycoprotein, and doxorubicin-resistant HL-60/ADR leukemia cells, which do not . METHODS: Cells were exposed to various concentrations of free doxorubicin and liposome-encapsulated doxorubicin . The cellular content of doxorubicin was determined by fluorescence analysis, and cytotoxicity was determined by cell growth inhibition . Photoaffinity-labeling studies of P-glycoprotein binding were performed on HL-60/VCR and HL-60/ADR cells and KB-GSV2 cells transfected with the MDR1 gene (also known as PGY1) . RESULTS: The concentrations that caused 50% inhibition of growth (IC50) for free doxorubicin in HL-60, HL-60/ADR, and HL-60/VCR cells were 30 nM, 9 microM, and 0.9 microM, respectively . The values for liposome-encapsulated doxorubicin in parental HL-60 cells and HL-60/ADR cells were 20 nM and 9 microM, respectively, indicating little or no sensitization . In contrast, HL-60/VCR cells were fivefold more sensitive to liposome-encapsulated doxorubicin than to free doxorubicin, and IC50 was reduced to 0.17 microM . In HL-60 cells exposed to liposome-encapsulated doxorubicin, intracellular doxorubicin accumulation was less than that seen with free drug . In contrast, in HL-60/VCR cells, accumulation was twofold to threefold higher than that with free doxorubicin . Liposome-encapsulated doxorubicin completely inhibited the photoaffinity labeling of P-glycoprotein by azidopine in membrane vesicles of HL-60/VCR cells, with a potency comparable to that of azidopine, suggesting that circumvention of MDR by liposomes is related to their specific interaction with P-glycoprotein . The studies with KB-GSV2 cells indicated that blank liposomes can directly inhibit photoaffinity labeling of P-glycoprotein . CONCLUSIONS: These results demonstrate the effectiveness of liposome-encapsulated doxorubicin in overcoming resistance in the multidrug-resistant phenotype of HL-60/VCR cells by direct interaction with P-glycoprotein . Furthermore, they indicate that liposome-encapsulated doxorubicin may be an effective treatment for human cancers.

J Biol Chem, 1992 Dec 15, 267(35), 24995 - 5002
Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles; Sinicrope FA et al.; P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues . P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport {3H}daunomycin . In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of {3H}daunomycin and {3H}vinblastine . Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics . Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) . Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity . Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of {3H}daunomycin . {3H}Vinblastine accumulation was also inhibited by A2C (p < 0.05) . Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation . Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited {3H}daunomycin accumulation and uncompetitively inhibited {3H}vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively . Verapamil competitively inhibited P-gp-mediated accumulation of {3H}daunomycin but failed to alter the fluidity of CMV . Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV . Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.

Cancer Res, 1992 Dec 15, 52(24), 6969 - 75
Expression and functional activity of P-glycoprotein in cultured cerebral capillary endothelial cells; Hegmann EJ et al.; Analysis of a panel of endothelial cells passaged between 5 and 25 times and derived from various organs and species demonstrated that murine and porcine cerebral capillary endothelial cells actively excluded the fluorescent dye rhodamine 123, a substrate of P-glycoprotein . In addition, rhodamine 123 accumulation could be enhanced by the multidrug resistance chemosensitizer verapamil, known to reduce P-glycoprotein-mediated drug efflux . Cloned murine and porcine cerebral capillary endothelial cells were immunoreactive with the C219 monoclonal antibody to P-glycoprotein, and a C219 epitope-specific blocking peptide could abolish staining . The antiproliferative and cytotoxic effects of vincristine, but not cis-platinum(II) diamminedichloride, were increased by the addition of either verapamil or cyclosporin A to brain endothelial cell cultures in a 72-h assay, as determined by {3H}thymidine incorporation and total protein measurement . Cyclosporin A was a more effective reversal agent than verapamil . Thus, a P-glycoprotein isoform may be constitutively expressed in brain endothelial cells in vitro and supports the available data on in situ immunohistochemical staining of P-glycoprotein at the blood-brain barrier . In addition, these findings may indicate that one function of P-glycoprotein in vivo at the blood-brain barrier is the exclusion of xenobiotics from central nervous system tissues.

Blood, 1992 Dec 15, 80(12), 3106 - 11
Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells; DelaFlor-Weiss E et al.; Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene . Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo . To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector . We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression . Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly . These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

J Biol Chem, 1992 Dec 5, 267(34), 24248 - 52
Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone; Ueda K et al.; We expressed human MDR1 cDNA isolated from the human adrenal gland in porcine LLC-PK1 cells . A highly polarized epithelium formed by LLC-GA5-COL300 cells that expressed human P-glycoprotein specifically on the apical surface showed a multidrug-resistant phenotype and had 8.3-, 3.4-, and 6.5-fold higher net basal to apical transport of 3H-labeled cortisol, aldosterone, and dexamethasone, respectively, compared with host cells . But progesterone was not transported, although it inhibited azidopine photoaffinity labeling of human P-glycoprotein and increased the sensitivity of multidrug-resistant cells to vinblastine . An excess of progesterone inhibited the transepithelial transport of cortisol by P-glycoprotein . These results suggest that cortisol and aldosterone are physiological substrates for P-glycoprotein in the human adrenal cortex and that substances that efficiently bind to P-glycoprotein are not necessarily transported by P-glycoprotein.

Science, 1992 Dec 4, 258(5088), 1650 - 4
Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line; Cole SP et al.; The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents . However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance . Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated . One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells . Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1 . Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression . The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

J Natl Cancer Inst, 1992 Dec 2, 84(23), 1811 - 6
High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine; Trump DL et al.; BACKGROUND: P-glycoprotein mediates resistance to natural-product anti-neoplastic agents like vinblastine through an active transport process resulting in reduced intracellular concentration of these agents . The triphenylethylene antiestrogen tamoxifen and its major metabolite N-desmethyltamoxifen at concentrations of 4-6 microM enhance the intracellular concentration of natural-product antineoplastics and augment the cytotoxicity of such drugs three-fold to 10-fold in a variety of human and murine cell lines . PURPOSE: On the basis of these preclinical findings, we conducted a phase I clinical trial of high-dose, oral tamoxifen administered in conjunction with a 5-day continuous infusion of vinblastine . METHODS: We studied 53 patients with advanced epithelial tumors . Tamoxifen was given orally as a loading dose on day 1, followed by two doses a day on days 2-13 . Vinblastine was given as a 120-hour continuous infusion (1.5 mg/m2 per day) on days 9-13 of each tamoxifen course . The starting dose of tamoxifen was 40 mg/m2 administered twice a day following a loading dose of 150 mg/m2 . The maximum dose was 260 mg/m2 twice a day following a loading dose of 680 mg/m2 . Treatment cycles were repeated every 28 days . RESULTS: The dose-limiting toxic effects of tamoxifen were neurologic and began within 3-5 days after the start of treatment . They consisted of tremor, hyperreflexia, dysmetria, unsteady gait, and dizziness . One patient experienced a grand mal seizure 24 hours after the last tamoxifen dose . Toxic effects were rapidly reversible . Asymptomatic prolongation of the QT interval on electrocardiogram occurred at doses of tamoxifen of 80 mg/m2 or higher given twice a day . No coagulation or ophthalmologic abnormalities occurred . Tamoxifen did not enhance the toxicity of vinblastine . Mean plasma concentrations of tamoxifen or N-desmethyltamoxifen at 260 mg/m2 tamoxifen given twice a day for 13 days were 6.04 and 6.56 microM, respectively . There was no relationship between plasma antiestrogen content and the development of neurotoxic effects . CONCLUSIONS: Tamoxifen at 150 mg/m2 given twice a day following a loading dose of 400 mg/m2 results in plasma levels of tamoxifen and N-desmethyltamoxifen of 4 and 6 microM, respectively, without dose-limiting toxicity . We recommend this dose for phase II trials of tamoxifen to modulate P-glycoprotein-mediated drug resistance . IMPLICATIONS: Our study demonstrates that high-dose tamoxifen can be safely administered and that plasma concentrations that may inhibit P-glycoprotein function can be achieved.

Clin Otolaryngol, 1992 Dec, 17(6), 501 - 4
A phase II study of cisplatinum versus cisplatinum + nifedipine in end-stage carcinoma of the head and neck; Jones AS et al.; Forty patients with end-stage carcinoma of the head and neck were admitted to a randomized double blind trial with cisplatinum in one arm and cisplatinum + nifedipine in the other . Nifedipine, a calcium channel blocking drug, inhibits the effect of acquired multidrug resistance in animal models . In the present study the addition of this agent had no effect on either response rate or survival in end-stage carcinoma of the head and neck . It is concluded that adding nifedipine to cisplatinum serves no useful purpose in the treatment of end-stage carcinoma of the head and neck.

Radiologe, 1992 Dec, 32(12), 579 - 83
{The pathology of soft tissue sarcomas}; Schmidt D; Soft tissue sarcomas are rare and can cause considerable difficulty in diagnosis and differential diagnosis as well as in estimation of the prognosis . These problems are due in part to the wide histological diversity, which is a consequence of intratumoral heterogeneity . The best known example of a very heterogeneous soft tissue tumor is malignant fibrous histiocytoma (MFH) . Regular application of ancillary techniques, including electron microscopy and immunohistochemistry, has made it possible to reduce the number of unclassified cases from more than 10% to about 5% . Further progress in this direction is to be expected from cytogenetic studies, since for some of the tumor types characteristic chromosomal abnormalities have been established . Prognosis has been related to the grade of malignancy, but recent studies show that in many soft tissue sarcomas it will also be possible to correlate prognosis with DNA ploidy . By contrast, overexpression of the multidrug resistance gene mdr-1 does not seem to play an essential role in soft tissue sarcomas.

Semin Oncol, 1992 Dec, 19(6), 670 - 86
The anthracyclines: will we ever find a better doxorubicin?
Weiss RB.
The anthracyclines are the class of antitumor drugs with the widest spectrum of activity in human cancers, and only a few cancers (eg, colon cancer) are unresponsive to them . The first two anthracyclines were developed in the 1960s . Doxorubicin (DOX) differs from daunorubicin (DNR) only by a single hydroxyl group . This fact has spurred researchers worldwide to find analogs of DOX that have less acute toxicity, cause less cardiomyopathy, can be administered orally, and/or have different, or greater, antitumor efficacy . Five DOX/DNR analogs are marketed in other countries, and one (idarubicin) is available in the United States . None of these analogs have stronger antitumor efficacy than the original two anthracyclines, but there are some differences in toxicity . Methods have been fashioned to keep the peak plasma level of DOX muted to minimize cardiotoxicity, but the only apparently effective method available so far (prolonged drug infusion) is cumbersome . The bisoxopiperazine class of drugs (especially dexrazoxane) provides protection against anthracycline-induced cardiomyopathy and has much promise for helping mitigate this major obstacle to prolonged use of the anthracyclines . The DOX analogs being evaluated in the 1990s have been selected for their ability to overcome multidrug resistance in cancer cells . Thirty years after discovery of the anticancer activity of the first anthracycline, some means of reducing anthracycline toxicity have been devised . Current studies are evaluating increased doses of epirubicin to improve anthracycline cytotoxicity, while limiting cardiotoxicity, but at present DOX still reigns in this drug class as the one having the most proven cancerocidal effect.

J Infect Dis, 1992 Dec, 166(6), 1393 - 400
High-dose mefloquine in the treatment of multidrug-resistant falciparum malaria; ter Kuile FO et al.; The therapeutic efficacy and toxicity of a high-dose (25 mg/kg) mefloquine regimen (M25) and the currently recommended regimen of 15 mg/kg (M15) were compared in 199 patients with acute falciparum malaria in an area with deteriorating multidrug resistance on the Thai-Burmese border . The clinical and parasitologic responses were significantly more rapid with M25 . The incidence of treatment failures by day 7-9 was 7% for M15 and 1% for M25 (P = .03) and had increased to 40% and 9%, respectively, by day 28 (P < .0001) . Overall failure rates were highest in children (P = .02) . Parasite clearance times were a good predictor of the therapeutic response; all patients with parasitemia persisting > 5 days after treatment experienced subsequent recrudescence . Side effects were dose-related and included dizziness, anorexia, nausea, vomiting, and fatigue . Although vomiting < 1 h after treatment was more likely in young children, children overall tolerated mefloquine better than adults, and men better than women . The optimum treatment dose of mefloquine in this area is 25 mg/kg.

Cancer Res, 1992 Dec 1, 52(23), 6666 - 70
Effect of buthionine sulfoximine and ethacrynic acid on cytotoxic activity of mitomycin C analogues BMY 25282 and BMY 25067; Xu BH et al.; BMY 25282 and BMY 25067, analogues of mitomycin C (MMC), were synthesized in an attempt to increase the therapeutic potential of the parent drug . The present studies were undertaken to determine if the cytotoxicity of MMC or its analogues is affected by cellular glutathione (GSH) and/or GSH transferase (GST) levels by using sensitive (P388/S) and multidrug resistant (P388/R-84) mouse leukemia cells as a model . P388/R-84 cells were cross-resistant to all three drugs . BMY 25067 was > 100 times more cytotoxic than MMC in both the cells . MMC and BMY 25282 produced significantly lower DNA interstrand cross-links (ISC) in P388/R-84 cells, whereas BMY 25067 induced ISC formation was comparable in these cells . GSH depletion with D,L-buthionine-S,R-sulfoximine (BSO) increased sensitivity to MMC, BMY 25282, and BMY 25067 by 3.4-, 4.1-, and 1.8-fold, respectively, in the resistant cells . Pretreatment of P388/R-84 cells with a nontoxic concentration of ethacrynic acid (EA) (10 micrograms/ml for 1 h), an inhibitor of GST activity, also resulted in a significant increase in the cytotoxic activities of MMC and BMY 25282 (3.8- and 3.1-fold, respectively), but not of BMY 25067 . Combined pretreatment of P388/R-84 cells with BSO and EA caused further increase in the cytotoxic activities of both MMC and BMY 25282 . Potentiation of BMY 25067 cytotoxicity by combined BSO and EA pretreatments was similar to that observed by BSO pretreatment alone . The ISC formation by MMC and BMY 25282 were also increased significantly by BSO or EA pretreatment in these cells . Whereas BSO treatment increased BMY 25067 induced ISC formation, it was not affected by EA pretreatment . These results suggest that (a) a potentiation of the cytotoxic activity of MMC or BMY 25282 can be achieved by GSH depletion and/or GST inhibition, (b) the enhanced cytotoxicity may be caused at least in part by the increased formation of drug-DNA cross-links, and (c) the mechanism of BMY 25067 cytotoxicity may be different from the other two drugs . The results of the present study also suggest that BMY 25067 may be seriously considered for further clinical development because it is much more active than MMC, and unlike the parent drug cytotoxicity of BMY 25067 does not seem to be affected by GST levels, which have been suggested to play an important role in cellular resistance to several cancer chemotherapy drugs.

Cancer Res, 1992 Dec 1, 52(23), 6440 - 6
Modulation of subcellular distribution of doxorubicin in multidrug-resistant P388/ADR mouse leukemia cells by the chemosensitizer ((2-isopropyl-1-(4-{3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino}propyloxy)-benzenesulfonyl))indolizine; Jaffrezou JP et al.; The impact of the novel chemosensitizer ((2-isopropyl-1-(4-{3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino}propyloxy)benzenesulfonyl))indolizine (SR33557) on the intracellular distribution of doxorubicin (DOX) within the multidrug-resistant murine P388/ADR leukemia cell line was studied by fluorescence microscopy . We found that under conditions which modulated multidrug-resistant (30 microM SR33557 for 1 h), P388/ADR cells presented an original sequestration of DOX in large intracellular vesicles, where SR33557 is itself sequestered, as seen by colocalization studies . Colocalization experiments with lysosomal and mitochondrial probes suggest that these vesicles are neither mitochondrial in nature nor functional lysosomes . To investigate the biochemical basis for this effect, we studied the impact of SR33557 on the sphingolipid metabolism of P388/ADR cells . We observed that although P388/ADR cells normally catabolized exogenous {3H}sphingomyelin, when pretreated with SR33557 they showed almost complete inhibition of sphingomyelin breakdown . Finally, in order to demonstrate that the inability of P388/ADR cells to degrade sphingomyelin in the presence of SR33557 (which is a potent inhibitor of acid lysosomal sphingomyelinase) leads to phospholipid accumulation, we performed electron microscopy where we observed laminated inclusions . These morphological modifications are similar to those observed in Niemann-Pick disease lymphoblastoid cell lines which are inherently deficient in acid sphingomyelinase activity . The observation that, in the absence of SR33557, these Niemann-Pick disease cell lines presented similar DOX sequestration to that of SR33557-treated P388/ADR cells strongly suggests that DOX accumulates in SR33557-induced myeloid bodies . The redistribution of DOX within these vesicles, perhaps by preventing its expulsion by P-glycoprotein, may be a key in discovering the mechanism of action of SR33557.

EMBO J, 1992 Dec, 11(12), 4291 - 303
The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression; Trezise AE et al.; The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities . We have compared their cell-specific expression in the rat by in situ hybridization . In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions . Additionally, a switch in expression from one gene to the other was observed in certain tissues . For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary . A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy . These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume . The patterns of expression suggest that the regulation of these two genes is coordinately controlled.

Pathol Biol (Paris), 1992 Dec, 40(9 Pt 2), 923 - 7
{How do cancers resist to chemotherapy?}; Poupon MF; Resistance is often defined as a lack of therapeutic response . Cellular resistance involves a decrease in intracellular levels of the antitumor agent due to a variety of mechanisms . These mechanisms are active in tumors with initial resistance as well as in those which respond initially but fail to be completely destroyed by chemotherapy . Although acquired forms of resistance seem to be the result of selection, some studies suggest that antitumor agents may induce resistance . Four main mechanisms of resistance are currently being investigated: 1) multidrug resistance, involving expression of a membrane P-glycoprotein, responsible for resistance to hydrophobic cationic agents; 2) detoxification of hydrophilic agents by the enzyme glutathione-S-transferase; 3) increased production of enzymes targeted by antimetabolites; 4) mutation or decreased synthesis of topoisomerases I and II which are the targets of very recent antitumor agents . New data were presented at the 1992 symposium of the American Association for Cancer Research; expression of P-glycoprotein is controlled by the mutant protein P53, the oncogene ras and differentiation agents . Physiological effects of this molecule are related to the chloride pump . Bone marrow stem cells from transgenic mice obtained by transfection of the gene MDR1 in germ cells exhibit resistance . Many agents can reverse P-glycoprotein-related resistance . Results from three phase I trials with Cyclosporin A as reversion agent were reported . It is essential to conduct clinical trials in order to assess the true value of these new data which hold promise for improving the performance of antitumor agents.

Anticancer Drugs, 1992 Dec, 3(6), 641 - 6
SDZ PSC-833--a novel potent in vitro chemosensitizer in multiple myeloma; Jonsson B et al.; Multiple myeloma cell lines and patient tumor samples with and without the expression of the classical multidrug resistance (MDR) phenotype were investigated in vitro for drug induced cytotoxicity and modulation of drug resistance . Overall there was a good correlation in the cell lines between MDR expression, as measured by immunocytochemistry with monoclonal antibodies against P-glycoprotein 170 (Pgp), and in vitro resistance to doxorubicin (dox) and vincristin (vcr) . Drug resistance in the cell line RPMI 8226 dox 40, expressing a high level of Pgp, was almost completely reversed by the novel non-immunosuppressive cyclosporin A (CsA) analog SDZ PSC-833 (PSC), while the chemosensitizers verapamil, CsA and quinine, in clinically achievable concentrations, were much less effective . In cell lines with low Pgp expression, PSC and the other chemosensitizers seem equally effective . The patient tumor samples were selected to represent different combinations of Pgp expression, drug resistance and effects of chemosensitizers . PSC and CsA appeared equally potent and resistance modulation was detected not only in Pgp positive, but also in Pgp negative tumor samples . Furthermore, in one case of a Pgp expression myeloma, chemosensitizers were without effect . These findings indicate the need to incorporate in vitro chemosensitivity assays with Pgp determination when the effects of MDR modulating chemosensitizers are to be studied in the clinic.

Mol Pharmacol, 1992 Dec, 42(6), 1004 - 9
Role of protein kinase C in the modulation of multidrug resistance: expression of the atypical gamma isoform of protein kinase C does not confer increased resistance to doxorubicin; Ahmad S et al.; Cross-resistance to anticancer drugs, termed multidrug resistance (MDR), is functionally associated with the expression of a plasma membrane, energy-dependent, drug efflux pump termed P-glycoprotein (PGP), the product of the mdr1 gene . We have shown previously that MCF-7 breast carcinoma cells transfected with the human mdr1 gene (BC-19 cells) exhibit greater MDR when stably transfected with protein kinase C alpha (PKC alpha) . We now demonstrate that transfection of BC-19 cells with the gamma isoform of PKC (BC-19/PKC gamma cells), which is not normally present in BC-19 cells, does not confer increased resistance to doxorubicin, despite a 19-fold increase in PKC activity . All of the increased PKC activity is accounted for by PKC gamma and it is rapidly down-regulated by phorbol dibutyrate, within 15 min of treatment . Endogenous PKC alpha and PKC epsilon activities are not affected by phorbol dibutyrate . The cytotoxicity of doxorubicin was similar in BC-19/neo or BC-19/PKC gamma cells after either 2-hr or continuous drug exposure, and co-treatment with phorbol dibutyrate increased resistance to doxorubicin 4-fold in both cell lines . Phosphorylation of PGP was similar in both cell lines and drug accumulation was not affected by overexpression of PKC gamma . These results demonstrate that transfection of PGP-expressing cells with an atypical isoform of PKC does not confer increased MDR, and they suggest that the regulation of PGP is phenotype specific with respect to the isoform of PKC.

Biochem Pharmacol, 1992 Dec 1, 44(11), 2259 - 62
Modulation of multidrug resistance gene expression in rat hepatocytes maintained under various culture conditions; Fardel O et al.; P-glycoprotein (P-gp), the multidrug resistance gene product, is overexpressed in normal adult rat hepatocytes under standard culture conditions . We have studied the modulation of P-gp expression in this in vitro model in the presence of both epidermal growth factor and pyruvate, which favor hepatocyte growth, as well as in the presence of either dimethyl sulfoxide (DMSO) or nicotinamide, which favors maintenance of differentiated functions . P-gp overexpression, estimated by northern blotting and doxorubicin-mediated drug efflux analyses, was similarly observed during culture in both standard and proliferating conditions, while it was delayed, but not inhibited, in the presence of DMSO or nicotinamide . These results suggest that the functional P-gp overexpression occurring in rat hepatocytes when exposed to an unfamiliar environment is at least partly not related to cell proliferation or the degree of cell differentiation in vitro.

Leuk Res, 1992 Dec, 16(12), 1165 - 73
The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells; Marks DC et al.; The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs . Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay . In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50 concentrations, although the resistance relative to the sensitive parent cell was correlated . Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.

Curr Opin Oncol, 1992 Dec, 4(6), 1065 - 72
Laboratory and phase I studies of new cancer drugs; Workman P et al.; New drug discovery continues to follow the time-honored paths of screening and novel target identification combined with analogue development and serendipity . The agents selected here reflect these various approaches . They include drugs already showing significant antitumor activity, eg, anthrapyrazoles, temozolomide, camptothecin analogues, and taxotere, and updated information is provided on their development . Other drugs just entering or currently in phase I trials include rhizoxin, which seems capable of overcoming multidrug resistance; the novel bioreductive agent EO9 {corrected}; and bryostatin, a highly potent protein kinase C agonist . Sequence specificity could well prove to be an important factor in the development of DNA-interactive agents, and information is provided on the progress with distamycin mustard and the new cyclopropylpyrroloindole analogues carzelesin and adozelesin.

Surgery, 1992 Dec, 112(6), 981 - 6
P-glycoprotein expression and multidrug resistance in adrenocortical carcinoma; Flynn SD et al.; BACKGROUND . The response of adrenocortical carcinoma (ACC) to adjuvant chemotherapy has been disappointing with no significant impact on survival . The normal adrenal cortex has very high levels of P-glycoprotein, an energy-dependent efflux pump of a variety of structurally unrelated chemotherapeutic agents . P-glycoprotein has been implicated as a cause of multidrug resistance in a variety of neoplasms . The purpose of this study was to evaluate P-glycoprotein expression in ACC . METHODS . Eleven patients with ACC had paraffin-embedded tumor evaluated for P-glycoprotein expression . These were analyzed by immunohistochemistry assay with a battery of four anti-P-glycoprotein antibodies (MRK-16, JSB-1, UIC-2, MDR) . RESULTS . All eleven cases showed intense, predominantly membrane immunoreactivity for P-glycoprotein . In 10 of the cases, most tumor cells were immunoreactive with at least three antibodies, and six of 11 cases were positive for all four antibodies . In this small series no correlation existed between P-glycoprotein expression and tumor grade, stage of disease, or survival . CONCLUSIONS . All 11 cases of ACC studied showed P-glycoprotein expression, which was similar to the normal adrenal cortex . This possible mechanism of multidrug resistance may help explain the significant chemoresistance seen in ACC.

Blood, 1992 Dec 1, 80(11), 2735 - 9
Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes; Chaudhary PM et al.; P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes . We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content . In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL . This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp . We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets . P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells . P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens . No significant P-gp activity was detectable in CD14+ monocytes . MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.

Blood, 1992 Dec 1, 80(11), 2729 - 34
Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype; Drach D et al.; The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells . Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes . To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR) . All subsets were positive by PCR, but only minimal MDR1 mRNA was detected in monocytes and granulocytes . Significant efflux of Rhodamine-123 (Rh-123), a measure of P-gp function, was detected in CD4+, CD8+, CD14+, CD19+, and CD56+ cells but not in granulocytes . Next, PCR-analysis was performed on FACS-sorted bone marrow (BM) cells to assess MDR1 expression in different maturational stages . Precursors (CD34+), early and late myeloid cells (CD33+/CD34+, CD33+/CD34-) as well as lymphocytes of the B-cell lineage (CD19+/CD10+, CD19+/CD10-) expressed the MDR1 gene . BM monocytic cells (CD33++/CD34-) were negative, and a very weak signal was detected in erythroid cells (glycophorin A+) . Significant Rh-123 efflux was found in CD34+, CD10+, CD33+, and CD33++ BM cells, but not in glycophorin A+ cells . We conclude that PB and BM lymphocytes, PB monocytes, BM progenitors, and immature myeloid cells, but not late BM monocytes, erythroid cells, and PB granulocytes, express MDR1 mRNA and a functional P-gp . These results have to be taken into account when MDR1 expression is determined in tumor samples containing normal blood cells.

Cancer Res, 1992 Dec 1, 52(23), 6692 - 5
A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells; Yeh GC et al.; We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump . In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen . We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells . Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil . Our findings suggest that P-gp may play a role in the cellular defense to carcinogens . The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.

Anticancer Drug Des, 1992 Dec, 7(6), 471 - 81
Further examination of 9-alkyl- and sugar-modified anthracyclines in the circumvention of multidrug resistance; Coley HM et al.; Anthracyclines possessing either a 9-alkyl modification in the A-ring of the tetracyclic aglycone and/or specific changes to the amino sugar moiety retain effective cytotoxic activity against multidrug resistant (MDR) cell lines . To obtain a better understanding of the structural features responsible for this potentially valuable behaviour, we used the MTT tetrazolium dye reduction assay to calculate resistance factors (RF = the ratio of ID50 for the drug-resistant line to that for the parental line) for the EMT6/P mouse mammary tumour and its MDR variant EMT6/AR1.0, and the H69/P human small cell lung cancer line and its MDR counterpart H69/LX4 . Both MDR lines exhibit marked resistance to doxorubicin, MDR 1 gene amplification, hyperexpression of the membrane P-glycoprotein and reduced drug accumulation . RF values for doxorubicin were 34 and 131 in the EMT6 and H69 cell line pairs, respectively . The 9-alkyl-substituted anthracyclines were confirmed as having RF values 9- to 15-fold lower than those for doxorubicin . The 9-ethyl analogues Ro 31-1966 (RF for EMT6 2.2, RF for H69 4.7) and Ro 31-1749 (RF for EMT6 3.9, RF for H69 9.5) were superior to the previously studied 9-methyl analogue Ro 31-1215 (RF for EMT6 8.1 RF for H69 12.4) . A clear trend for RF values to decrease with increasing 9-alkyl chain length was also noted in the structurally more complex aclacinomycin series . For example, 13-methyl-aclacinomycin (RF for EMT6 1.0, RF for H69 2.2) featuring a 9-isopropyl moiety was superior to the 9-alkyl-containing aclacinomycin A (RF for EMT6 4.7, RF for H69 5.8), and this was in turn more effective than the 9-methyl analogue sulfurmycin A (RF for EMT6 6.4, RF for H69 14.2) . The trisaccharide moiety was not an essential feature for activity against MDR lines in the aclacinomycins, as shown by the low RF value with aklavine (RF for EMT6 2.1, RF for H69 2.5) . However, a small change in one of the sugar moieties of aclacinomycin A, as in marcellomycin, resulted in a considerable increase in RF values (RF for EMT6 18.5, RF for H69 25.3) . The complex anthracyclines AD 32 (RF for EMT6 6.5, RF for H69 11.7) and particularly tetrahydropyranyl-doxorubicin (RF for EMT6 1.4, RF for H69 3.2) were effective against MDR lines.(ABSTRACT TRUNCATED AT 400 WORDS)

Kobe J Med Sci, 1992 Dec, 38(6), 347 - 63
FK506 reverses adriamycin resistance in a multidrug-resistant human leukemia cell line; Natazuka T; We have examined the effect of FK506 on the Adriamycin sensitivity of the multidrug resistant human chronic myelocytic leukemia cell line (K562/ADM) . In K562/ADM cells, 1.0 microgram/ml FK506 reversed the resistance of Adriamycin, and increased the IC50 value for Adriamycin up to 17 fold . However, IC50 value for the parent cells (K562) increased only 1.5 fold . By cell cycle analysis, the accumulation in late S-G2M phase was confirmed on K562/ADM cells, treated with 1.0 microgram/ml FK506 and low-dose of Adriamycin . Cyclosporin A (CsA) could also restored the Adriamycin sensitivity in the K562/ADM cells, as previously reported . 1.0 microgram/ml FK506 as well as CsA significantly increased radioactive Adriamycin accumulation in K562/ADM cells and blocked {3H}azidopien photoaffinity labeling of P-glycoprotein . These results suggest that 1.0 microgram/ml FK506 could reverse the Adriamycin resistance in a MDR human leukemia cells through the interaction with P-glycoprotein.

Biochem Biophys Res Commun, 1992 Nov 30, 189(1), 92 - 100
Multidrug resistant HOB1 lymphoma cells express P-glycoprotein that does not play the major role in the development of drug resistance to adriamycin; Lee WP et al.; Two cell lines resistant to 0.1 microM vincristine (VCR) and 2.0 microM adriamycin (ADR), respectively, (designated HOB1/VCR0.1 and HOB1/ADR2.0) were established from a human immunoblastic B lymphoma cell line . These cell lines showed the typical MDR phenotype with overexpression of P-glycoprotein and decreased {3H}VCR accumulation . The retention amounts of intracellular {3H}VCR in these two cell lines could be augmented by verapamil . However, in spite of the overproduction of P-glycoprotein, both HOB1/VCR1.0 and HOB1/ADR2.0 cells did not exhibit decreased accumulation of intracellular {14C}ADR . And the retention of {14C}ADR was not affected by verapamil . Our data support that P-glycoprotein is a drug transporter more important for the development of drug resistance to VCR than to ADR.

Biochem Biophys Res Commun, 1992 Nov 30, 189(1), 551 - 7
The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin; de Lannoy IA et al.; Digoxin, a widely used cardiac glycoside with a low therapeutic index, is known to interact with a large and diverse group of co-administered drugs, frequently leading to toxic accumulation of the glycoside . Establishing the mechanism(s) of these interactions, therefore, has potential clinical significance . The present studies implicate P-glycoprotein, the MDR1 gene product overexpressed in multidrug resistant cells, as the apical membrane protein responsible for the renal secretion of digoxin and provide an explanation for the occurrence of digoxin toxicity in the presence of certain co-administered medications . Since digoxin is considered a prototype for endogenous digitalis-like glycosides, the results also allow for speculation that endogenous digitalis-like glycosides may be the natural substrates for P-gp.

J Mol Biol, 1992 Nov 20, 228(2), 701 - 11
The P-glycoprotein gene family of Caenorhabditis elegans . Cloning and characterization of genomic and complementary DNA sequences; Lincke CR et al.; P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells . To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively . The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4) . Phenotypic mutants corresponding to these loci have not yet been described . Two of the genes, pgp-1 and pgp-3, were analyzed in detail . They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far . Intra-species divergence of P-glycoprotein genes is more pronounced in C . elegans than in mammals . Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3 . pgp-1 consists of 14 exons, pgp-3 of 13 . The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes . pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes . pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends . Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types . Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA . Thus, in C . elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.

Cancer Res, 1992 Nov 15, 52(22), 6385 - 9
Structural requirements of simple organic cations for recognition by multidrug-resistant cells; Dellinger M et al.; We previously noted that a wide variety of drugs which are recognized by multidrug-resistant cells (MDR+) are positively charged . However, it remains unclear why and how such a large number of structurally different compounds can be distinguished by MDR+ cells . The majority of the diverse compounds subject to MDR are complex and thereby complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism . Using a series of simple aromatic (alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations differing in their lipophilicity by stepwise additions of single alkyl carbons, we demonstrate by growth inhibition studies that a single aromatic moiety and a critical degree of lipophilicity (log P > -1) are required for recognition of these simple organic cations by MDR+ cells . Thus, MDR+ cells are not cross-resistant to the nonaromatic guanidiniums but do show cross-resistance to those aromatic pyridiniums with chain lengths > four . Resistance ratios, as determined by comparison of 50% inhibitory doses in MDR- versus MDR+ cells, increase as a function of increasing chain lengths of these latter simple aromatic compounds . Resistance to pyridinium analogues in MDR+ cells is reversible by co-treatment with nontoxic doses of verapamil . Preliminary uptake data with radioactive analogues further implicate the MDR mechanism of lowered drug accumulation in accounting for resistance to the pyridinium homologues . Utilization of these simple organic cations provides a rational basis for better defining the physical chemical properties of more complex compounds processed by the MDR mechanism and suggests a strategy for designing chemotherapeutic agents with reduced susceptibility to MDR.

Cancer Res, 1992 Nov 15, 52(22), 6175 - 81
Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells; Nakagawa M et al.; A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug . MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine . Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells . This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process . Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells . Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro . However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT . Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells . Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.

Cancer, 1992 Nov 15, 70(10), 2402 - 9
Different sensitivities of human esophageal cancer cells to multiple anti-cancer agents and related mechanisms; Saito T et al.; Mechanisms responsible for drug resistance in human esophageal cancer cell lines were investigated . Three cell lines established from human esophageal carcinoma (TE-1, SH-1, and TH) showed different sensitivities to vindesine, vincristine, cisplatin (CDDP), etoposide (VP-16), and pepleomycin . Both SH-1 and TH cell lines were twofold to sevenfold more resistant to pepleomycin, vindesine, and vincristine than TE-1 was . SH-1 showed twofold more resistance to CDDP than either TE-1 or TH did, and TH and TE-1 showed a 3-fold or 1.5-fold more resistance, respectively, to VP-16 than SH-1 did . The accumulation of tritiated vincristine in SH-1 and TH was approximately 50% that in TE-1 . Two multidrug resistance reversal agents, cepharanthine and a synthetic dihydropyridine analogue (NK-252; Nikken Chemicals, Saitama, Japan), potentiated the cytocidal actions of vindesine against SH-1, TH, and TE-1 cells, with no apparent expression of P-glycoprotein in the three cell lines . The glutathione S-transferase pi gene was expressed in all three cell lines . DNA topoisomerase II levels were lowest in TE-1, followed by SH-1 and TH, although the accumulation of tritiated VP-16 was less in both TH and SH-1 than in TE-1 . Differential sensitivities to anti-cancer drugs appear to be mediated through pleiotropic mechanisms.

Biochim Biophys Acta, 1992 Nov 15, 1171(1), 65 - 72
P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of two types of genomic clones carrying 3' terminal exons; Chan KM et al.; In mammals, P-glycoprotein (P-gp) is encoded by two or more highly conserved genes that differ in their abilities to transport drugs . One isoform class (class I) is consistently associated with the multidrug resistance phenotype, while the other (class III) is not . This study was designed to enumerate the P-gp genes in fish and determine how they are related to the two functional classes already defined in mammals . Southern blot analysis using a conserved single exon from the 3' terminal region of hamster P-gp cDNA (pEX1-172) as a probe indicated that there were two P-gp genes in right-eye flounders . Subsequently, two sets of clones were isolated from a winter flounder genomic library that correspond to the 3' ends of the two flounder P-gp genes . Sequence analysis was done on two key areas: the 3' ATP binding site and the 3' terminal exon, both of which were found to be homologous with their mammalian counterparts . Despite high levels of sequence identity in the predicted coding regions of the gene fragments it has not been possible to use these sequences to relate the homologs to particular mammalian classes of P-gp genes, perhaps because of gene conversion between mammalian P-gp genes . These cloned sequences are the first set of P-gp genes reported in lower vertebrates and will be useful for delineating the expression of P-gp genes in fish and understanding the role of P-gp in fish physiology.

Med Hypotheses, 1992 Nov, 39(3), 229 - 37
The Na+/H+ antiporter in oncology in the light of the spontaneous regression of cancer and cell metabolism; Harguindey S et al.; Multiple metabolic and biochemical interrelationships, as well as the most recent views on mechanisms of malignant cell growth, proliferation, and oncogen activity mediated by the Na+/H+ antiporter, can be integrated from the unitarian point of view of the dynamics of the hydrogen ion to parallel pH-related mechanisms involved in the Spontaneous Regression (SR) of some malignant tumors . Also, pH-related growth inhibitors of the amiloride series are considered as possible agents to be used in the adjuvant and co-adjuvant treatment of some human tumors as well as in the control of the metastatic process and in overcoming cancer multidrug resistance (MDR).

Int J Cell Cloning, 1992 Nov, 10(6), 359 - 68
Pharmacologic validation of human tumor clonogenic assays based on pleiotropic drug resistance: implications for individualized chemotherapy and new drug screening programs; Parchment RE et al.; Because of the bias toward successful cloning of human tumor cells from more advanced malignancies, alternative approaches to clinical correlations of drug resistance are needed to determine the validity of the human tumor clonogenic assay (HTCA) as a clinically useful test . Capitalizing on the prevalence of clinical drug resistance among these advanced malignancies, we have taken an independent approach to testing the validity of HTCAs based upon pharmacologic principles rather than tumor response . A database of results from drug sensitivity/resistance testing in 1,777 HTCAs has been examined retrospectively for specimens exhibiting either the MDR1 or Topo-II pleiotropic drug resistance phenotype . Twenty specimens were identified as MDR1 based upon test results showing resistance to adriamycin, vinca alkaloid, and etoposide . Test results with mitomycin-c confirmed the MDR1 phenotype in eight out of nine of these specimens . Seven out of eight of the confirmed MDR1 samples were resistant to either cis-platinum or alkylating agents or to both . There was no significant difference in the 5-fluorouracil resistance of these MDR1 specimens and the database as a whole, demonstrating the specific nature of this drug resistance phenotype in vitro . One specimen, a squamous carcinoma of the lung, was mitomycin-c sensitive, even though it exhibited all the other drug resistances characteristic of the MDR1 phenotype . Six specimens with the Topo-II phenotype were identified based upon resistance to adriamycin and etoposide with sensitivity to vinca alkaloids . Surprisingly, the Topo-II phenotype showed a strong association with increased cis-platinum resistance and a weaker one with decreased 5-fluorouracil resistance . Thus, 26/30 (87%) of analyzable specimens showed some form of clinically characterized multidrug resistance, illustrating how easily one can obtain 90% accuracy in predicting clinical drug resistance with HTCAs that are heavily biased by a disproportionate number of successful cloning assays with advanced malignancies . The data analysis also shows that prediction of adriamycin resistance based on lack of Topoisomerase II expression will not be very accurate, in contrast to a previous claim . Until cell culture technology can facilitate frequent successes in the cloning of early detected, drug-sensitive lesions, this bias will remain in HTCA databases, and studies comparing HTCA results with clinical response will continue to be uninformative . However, the in vitro identification of pleiotropic drug resistance phenotypes exactly analogous to those previously observed in patients provides pharmacologic validation at least for the prediction of drug resistance as measured by current HTCAs using suprapharmacologic drug concentrations.

Nucleic Acids Res, 1992 Nov 11, 20(21), 5811 - 7
Purification and characterization of NF-R1 that regulates the expression of the human multidrug resistance (MDR1) gene; Ogura M et al.; We have purified a protein, NF-R1, that specifically binds to two unrelated motifs--the ATTCAGTCA motif and the GC-box motif--on the MDR1 proximal promoter . Purified NF-R1 has been confirmed by southwestern blotting to be a 110-kDa protein . Methylation interference analysis revealed that the nucleotides were in close contact with purified NF-R1 on the ATTCAGTCA and GC-box motifs . The nucleotides were required for the binding of NF-R1, as seen by competition gel mobility shift assay using point mutated oligonucleotides . In a CAT expression assay using the corresponding point-mutated MDR1 promoter fused to a CAT gene, binding inhibition of NF-R1 to the promoter resulted in 2- to 3-fold increases of CAT activity, as compared to the intact promoter in Adriamycin-resistant K562 cells . Thus NF-R1 has a relation to the negative regulation of the MDR1 gene transcription.

J Natl Cancer Inst, 1992 Nov 4, 84(21), 1653 - 60
Effects of verapamil on the acute toxicity of doxorubicin in vivo; Sridhar R et al.; BACKGROUND: Studies indicating that verapamil substantially enhances doxorubicin levels in certain drug-resistant tumor cells have led to the use of verapamil in combination with doxorubicin in animal and clinical studies of multidrug-resistant tumors . These studies have shown this drug combination to be associated with severe toxic effects . It is important to determine whether verapamil modulates the dose-limiting and potentially lethal cardiotoxicity of doxorubicin and to elucidate possible mechanisms . PURPOSE: The aims of this study were to evaluate the in vivo effects of verapamil on (a) doxorubicin-stimulated cardiac lipid peroxidation and cardiac damage, (b) doxorubicin-induced animal mortality, and (c) biodistribution of doxorubicin to the heart . METHODS: Male (BALB/c x DBA/2)F1 mice were treated with a high dose of doxorubicin (15 mg/kg, injected intraperitoneally), verapamil (25 mg/kg, injected intraperitoneally), or combinations of the two . Lipid peroxidation was determined using the 2-thiobarbituric acid assay for malonaldehyde . Light microscopy was used for histopathologic examination of cardiac tissue . A fluorometric assay procedure was employed to determine doxorubicin levels in the heart . RESULTS: Verapamil was an effective inhibitor of peroxidative damage to myocardial lipids following a high dose of doxorubicin (15 mg/kg, injected intraperitoneally) . However, mice treated with verapamil and doxorubicin had a lower survival rate and a higher initial peak concentration of doxorubicin in the heart than those treated with doxorubicin alone . They also demonstrated a higher incidence and severity of degenerative changes in cardiac tissue . CONCLUSIONS: Our findings suggest that verapamil effectively inhibits doxorubicin-mediated lipid peroxidation in vivo but that cardiac lipid peroxidation is not the major limiting mechanism underlying doxorubicin-induced toxicity . A possible explanation for the excess mortality and cardiac injury in mice treated with verapamil plus doxorubicin is that verapamil alters the pharmacokinetics of doxorubicin . IMPLICATIONS: Further studies are necessary for development of safer protocols and/or drug combinations to treat multidrug-resistant tumors . We are currently studying treatment of tumor-bearing animals with a cumulative dosage regimen of doxorubicin in the presence and absence of verapamil.

Biochem Pharmacol, 1992 Nov 3, 44(9), 1869 - 77
An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8); Zhang Y et al.; Multidrug-resistant LZ-8 cells are 9000-fold more resistant to Adriamycin (ADRM) exposure than wild-type V79 cells . To understand more about the mechanisms producing such high level resistance, we tested whether LZ-8 cells inactivate ADRM toxicity to a greater extent than wild-type V79 cells . ADRM was recovered from (1) culture media of wild-type V79 and ADRM-resistant LZ-8 cells; (2) V79 and LZ-8 cells; and (3) LZ-8 cell plasma membrane, and the cytotoxicity was determined by treating V79 cells for 1 hr with a known concentration of the recovered ADRM . ADRM obtained from LZ-8 cells or its culture medium exhibited less cytotoxicity than that recovered from V79 cells or its culture medium . ADRM extracted from LZ-8 cell plasma membrane was noncytotoxic . HPLC analysis revealed that the extracted ADRM was structurally changed compared to stock ADRM . The retention time in the column was 7 min for stock ADRM, and 23 min for the recovered ADRM . Thus, LZ-8 cells have an increased ability to transform ADRM into a noncytotoxic form compared to wild-type V79 cells . This transformation involves structural conversion into a previously unidentified ADRM metabolite . The greatly increased survival of LZ-8 cells compared to V79 cells after ADRM treatment is due to at least two mechanisms: (1) an enhanced ability to inactivate the cytotoxicity of ADRM, and (2) increased drug efflux resulting from the amplification and overexpression of the pgp 1 gene in these cells . Our results suggest the possibility that P-glycoprotein participates in drug binding/inactivation in addition to serving as a drug efflux pump.

Biochem Pharmacol, 1992 Nov 3, 44(9), 1859 - 68
Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines; Sognier MA et al.; A series of cell lines derived from Chinese hamster V79 cells by selection in increasing concentrations of Adriamycin (ADRM) was developed to study the mechanisms of drug resistance and its relationship to radiation response . Survival studies revealed that selection in increasingly higher concentrations of ADRM positively correlated with increased cellular drug resistance . Increased cellular resistance correlated positively with amplification of the hamster multidrug-resistance gene (pgp 1) as detected with dot blot analysis using the pCHP1 probe . Southern blot analysis of restriction endonuclease digested DNA (Eco RI, Hind III, Pst I, or Bam HI) showed that (1) some fragments were preferentially amplified compared to others in the ADRM-resistant lines; and (2) no major gene rearrangement appeared to have occurred during the selection for greater ADRM resistance . Levels of pgp 1 gene expression assayed with dot blot and Northern analysis showed a parallel increase of mRNA with gene amplification and increased ADRM resistance . The amounts of the pgp 1 gene product, P-glycoprotein (P-gp), in the cell membrane of the ADRM-resistant cells correlated with the amount of gene amplification/expression . However, levels of P-gp only correlated with degree of drug resistance as measured by cell survival in earlier selection stages (77A and LZ-3) . In later selection stages (LZ-8 and LZ-24), higher levels of ADRM resistance were achieved but levels of P-gp did not increase beyond approximately 20% of plasma membrane proteins . These results suggest that (1) the LZ cell plasma membrane may have a physical limit as to the amount of P-gp it can accommodate and/or there is a cellular mechanism for regulating the amount of P-gp in the plasma membrane, and (2) additional resistance mechanisms are present in LZ-8 and LZ-24 cells . Microscopic observations of intracellular drug distribution in these cell lines revealed that (1) ADRM appeared to be sequestered in cytoplasmic vesicles, and (2) the amount of sequestration (number of vesicles) exhibited correlated with the degree of drug resistance attained by the cell lines . These results suggest that drug sequestration is another mechanism of resistance in LZ cells in addition to P-gp-mediated drug efflux.

Biochem Pharmacol, 1992 Nov 3, 44(9), 1707 - 15
Effects of a new triazinoaminopiperidine derivative on adriamycin accumulation and retention in cells displaying P-glycoprotein-mediated multidrug resistance; Leonce S et al.; A new triazinoaminopiperidine derivative, Servier 9788 (S9788), was investigated for its ability to increase Adriamycin (ADR) accumulation and retention in two rodent (P388/ADR and DC-3F/AD) and three human (KB-A1, K562/R and COLO 320DM) cell lines displaying the P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) phenotype . Depending on the cell line S9788 was shown to be two to five times more active and five to 15 times more potent than Verapamil (VRP) in increasing ADR accumulation in resistant cells . ADR retention in KB-A1 cells maintained in a concentration of 10 microM S9788 was twice that in VRP-treated cells, and similar to that measured in the untreated sensitive KB-3-1 cells . Although 5 microM S9788 and 50 microM VRP gave the same values of ADR uptake in KB-A1 cells, S9788 was shown to induce a greater ADR retention following cell wash and post-incubation in resistance modifier- and ADR-free medium . Taking into account that S9788 had no effects on ADR accumulation and retention in sensitive KB-3-1 cells, it can be suggested that S9788 inhibits specifically the P-gp dependent ADR efflux, and in a manner less reversible than that observed with VRP . Moreover, {3H}azidopine photolabeling of P-gp, in P388/ADR plasma membranes, was completely inhibited by 100 microM S9788 . Although S9788, as VRP, had no effect on the cell cycle of P388 cells, 5 microM S9788 increased 700-fold the efficacy of ADR to block P388/ADR cells in the G2+M phase of the cell cycle . Together, these results show that the sensitization, by S9788, of cell lines resistant to ADR is mainly due to an increase in ADR accumulation and retention, leading to an increase in the number of resistant cells blocked in the G2+M phase.

Hinyokika Kiyo, 1992 Nov, 38(11), 1319 - 24
{Circumvention of the intrinsic multidrug-resistance in renal cell carcinoma}; Kakehi Y et al.; Most of renal cell carcinomas (RCCs) are refractory at the start of chemotherapy . We have demonstrated that the frequently elevated expression of the multidrug resistance gene (MDR) in RCCs is associated with the intrinsic vinca alkaloids and anthracyclines resistance . The preliminary clinical trials using verapamil or amiodarone in combination with vinblastine or doxorubicin to overcome multidrug-resistant (MDR) tumors could not achieve satisfactory results owing to severe cardiovascular toxicities of such reversing agents . In the present study, we studied the sensitizing ability of bis-benzyl-isoquinoline (cepharanthine) and SDB-ethylenediamine (N-1379) in natural MDR kidney cancer cells . Cepharanthine remarkably sensitized vinblastine and doxorubicin sensitivities in those cells with high MDR RNA levels . From a clinical point of view, cepharanthine seems to be a potent and less toxic agent to treat natural MDR kidney cancers.

Public Health Rep, 1992 Nov-Dec, 107(6), 616 - 25
The multidrug-resistant tuberculosis challenge to public health efforts to control tuberculosis; Villarino ME et al.; After years of steady decline, there has been an unprecedented resurgence of tuberculosis (TB) in the United States and outbreaks of multidrug-resistant tuberculosis (MDR-TB) . The authors assess the nature, epidemiology, and implications of MDR-TB; provide suggestions for preventing drug resistance among patients with drug-susceptible TB; and offer recommendations for managing patients with MDR-TB . They outline the National Action Plan to Combat MDR-TB . Close collaboration among medical practitioners and staff members of TB control programs is needed to ensure the most effective management of patients with TB and their contacts . This collaboration is one of the most important steps for successful control of MDR-TB.

Pediatr Infect Dis J, 1992 Nov, 11(11), 950 - 5
Mycobacterium tuberculosis in children with human immunodeficiency virus type 1 infection; Khouri YF et al.; A retrospective study was conducted at the Childrens Hospital Center at Jackson Memorial Hospital in Miami, FL, to evaluate the natural history of Mycobacterium tuberculosis infection in nine children with vertically acquired human immunodeficiency virus type 1 infection . The patients' ages ranged from 6 months to 7 years (median age, 42 months) . Common presenting symptoms included prolonged fever, cough and anorexia . Only one patient had a positive tuberculin test . Five patients evidenced only pulmonary disease, three patients had pulmonary and extrapulmonary disease and one patient developed extrapulmonary tuberculosis (mastoiditis) and pulmonary interstitial disease that could not be attributed to mycobacterial infection because of lack of information . Organisms isolated before January, 1989, were susceptible to isoniazid and rifampin whereas isolates from three patients cultured after that time were resistant to multiple antituberculosis drugs . The median survival time after M . tuberculosis diagnosis for all children was 20 months . Our study suggests that children with human immunodeficiency virus type 1 infection who have tuberculosis have an increased risk for extrapulmonary disease . A high index of suspicion for the diagnosis of M . tuberculosis should be maintained in human immunodeficiency virus type 1-infected children with prolonged fever and respiratory symptoms . In areas of high endemicity of multidrug-resistant organisms, therapy with a broader panel of drugs may need to be instituted until susceptibility testing becomes available.

Anticancer Res, 1992 Nov-Dec, 12(6B), 2127 - 32
Lack of reversal of daunorubicin resistance in HL60/AR cells by cyclosporin A; Gollapudi S et al.; Cyclosporin A and verapamil are substrates for P-glycoprotein . Both agents are known to reverse multidrug resistance in cells overexpressing P-glycoprotein . In this investigation, we have examined the effects of cyclosporin A and verapamil on multidrug resistance in HL60/AR cells that lack P-glycoprotein . In addition, a correlation was sought between an alteration in plasma membrane potential as measured with cationic dye DIOC5 and overexpression of P-glycoprotein . HL60/AR cells accumulated 3 fold less daunorubicin than HL60 cells . The drug accumulation defect and drug resistance in HL60/AR cells were partially corrected by verapamil and buthionine sulfoximine . However, cyclosporin A had no detectable effect on daunorubicin accumulation or drug resistance in HL60/AR cells . The multidrug resistant P338/ADR cell line overexpressed P-glycoprotein and exhibited depolarization of plasma membrane when compared to its corresponding drug sensitive parental cell line . In contrast, HL60/AR cells lacked P-glycoprotein and plasma membrane potentials were similar to those of drug sensitive HL60 cells . These results suggest that {1} verapamil modulates daunorubicin transport by a mechanism independent of P-glycoprotein, {2} the mechanisms of reversal of multidrug resistance by verapamil and cyclosporin A are distinct, and {3} the plasma membrane depolarization in multidrug resistant cell lines that overexpress P-glycoprotein, as determined by DIOC5, may be due to an increased efflux of cationic dye by P-glycoprotein, rather than a true measurement of plasma membrane potential in multidrug resistant cells.

Anticancer Res, 1992 Nov-Dec, 12(6B), 2021 - 4
Increase of vinblastine accumulation by inhibitors of calmodulin-dependent cell functions in rat ascites hepatoma AH66 cells; Wakusawa S et al.; ML-9, an inhibitor for myosin light chain kinase, and W-7, a calmodulin inhibitor, suppressed the efflux of vinblastine and increased the intracellular accumulation of vinblastine, but W-5, an inactive compound for calmodulin, did not so in rat ascites hepatoma AH66 cells, which have a multidrug-resistant phenotype . In sensitive counterpart AH66F cells, W-7 and ML-9 were less effective . W-7 and ML-9 did not interfere with {3H}azidopine photolabeling of P-glycoprotein in the plasma membrane from AH66 cells . While P-glycoprotein is reported to be superphosphorylated by protein kinases, W-7 did not influence the phosphorylation of the P-glycoprotein in AH66 cells . There may be an unknown Ca(2+)-calmodulin-dependent mechanism in the extrusion of vinblastine from AH66 cells.

Leuk Lymphoma, 1992 Nov, 8(4-5), 261 - 5
Multidrug resistance (MDR) gene expression in acute non lymphoblastic leukemia: sequential analysis; Marie JP et al.; Sequential evaluation of P-glycoprotein expression was performed in 29 patients with acute nonlymphoblastic leukemia using immunocytochemistry with the C219 antibody . At diagnosis, 32% of the patients exhibited more than 5% of the P-gp(+) leukemic cells . Under chemotherapy, 62% of the patients eventually expressed a subset of P-gp positive leukemic cells . After conventional doses of cytosine-arabinoside (Ara-C) and daunorubicin or mitoxantrone, positive P-gp cells were noted in 65% of the cases . This percentage was significantly higher (p = 0.002) than the proportion of positive cases (15%) observed after regimens containing either intermediate doses of Ara-C or cyclosporine A, a P-gp modulator.

Haematologica, 1992 Nov-Dec, 77(6), 470 - 2
Cancer chemotherapy does not enhance MDR-associated 170 kd glycoprotein expression in normal blood mononuclear cells; Geromin A et al.; BACKGROUND . The multidrug resistance (MDR) associated 170kd glycoprotein (P170) is expressed at a low level in normal and malignant cells, but in the latter it becomes frequently overexpressed after chemotherapy . This study evaluated P170 expression in normal blood mononuclear cells after and during cancer chemotherapy . METHODS . P170 was detected by immunocytochemistry with two monoclonal antibodies (MRK-16 and JSB-1) . RESULTS . P170 expression was low in all samples before, during and after chemotherapy . CONCLUSIONS . Cancer chemotherapy did not enhance P170 expression in normal peripheral blood mononuclear cells . The mechanisms enhancing P170 expression are likely to be more operative in tumor cells than in normal cells.

J Clin Immunol, 1992 Nov, 12(6), 451 - 8
Preferential expression and activity of multidrug resistance gene 1 product (P-glycoprotein), a functionally active efflux pump, in human CD8+ T cells: a role in cytotoxic effector function; Gupta S et al.; The multidrug resistance gene 1 (mdr 1) product, the P-glycoprotein (Pgp), is a 170-kD transmembrane transport protein, whose overexpression is associated with multidrug resistance in cancer cells and in chloroquine-resistant Plasmodium falciparum infection . In this study we show that normal freshly isolated human lymphocytes express low levels of mdr 1 mRNA and membrane Pgp . Although Pgp is expressed in both CD4+ and CD8+ T cells, it is preferentially expressed in CD8+ T cells . Activation of T lymphocytes with phytohemagglutinin leads to an amplification of both mdr 1 mRNA and membrane Pgp in T cells . P-glycoprotein in T cells is a functionally active efflux pump as demonstrated by decreased retention of rhodamine-123 and its increased accumulation by cyclosporin A, an inhibitor of Pgp function . In addition, MRK-16 antibody increased accumulation of Rh123 in CD8+ T cells . Furthermore, MRK16 anti-P-glycoprotein monoclonal antibody, in a concentration-dependent manner, inhibited T lymphocyte-mediated cytotoxicity . These data suggest a physiologic role of P-glycoprotein in cytotoxic T-lymphocyte effector function.

Am J Pathol, 1992 Nov, 141(5), 1063 - 72
Multidrug resistance gene (P-glycoprotein) expression in the human fetus; van Kalken C et al.; P-glycoprotein, a transmembrane protein associated with multidrug resistance in cancer cells, is also expressed in normal tissues . To get more insight into the physiologic role of mdr1/P-glycoprotein, we investigated its expression in human fetal tissues after 7 to 38 weeks of gestation by an immunohistochemical technique, using three different monoclonal antibodies, and by a sensitive RNAse protection assay . Expression of mdr1-mRNA could already be demonstrated in the embryonal phase of human development, after 7 weeks of gestation . Comparing the adult with the fetal tissue distribution, differences were found in specific organs, such as adrenal, intestine, respiratory epithelium, and brain capillaries . In the fetal zone cells of the fetal adrenal cortex no staining was observed by immunohistochemistry, whereas the definitive zone showed increasing expression throughout gestation . Prenatal intestine did not show staining of the epithelium, although definite mdr1-mRNA expression was observed in late specimens . Interestingly, respiratory epithelium of main bronchi and pharynx, not expressing P-gp in adults, did stain positive . Expression of P-gp in brain capillaries was not observed before the third trimester of pregnancy, whereas in kidney and liver, mdr1-mRNA expression and staining for P-glycoprotein were detected in early fetal life (11 to 14 weeks) . These findings suggest a pivotal role of P-glycoprotein in physiology of various organs already in early phases of human development and may help to identify its physiologic substrates.

Br J Cancer, 1992 Nov, 66(5), 833 - 9
High-dose tamoxifen as an enhancer of etoposide cytotoxicity . Clinical effects and in vitro assessment in p-glycoprotein expressing cell lines; Stuart NS et al.; Twenty-six patients with relapsed or drug-resistant cancer were treated with a combination of oral etoposide (300 mg day-1 for 3 days) and high-dose oral tamoxifen as a potential modulator of drug resistance (480 or 720 mg day-1 for 6 days beginning 3 days before etoposide) . One patient with relapsed high-grade lymphoma and one with adenocarcinoma of unknown primary site has a partial response . Toxicity consisting of nausea, vomiting and subjective dizziness, unsteadiness of gait and malaise occurred during tamoxifen treatment . Serum levels of tamoxifen averaged 3-3.5 microM on day 4 of all courses of treatment at both 480 and 720 mg day-1 . N-desmethyltamoxifen levels were lower than tamoxifen during the first course (2 microM) but increased to equal tamoxifen levels during the second course . Didesmethyltamoxifen levels remained below 1 microM . In vitro, both tamoxifen and the standard modulator of multidrug resistance, verapamil, produced minor enhancement of etoposide cytotoxicity in the MCF-7 wt cell line but produced no enhancement with any other cell line . High, intermittent doses of tamoxifen can be given with acceptable toxicity and produce serum levels that have been shown to modulate drug resistance in vitro . In vitro, however, such levels have no significant effect on etoposide cytotoxicity towards a range of wild-type and MDR cell lines.

J Clin Oncol, 1992 Nov, 10(11), 1730 - 6
Feasibility of using quinine, a potential multidrug resistance-reversing agent, in combination with mitoxantrone and cytarabine for the treatment of acute leukemia; Solary E et al.; PURPOSE: We demonstrated previously that sera from quinine-treated patients reversed the multidrug resistance (MDR) of a human leukemic cell line . We now report a phase I and II clinical study that examined the toxicity of the combination of quinine with mitoxantrone and cytarabine (Ara-C) . PATIENTS AND METHODS: Fifteen adult patients with relapsed or refractory acute leukemia were treated with quinine formiate (30 mg/kg/d in continuous intravenous (IV) infusion from day 1 through day 5 or 6) associated with Ara-C (1 g/m2 in 3-hour IV infusion twice a day for 5 days) and five increasing doses of mitoxantrone (from 8 mg/m2/d for 4 days to 12 mg/m2/d for 5 days) . RESULTS: The main toxicity was severe myelosuppression: the mean times to leukocyte recovery (> 500/microL), granulocytes recovery (> 500/microL), and platelet count recovery (> 50,000/microL) were 23 days (range, 17 to 29 days), 30.6 days (range, 17 to 48 days), and 35.4 days (range, 14 to 75 days), respectively . The nonhematopoietic toxicity of this regimen was acceptable . Nausea and vomiting were common, but severe mucositis was observed in only two patients . Cardiotoxicity was limited to transient episodes of moderate supraventricular tachycardia and a clinically well-tolerated bradycardia . Tinnitus and vertigo were observed in 10 cases (67%), and mild hearing loss and transient increase of serum bilirubin were observed in six patients (40%) . Total quinine serum levels reached a steady-state concentration between 6.4 and 18 mg/L in 24 hours . Complete remission (CR) was achieved in eight of 14 (57%) assessable patients, and partial response (PR) was achieved in two additional patients (14%) . P-glycoprotein expression was detected on blast cells from five of 13 studied patients before treatment . A response was observed in all P-glycoprotein-positive cases . CONCLUSION: Quinine can be used safely as a potential reversing agent of MDR for the treatment of clinically resistant acute leukemias.

Cancer Res, 1992 Nov 1, 52(21), 5893 - 9
Decreased P-glycoprotein expression in multidrug-sensitive and -resistant human myeloma cells induced by the cytokine leukoregulin; Evans CH et al.; Modulation of the expression of P-glycoprotein, a plasma membrane protein associated with multidrug resistance, was examined in drug-sensitive and drug-resistant tumor cells treated with leukoregulin, a M(r) 50,000 cytokine from human lymphocytes that rapidly permeabilizes the plasma membrane of many tumor cells facilitating the uptake of doxorubicin and other tumor-inhibitory antibiotics . P-glycoprotein expression was measured flow cytometrically by the binding of C219 or MRK16 monoclonal antibody to multidrug-sensitive human K562 erythroleukemia and 8226/S myeloma cells, compared to multidrug-resistant 8226/DOX40 myeloma cells . Cells were treated for up to 2 h with up to 80 units of leukoregulin/ml or one of a variety of unrelated cytokines including interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, colony-stimulating factor, macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, tumor necrosis factor alpha, gamma-interferon, alpha-interferon, epidermal growth factor, platelet-derived growth factor AA, platelet-derived growth factor BB, insulin-like growth factor I, insulin-like growth factor II, fibroblast growth factor, or transforming growth factor beta . Leukoregulin caused a concentration-dependent decrease in P-glycoprotein expression; however, P-glycoprotein expression was unaffected by the other cytokines (< 12% decrease in expression) . Leukoregulin-induced membrane permeabilization, determined flow cytometrically by intracellular fluorescein efflux, and decreased P-glycoprotein expression occurred simultaneously within 15 min in drug-sensitive and -resistant cells . Enhanced doxorubicin uptake, measured flow cytometrically by doxorubicin influx, was also present within 15 min . Leukoregulin enhancement of doxorubicin uptake and increased membrane permeability varied directly with the decrease in P-glycoprotein expression . Leukoregulin in combination with doxorubicin enhanced the inhibition of cell proliferation in 8226/DOX40 multidrug-resistant cells over expressing P-glycoprotein . In contrast, combined treatment of HL-60/MX2 multidrug-resistant human promyelocytic leukemia cells that do not overexpress P-glycoprotein in association with their multidrug resistance resulted in no greater growth inhibition than observed with HL-60/MX2 cells treated with doxorubicin alone . This is the first demonstration that a naturally occurring macromolecule with anticancer activities can modulate the expression of P-glycoprotein concomitant with enhanced drug uptake and inhibition of cell proliferation.

Bull Math Biol, 1992 Nov, 54(6), 1023 - 38
A mathematical model of the P-glycoprotein pump as a mediator of multidrug resistance; Michelson S et al.; Cells displaying the classic multidrug resistant (MDR) phenotype possess a transmembrane protein (p170 or P-glycoprotein) which can actively extrude cytotoxic agents from the cytoplasm . A mathematical model of this drug efflux pump has been developed . Outward transport is modeled as a facilitated diffusion process . Since energy-dependent efflux of cytotoxic agents requires that ATP also bind to p170, the model includes a dynamic calculation for efflux rate which considers Michaelis-Menten kinetics for both the substrate agent and ATP . The final system consists of one partial differential equation (PDE) for the facilitated diffusion of substrate agents out of the cell, a 2 x 2 ordinary differential equation (ODE) system for the dynamic calculation of the ATP-ADP pool, and a dynamic algebraic calculation of the efflux rate given substrate levels at the interior cell membrane interface and ATP levels in the cell . A stability analysis of the ATP-ADP pool distribution and a simplistic closed form solution of the linearized PDE are included . Numerical simulations are also provided.

Anticancer Res, 1992 Nov-Dec, 12(6B), 2253 - 6
Beta-adrenergic influences on doxorubicin-sensitive or -resistant P388 leukemia cells; D'Amico C et al.; Taking into account the possible regulatory influences of the beta-adrenergic system on lymphocyte proliferation as well as the proposed role of cyclic 3'-5'-adenosine monophosphate (cAMP) in the modulation of multidrug resistance (MDR) in tumour cells, we have tried to assess the status of the interactions between the beta-adrenergic system and a mouse lymphocytic leukemia, the P388, both as a doxorubicin-sensitive (P388) and -resistant (MDR) variant (P388/DXR) . P388 showed a low total number of high affinity 125I-pindolol binding sites (340 +/- 33/cell, Kd 108 pM) when compared with normal splenocytes (1221 +/- 67 sites/cell, Kd 97 pM) . The number of beta-adrenergic receptors was even lower in P388/DXR cells (230 +/- 41 sites/cell, Kd 101 pM) . In addition, these receptors were subnormally expressed on the cell surface: only 26% and 52% of the total receptors were surface receptors in P388 and P388/DXR, respectively, whereas it was 87% in normal splenocytes . Isoproterenol slightly (less than 1-fold) stimulated cAMP accumulation in P388 and P388/DXR; the stimulation observed in splenocytes was 2.5-fold . In addition, the basal levels of cAMP appeared to be low (0.48 +/- 0.05 pmoles/10(6) cells in P388 and 0.71 +/- 0.08 pmoles/10(6) cells in P388/DXR; 3.47 +/- 0.28 pmoles/10(6) cells in splenocytes) in the two leukemias and they were only slightly (less than 2-fold) increased by forskolin, which otherwise stimulated about 15-fold cAMP accumulation in splenocytes; thus, P388 and P388/DXR were probably also defective in their adenylate cyclase activity . It can be concluded that, owing to multifactorial mechanisms, the lymphocytic leukemia P388, also as an MDR variant, is minimally sensitive to the direct influences of the beta-adrenergic system, probably including any effect of this system on drug-sensitivity.

Anticancer Res, 1992 Nov-Dec, 12(6B), 2297 - 302
MDR hamster cells exhibiting multiple altered gene expression: effects of dexniguldipine-HCl (B859-35), cyclosporin A and buthionine sulfoximine; Neumann M et al.; An actinomycin D selected, multidrug-resistant (MDR) hamster CHO subline showed strong expression of the P-glycoprotein and sorcin genes together with several other alterations such as a: (i) reduced growth rate, (ii) lowered topoisomerase II, (iii) lowered glutathione-S-transferase-P gene expression, and (iv) the emergence of a 15.5 kDa protein . Besides high resistances to adriamycin, actinomycin D, and vincristine, we observed a lowered sensitivity towards bleomycin, a rather hydrophilic drug usually not involved in P-glycoprotein associated MDR . Moreover, the MDR subline showed a pronounced collateral (enhanced) sensitivity towards the sterically pure dihydropyridine anticancer drug dexniguldipine-HCl (B859-35) preventing its characterization for MDR modulation here . At a non-cytotoxic dose (10 microM) the immunosuppressive cyclic peptide cyclosporin A completely abolished the resistance to vincristine, partially reversed the resistance to teniposide and strongly enhanced the sensitivity towards bleomycin, while not influencing the drug sensitivities of the parental cell line . Buthionine sulfoximine (BSO), an agent depleting cellular glutathione levels, distinctly increased the sensitivity towards teniposide at nontoxic doses (50 microM) exclusively in the MDR subline, while it did not alter vincristine or bleomycin cytotoxicity.

Antimicrob Agents Chemother, 1992 Nov, 36(11), 2392 - 7
Susceptibility of an emetine-resistant mutant of Entamoeba histolytica to multiple drugs and to channel blockers; Samuelson JC et al.; Previously a cloned emetine-resistant mutant of the protozoal parasite Entamoeba histolytica was shown to overexpress a gene for an ameba homolog of the mammalian P-glycoprotein, a plasma membrane pump that removes hydrophobic drugs from multidrug-resistant tumor cells . Three sets of experiments were performed to better characterize the multidrug-resistant phenotype of the emetine-resistant amebae . First, the emetine resistance of the mutant amebae was reversed by concentrations of calcium and sodium channel blockers effective in reversing drug resistance by multidrug-resistant tumor cells, but it was reversed only in the presence of very high concentrations of the tricyclic antidepressants . Second, the mutant amebae showed cross-resistance to antiamebic drugs used to treat luminal infection (iodoquinol and diloxanide) but were not cross-resistant to drugs used to treat invasive disease (chloroquine and metronidazole) . Third, when amebae were loaded with radiolabeled emetine, the mutant parasites released the drug at approximately 1.6 times the rate of the wild-type organisms . We conclude that the emetine-resistant E . histolytica parasites have some but not all the features of the multidrug-resistant phenotype.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 440 - 5
P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is alloserically coupled to a vinca-alkaloid-selective binding site; Ferry DR et al.; {3H}Vinblastine bound with high affinity to surface membranes prepared from H69/LX4 cells which express P-glycoprotein (P-gp) and as a consequence are multidrug resistant (MDR) . The KD was 9.8 +/- 1.5 nM and density of sites 31.2 +/- 8.6 pmol/mg of protein . {3H}Vinblastine binding was inhibited by cytotoxics and agents known to reverse MDR . 1,4-Dihydropyridine MDR reversing agents including nicardipine and nifedipine accelerated the dissociation of {3H}vinblastine from P-gp indicating a negative heterotropic allosteric effect . Cyclosporin A, vincristine and actinomycin D did not alter {3H}vinblastine dissociation kinetics . It is concluded that P-gp possesses at least two allosterically coupled drug acceptor sites, receptor site-1 that is selective for vinca alkaloids and cyclosporin A, and receptor site-2 that is selective for 1,4-dihydropyridines.

J Biol Chem, 1992 Oct 15, 267(29), 21020 - 6
Characterization of the azidopine and vinblastine binding site of P-glycoprotein; Bruggemann EP et al.; To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein . Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein . Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein . We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein . Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling . These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins . We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.

Cancer Res, 1992 Oct 15, 52(20), 5759 - 64
N-(4'-hydroxyphenylacetyl)palytoxin: a palytoxin prodrug that can be activated by a monoclonal antibody-penicillin G amidase conjugate; Bignami GS et al.; Palytoxin (PTX), one of the most toxic nonprotein molecules known, is cytotoxic at picomolar concentrations against a wide variety of cell types . In contrast to most cytotoxins, PTX exerts its activity extracellularly . A method for targeting PTX to tumor cells is described in which a monoclonal antibody-enzyme conjugate activates a PTX prodrug at surfaces of tumor cells . The prodrug, N-(4'-hydroxyphenylacetyl)palytoxin (NHPAP), was prepared by reacting PTX with an active ester of 4-hydroxyphenylacetic acid . NHPAP was 1000 times less toxic than PTX to a panel of carcinoma and lymphoma cell lines . The cytotoxic activity of the combination of penicillin G amidase from Escherichia coli with NHPAP was equal to PTX . Two cell lines that were multidrug resistant showed no enhanced resistance to NHPAP +/- penicillin G amidase . Immunologically specific activation of NHPAP took place when H2981 cells (L6 antigen positive) were treated with the monoclonal antibody conjugate L6-penicillin G amidase followed by NHPAP . This system is distinguished from other prodrug activation schemes, since the released drug exerts its activity extracellularly, has high potency, and may be able to overcome the multidrug resistant phenotype.

Cancer Res, 1992 Oct 15, 52(20), 5701 - 6
Characterization of tumor cell resistance to 4'-deoxy-4'-iododoxorubicin developed in Ehrlich ascites cells in vivo; Friche E et al.; Reduced drug accumulation is the most common functional change accompanying development of P-glycoprotein-associated multidrug resistance . One of our laboratories showed earlier that the anthracycline analogue 4'-deoxy-4'-iododoxorubicin (DIDOX) was accumulated to identical levels in Ehrlich ascites tumor (EHR2) and daunorubicin (DNR)-resistant EHR2/DNR+ cells (E . Friche, P . B . Jensen, T . Skovsgaard, and N . I . Nissen, J . Cell . Pharmacol., 1:57-65, 1990) . In this communication, we show that weekly treatment of EHR2-bearing mice with 4, 8, or 12 mg of DIDOX/kg/week led to the development of three DIDOX-resistant cell lines, EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3 . The levels of DIDOX accumulation and retention and its outward transport were similar in the drug-sensitive and three drug-resistant cell lines . By contrast, the accumulation of the active DIDOX metabolite, 13-dihydro-DIDOX (13-OH-DIDOX), the parent compound doxorubicin, and daunorubicin were all decreased in proportion to the resistance of the cells . In EHR2/DIDOX-3 cells, the reduction in daunorubicin accumulation coincided with the development of P-glycoprotein as demonstrated by Western blot and flow cytometry with C219 antibody . DIDOX had no effect on the photolabeling of P-glycoprotein by {3H}azidopine, whereas 13-OH-DIDOX inhibited this labeling in a concentration-dependent manner . Subsequent analysis of topoisomerase II activities and amounts in EHR2/DIDOX-3 cells revealed decreased DNA topoisomerase II catalytic activity . The amounts of immunoreactive DNA topoisomerase II from EHR2/DIDOX-1, EHR2/DIDOX-2, and EHR2/DIDOX-3 cells were about 89%, 73%, and 52%, respectively, of that seen in the drug-sensitive cells . We also found that teniposide stabilized DNA-protein complexes in EHR2/DIDOX-3 but they never reached the level seen in EHR2 cells . Because it has been reported that DIDOX is rapidly metabolized to 13-OH-DIDOX, we postulate that the development of resistance to DIDOX in vivo is due in part to its metabolite, 13-OH-DIDOX, which is a substrate for plasma membrane glycoprotein, and in part to DIDOX, which is an inhibitor of topoisomerase II.

Cancer Res, 1992 Oct 1, 52(19), 5244 - 9
Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification; Gervasoni JE Jr et al.; Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp) . The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986 . This change did not affect drug sensitivity . The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype . The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere . Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome . Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp . Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

J Natl Cancer Inst, 1992 Oct 7, 84(19), 1506 - 12
Effect of P-glycoprotein expression on sensitivity to hormones in MCF-7 human breast cancer cells; Clarke R et al.; BACKGROUND: Data obtained from studies of primary human breast cancers and established cell lines indicate that overexpression of the MDR1 gene (also known as PGY1) is associated with decreased expression of steroid hormone receptors and increased expression of epidermal growth factor (EGF) receptors . Other study results indicate that both progestins and triphenylethylene antiestrogens may be substrates for P-glycoprotein, the product of the MDR1 gene . These findings together suggest an association between overexpression of the MDR1 gene and cross-resistance to progestin and antiestrogen therapies . PURPOSE: This study was designed to determine (a) the ability of MDR1 expression to alter tumor sensitivity to hormone therapy and (b) the role of MDR1 expression in expression of functional hormone receptors in human breast cancer . METHODS: We transduced MCF-7 cells with MDR1 complementary DNA, using a retroviral vector directing the constitutive expression of the MDR1 gene . Transduced cells (MCF-7MDR1) were examined for ability to produce P-glycoprotein, expression of steroid hormone receptors, and responsivity to antiestrogens . For comparison, we used MCF-7ADR human breast cancer cells, which overexpress MDR1 and have also lost the requirement for 17 beta-estradiol supplementation to form tumors in nude mice . We also investigated the level of EGF-R mRNA expression by using a sensitive RNase protection analysis . RESULTS: MCF-7MDR1 cells retained both estrogen receptor and progesterone receptor expression as well as sensitivity to 4-hydroxytamoxifen . Expression of the estrogen-inducible pS2 and EGF receptor genes was similar in parental MCF-7 and transduced MCF-7MDR1 cells . EGF receptor expression was increased, and pS2 expression was lost (undetectable) in MCF-7ADR cells . CONCLUSIONS: The data indicate that overexpression of the MDR1 gene alone confers a multidrug-resistant phenotype, but it does not directly result in either cross-resistance to antiestrogens or a loss of steroid hormone receptor expression . IMPLICATIONS: MCF-7MDR1 cells provide an important model for study of the interactions of cytotoxic drugs, hormones, and the MDR1 glycoprotein in human hormone-responsive breast cancer cells.

J Biol Chem, 1992 Oct 5, 267(28), 20383 - 91
Functional involvement of P-glycoprotein in blood-brain barrier; Tatsuta T et al.; P-glycoprotein, an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in various normal tissues, including brain capillaries . To study the physiological function of P-glycoprotein expressed in brain capillary endothelium, we established nine mouse brain capillary endothelial cell (MBEC) lines and examined the transport of antitumor agents across the monolayer of MBEC epithelia . In the MBECs, the activities of alkaline phosphatase and gamma-glutamyl transpeptidase, specific markers for brain capillary endothelial cells, were about three times higher than those in other cells including human umbilical vein endothelial cells . By immunoblot analysis, P-glycoprotein was detected in all of the nine MBEC clones . The P-glycoprotein expressed in MBECs specifically bound {125I}iodoaryl azidoprazosin as that in multidrug-resistant cells, and efflux of vincristine was observed in the MBECs . When MBECs were grown on a porous filter membrane, they formed a monolayer of epithelium . By immunoelectron microscopic analysis, P-glycoprotein in MBEC epithelia was shown to be localized to the apical surface of the cells . Moreover, the unidirectional transepithelial transport of vincristine from basal side to apical side was demonstrated in vitro . These observations indicate that P-glycoprotein in brain capillary endothelium prevents vincristine from entering the central nervous system and thus may be one of the functional components of the blood-brain barrier.

J Biol Chem, 1992 Oct 5, 267(28), 20248 - 54
The involvement of a LINE-1 element in a DNA rearrangement upstream of the mdr1a gene in a taxol multidrug-resistant murine cell line; Cohen D et al.; Two closely related but functionally distinct P-glycoprotein isoforms are encoded by the murine multidrug-resistance genes mdr1a and mdr1b . In a series of independently selected multidrug-resistant (MDR) J774.2 cell lines, mdr gene amplification and/or overexpression and overproduction of either the mdr1a or mdr1b products, or both gene products, correlates with the MDR phenotype . To investigate the possibility that mutations in the promoter regions of the mdr1a or mdr1b genes could influence their differential expression, mdr promoter-specific probes were used to detect and map potential structural alterations . An unusual structural rearrangement was found in the 5'-region of the amplified mdr1a allele in J7.T1, a cell line selected with taxol . To characterize this rearrangement, the regulatory regions of the mdr1a and mdr1b genes were analyzed . Whereas no gross structural alterations were detected by Southern blot hybridization using the mdr1b promoter probe, a novel amplified EcoRI fragment was detected by the mdr1a promoter probe . To determine the precise nature of this mutation, an mdr1a 5'-genomic clone was isolated from J7.T1 cells . Sequence analysis revealed an unusual DNA rearrangement consisting of the mdr1b gene, from its fourth intron toward its 3'-end, upstream of an intact mdr1a promoter on the amplified allele . We propose that this event occurred by an unequal sister chromatid exchange that was mediated by LINE-1 repetitive elements.

Cell, 1992 Oct 2, 71(1), 23 - 32
Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein; Gill DR et al.; The human multidrug resistance P-glycoprotein is an active transporter that pumps cytotoxic drugs out of cells . Expression of P-glycoprotein is also associated with a volume-activated chloride channel . Here we address the relationship between these two functions . Drug transport requires ATP hydrolysis while, in contrast, ATP binding is sufficient to enable activation of the chloride channel . The chloride channel and drug transport activities of P-glycoprotein appear to reflect two distinct functional states of the protein that can be interconverted by changes in tonicity . Transportable drugs prevent channel activation but have no effect on channel activity once it has been preactivated by hypotonicity . The transport and channel functions of P-glycoprotein have been separated by directed mutations in the nucleotide-binding domains of the protein . These data provide further evidence that P-glycoprotein is bifunctional with both transport and channel activities . Implications for the design of chemotherapeutic drugs and for the function of the related cystic fibrosis gene product, CFTR, are discussed.

Med Trop (Mars), 1992 Oct-Dec, 52(4), 377 - 84
{Angkor . The mystery of the dead city and Anopheles dirus}; Verdrager J; The desertion of Angkor, which during more than five centuries was the center of a glorious civilization, has long been a matter of mystery and conjecture . The discovery of the vectorial capacity of the jungle mosquito Anopheles dirus, its epidemiological importance in the emergence and spread of multidrug resistance in Plasmodium falciparum malaria, the wiping out of large populations after transfer or deportation of non-immune Khmers into forest areas can now easily explain the desertion of Angkor . In 1431, Angkor Thom, the capital of the Khmer kingdom surrendered to the Thai conquerors . Soon afterwards, the young king left the city in search of a new capital . As a result of the population decrease large surfaces of rice fields were abandoned and reinvaded by the jungle, the typical biotope of Anopheles dirus . Severe epidemics of Plasmodium falciparum then occurred in the non-immune population with very high mortality decreasing again the number of workers and, thus, creating a vicious circle resulting in the progressive but complete desertion of Angkor.

J Chemother, 1992 Oct, 4(5), 306 - 11
Evaluation of the in vitro antiproliferative properties of four novel anthracyclines YM1, 3, 4 and 6 in human leukemia cell lines; Jiang XR et al.; The antitumor activity of novel doxorubicin analogues YM1, YM3, YM4 and YM6 was evaluated against drug sensitive U937 monocytic leukemia and CCRF-CEM lymphoid leukemia cell lines, as well as drug resistant CEM/VLB100 lymphoid multidrug resistant leukemia cell line by a {3H}thymidine incorporation assay . Different antileukemic activities of these new anthracyclines were observed in our studies . These novel anthracyclines produced a dose- and time-dependent inhibition in all the leukemic cell lines tested, while YM1 and YM3 were more effective than YM4 and YM6 against all the leukemic cell lines . The antitumor activity of all these novel analogues was lower than that of doxorubicin or epidoxorubicin in drug sensitive leukemic cells . The relative resistance values (IC50 of resistant cell line/IC50 of sensitive parental cell line) of YM1, 3, 4 and 6 were 27, 7, 5 and 14 respectively . These were lower than the resistance values for ADM and EDR which were 45 and 40 respectively . YM3 had a similar antileukemic activity against the CEM/VLB100 drug resistant leukemic cell line to ADM or EDR with a lower relative resistance value and a slightly increased IC50 value . Our results suggest that YM3 may be used in high dose for the clinical treatment of leukemias with possible less cardiotoxicity as well as less drug resistance.

Iowa Med, 1992 Oct, 82(10), 411 - 4
Resurgence of tuberculosis; Hornick D et al.; Since 1985 there has been an 18% increase in the number of cases of tuberculosis nationally . Even more alarming is the increasing incidence of multidrug-resistant tuberculosis which presents a serious public health problem, particularly for HIV-infected patients.

Exp Hematol, 1992 Oct, 20(9), 1048 - 54
Use of etoposide in combination with cyclosporin for purging multidrug-resistant leukemic cells from bone marrow in a mouse model; Kuhl JS et al.; Cyclosporin (CsA) is a potent modulator of multidrug resistance (MDR) and has been combined with etoposide (VP-16) to purge MDR leukemic cells from human bone marrow (BM) in vitro . We studied the feasibility of this approach in an in vivo model for autologous BM transplantation using the murine leukemia cell line P388 and its MDR variant P388/ADR . Colony-forming assays with 2-h drug exposure revealed a tumor selectivity of VP-16 for P388 cells compared to normal murine marrow granulocyte-macrophage colony-forming units (CFU-GM), whereas P388/ADR cells were resistant to VP-16 . Simultaneous incubation with CsA restored sensitivity in these cells . Almost 4 logs of cell kill were achieved by treating P388/ADR cells with 60 microM VP-16 plus 2.5 microM CsA (combination A) or 40 microM VP-16 plus 10 microM CsA (combination B), whereas there was a 2.5-log reduction of CFU-GM at these doses . Even though the myelotoxicity of VP-16 was increased by the addition of CsA, this effect was nonspecific as shown by a similar chemosensitization in sensitive P388 as well as in P388/VP 2.5 cells, an atypical MDR variant lacking P-glycoprotein . In vivo experiments addressed the ability of BM treated with VP-16 and CsA to rescue lethally irradiated mice and to purge leukemic cells . In total, 1/14 lethally irradiated mice died due to sepsis within 10 days after receiving 15 x 10(6) BM cells treated ex vivo with combination A in contrast to 1/4 for combination B . All 16 surviving animals demonstrated long-term engraftment . When simulated remission marrow contaminated with 0.1% P388/ADR was purged with VP-16 (60 microM) or CsA (2.5 microM) alone, all mice died from leukemia before day 16 after transplantation (median 14.3 and 12.2 days) . In contrast, nine of ten animals receiving similar marrow purged with combination A survived > 60 days without any evidence of disease (p < 0.01) . We conclude that combining VP-16 and CsA was effective in purging MDR leukemia cells from transplanted BM in this murine model.

J Clin Oncol, 1992 Oct, 10(10), 1635 - 42
Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a phase I trial to modulate multidrug resistance; Lum BL et al.; PURPOSE: To determine the effects of high-dose cyclosporine (CsA) infusion on the pharmacokinetics of etoposide in patients with cancer . PATIENTS AND METHODS: Sixteen patients were administered 20 paired courses of etoposide and CsA/etoposide . Etoposide was administered daily for three days, alone or with CsA, which was delivered by a loading dose and 3-day infusion . Etoposide was measured by high-performance liquid chromatography (HPLC) and serum CsA by nonspecific immunoassay . Etoposide pharmacokinetics included area under the concentration-time curve (AUC), total and renal clearance (CL), half-life (T1/2), and volume of distribution at steady state (Vss) . RESULTS: CsA concentrations more than 2,000 ng/mL produced an increase in etoposide AUC of 80% (P less than .001), a 38% decrease in total CL (P < .01), a > twofold increase in T1/2 (P < .01), and a 46% larger Vss (P = .01) compared with etoposide alone . CsA levels ranged from 297 to 5,073 ng/mL . Higher CsA levels (< 2,000 ng/mL v > 2,000 ng/mL) resulted in greater changes in etoposide kinetics: Vss (1.4% v 46%) and T1/2 (40% v 108%) . CsA produced a 38% decrease in renal and a 52% decrease in nonrenal CL of etoposide . Etoposide with CsA levels > 2,000 ng/mL produced a lower WBC count nadir (900/mm3 v 1,600/mm3) compared with baseline etoposide cycles . CONCLUSIONS: High-dose CsA produces significant increases in etoposide systemic exposure and leukopenia . These pharmacokinetic changes are consistent with inhibition by CsA of the multidrug transporter P-glycoprotein in normal tissues . Etoposide doses should be reduced by 50% when used with high-dose CsA in patients with normal renal and liver function . Alterations in the disposition of other multidrug resistance (MDR)-related drugs should be expected to occur with modulation of P-glycoprotein function in clinical trials.

J Clin Oncol, 1992 Oct, 10(10), 1624 - 34
Phase I trial of etoposide with cyclosporine as a modulator of multidrug resistance; Yahanda AM et al.; PURPOSE: To determine the maximum-tolerated dose (MTD) of cyclosporine (CsA) infusion administered with etoposide for 3 days in patients with cancer . PATIENTS AND METHODS: Of the 72 registered patients, 26 were treated initially with CsA and etoposide . Forty-six received etoposide alone until disease progression, and 31 of these proceeded to CsA and etoposide . CsA was administered as a 2-hour loading dose (LD) and as a 3-day continuous infusion (CI); doses were escalated from 2 to 8 mg/kg LD and 5 to 24 mg/kg/d CI . RESULTS: Fifty-seven patients were treated with 113 cycles of CsA with etoposide . Steady-state serum CsA levels (nonspecific immunoassay) more than 2,000 ng/mL were achieved in 91% of the cycles at CsA doses > or = 5 mg/kg LD and > or = 15 mg/kg/d CI . The major dose-related toxicity of CsA was reversible hyperbilirubinemia, which occurred in 78% of the courses with CsA levels > 2,000 ng/mL . Myelosuppression and nausea were more severe with CsA and etoposide . Other CsA toxicities included hypomagnesemia, 60%; hypertension, 29%; and headache, 21% . Nephrotoxicity was mild in 12% and severe in 2% of the cycles . Tumor regressions occurred in four patients after the addition of CsA (one non-Hodgkin's lymphoma, one Hodgkin's disease, and two ovarian carcinomas) . Biopsy procedures for tumors from three of the four patients who responded were performed, and the results were positive for mdr1 expression . CONCLUSIONS: Serum CsA levels of up to 4 mumol/L (4,800 ng/mL) are achievable during a short-term administration with acceptable toxicities when administered in combination with etoposide . The CsA dose that is recommended in adults is a LD of 5 to 6 mg/kg, followed by a CI of 15 to 18 mg/kg/d for 60 hours . CsA blood levels should be monitored and the doses should be adjusted to achieve CsA levels of 2.5 to 4 mumol/L (3,000 to 4,800 ng/mL) . Reversible hyperbilirubinemia may be a useful marker of inhibition by CsA of P-glycoprotein function . When used with high-dose CsA, etoposide doses should be reduced by approximately 50% to compensate for the pharmacokinetic effects of CsA on etoposide (Lum et al, J Clin Oncol, 10:1635-1642, 1992).

Arch Pathol Lab Med, 1992 Oct, 116(10), 1055 - 61
Expression of P-glycoprotein in normal muscle cells and myogenic tumors; Garberoglio C et al.; We have analyzed the expression of the multidrug resistance (mdr-1) gene product, P-glycoprotein, by immunohistochemical staining of frozen tissue sections of human normal muscle fibers and 31 tissue specimens of cases of myogenic sarcomas . The objective of this study was to further characterize what appears to be a variety of responses to therapy in like-appearing but distinct tumors . We have used two mouse monoclonal antibodies that recognize two different epitopes of P-glycoprotein . Mouse monoclonal antibody HYB-241 detects an extracellular epitope of P-glycoprotein, whereas C219 detects a carboxy-terminal intracellular epitope and has recently been reported to cross-react with the mdr-3 gene product . Differential epitope expression was observed among normal muscle fibers with the two antibodies used . Smooth-muscle cells were unreactive for the two antibodies, whereas cardiac and a subgroup of skeletal muscle fibers were intensely stained by C219, but not by HYB-241 . P-glycoprotein expression was observed in 23% of the 31 myogenic sarcomas analyzed . Our study was conducted mainly using adult myogenic sarcomas (28 out of 31 cases), with a few cases (three out of 31 cases) of childhood sarcomas . Nineteen tumors were leiomyosarcomas, seven cases were embryonal rhabdomyosarcomas, and five cases were rhabdomyosarcomas . We have considered expression of the mdr-1-coded P-glycoprotein when we observed either HYB-241 and C219 staining, or just HYB-241 immunoreactivities . Although P-glycoprotein expression can now be detected in human sarcomas, further studies are needed, mainly comparing tumor samples before, during, and after therapy, to establish the possible significance of the P-glycoprotein expression in clinical drug resistance.

Cancer Res, 1992 Oct 1, 52(19), 5154 - 61
P-glycoprotein expression during tumor progression in the rat liver; Bradley G et al.; P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines . Overexpression of Pgp has been detected in clinical cancer samples of many histological types . The basis and biological significance of such increases in Pgp expression are not well understood . In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis . An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas . The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions . In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions . A particularly striking finding was the consistent expression of Pgp in the lung metastases . These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.

Baillieres Clin Haematol, 1992 Oct, 5(4), 943 - 60
Multidrug resistance in leukaemia; Baines P et al.; Multidrug resistance hampers successful chemotherapy in many haematological neoplasms and is mediated by several cellular proteins . In some cases, the genes encoding these proteins have been shown to confer resistance on transfer to drug-sensitive cell lines . This is true for the efflux pump product of the MDR1 gene, P-170 . Upregulation of enzymes such as GST has been observed, although the contribution of this enzyme in drug resistance expressed by malignant haematopoietic cells is still uncertain . Cells also appear to be able to downregulate enzymes which are drug targets . Examples include the decrease in Topo II which accompanies the resistance shown by cells to VP-16 and VM-26 . Although many reports include both presentation and relapsed patients, there are few data on samples drawn from the same patients before and after chemotherapy . While P-170 and GST appear to be raised more often in cells from resistant and relapsed disease, it is quite clear that such mechanisms can be active in de novo malignancy and do not necessarily emerge as a consequence of prior chemotherapy . Methods of detecting drug resistance are reviewed here; these include in vitro cellular assays for drug toxicity, and molecular, immunological and functional detection of P-170 or Topo II . The clinical evaluation of such assays is only just beginning and some of the data are contradictory . To some extent, this may reflect the complex way in which the various resistance mechanisms may interact . Nevertheless, there are some encouraging early signs that the application of these assays to clinical material will yield valuable data on the relative contributions of these mechanisms and on ways in which they may be overcome . At present, much attention has focused on the potential of agents which prevent the P-170 efflux pump from exporting cytotoxics from the cell . This is likely to be only the first of new therapies arising from an improved understanding of multidrug resistance . More immediately, assays for multidrug resistance and its parameters may find their place as routine diagnostic and prognostic tools in the laboratory.

AIDS Res Hum Retroviruses, 1992 Oct, 8(10), 1839 - 44
Resistance of HIV-1 to AZT might also involve the cellular expression of multidrug resistance P-glycoprotein; Antonelli G et al.; Resistance of tumor cells to the antigrowth activity of several cytotoxic compounds has been associated with the expression of the so-called multidrug resistance protein or P-glycoprotein . This article addresses the question whether the expression of such protein could also affect the sensitivity of HIV to AZT . Our data indicate that this possibility does exist . In fact, multidrug-resistant CEM VBL100 cells, which express high levels of P-glycoprotein, are less sensitive to both the antiproliferative activity and the antiviral action of AZT . Additionally, our data suggest that this phenomenon is specifically mediated by P-glycoprotein since trifluoroperazine, which is known to circumvent multidrug resistance due to the action on P-glycoprotein, increases the intracellular accumulation of AZT and affects the sensitivity of HIV to AZT . Although the biological and clinical significance of these observations has still to be established, this study suggests that cellular factors, other than virus itself, should be taken into account to address the phenomenon of drug resistance of HIV.

Ann Trop Med Parasitol, 1992 Oct, 86(5), 467 - 73
Treatment of chloroquine-resistant malaria in monkeys with a drug combination that reverses resistance in vitro; Williams HL et al.; Compounds that inhibit the P-glycoprotein-related efflux mechanism of multidrug-resistant cells reverse chloroquine resistance in vitro . Hence, the co-administration of chloroquine and an efflux-blocking drug could potentially treat chloroquine-resistant malaria infections . We administered a drug combination (chloroquine and a tiapamil analogue), that has been shown to reverse chloroquine resistance in vitro, to Aotus monkeys but failed to safely clear experimentally-induced chloroquine-resistant Plasmodium falciparum parasitaemias.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 677 - 84
Activation of human multidrug resistance-1 gene promoter in response to heat shock stress; Miyazaki M et al.; The multidrug resistance (MDR1) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents . Various environmental stresses including heat shock can induce the expression of endogenous MDR1 genes . In order to study the regulatory mechanisms of MDR1 gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1 promoter . Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDR1 promoter of -2 kilobases . The MDR1 gene promoter contains a typical heat shock element (HSE) motif located -152 bp to -178 bp from the initiation site . Heat shock at 45 degrees C for 90 min significantly induced CAT activity in Kst-6 cells . Northern blot analysis showed a 4-5 fold increase in CAT mRNA levels in Kst-6 cells . Deletion analysis of the MDR1 promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDR1 promoter construct, pMDRCAT7 . However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing -76 approximately +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between -136 and -76 . Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at -152 bp to -178 bp in the MDR1 promoter . We also found that one distinct DNA-protein complex formed specifically within the MDR1 promoter region -99 to -66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells . In our present assay system, activation of the MDR1 promoter in response to heat shock appears to be mediated through both a new heat shock responsive element and MDR1 specific transcription factor.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 1098 - 105
Effect of liposomes on P-glycoprotein function in multidrug resistant cells; Thierry AR et al.; Presentation of doxorubicin in liposomes has shown to enhance the sensitivity of multidrug resistant CH LZ cells to the drug (Thierry et al . Cancer Commun . 1:311-316, 1989) . We confirmed that liposomally encapsulated doxorubicin may partially overcome multidrug resistance in the human ovarian carcinoma SKVLB cell line and that this effect is, at least in part, due to an increase of cellular drug accumulation . When used at high concentration, empty liposomes appear to be specifically cytotoxic in the MDR SKVLB and CH LZ cells . As observed with certain multidrug resistance modulators, empty liposomes inhibited the specific {3H}-vincristine binding to P-glycoprotein-enriched membranes isolated from CH LZ cells (60% at 0.2 mg lipid/ml) . Our data suggest that liposomes may alter the P-glycoprotein function by direct interaction.

Cancer Res, 1992 Sep 15, 52(18), 5013 - 7
Collateral sensitivity to nitrosoureas in multidrug-resistant cells selected with verapamil; Futscher BW et al.; We have examined the effects of the nitrosoureas, streptozotocin (STZ) and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), on a human multiple myeloma cell line, RPMI 8226, and its drug-resistant variants . Cell lines selected for doxorubicin (DOX) resistance alone displayed a STZ and BCNU cytotoxicity profile similar to that of the parent cell line . In contrast, two of the drug-resistant variants selected with DOX plus verapamil, an agent which inhibits P-glycoprotein-mediated multidrug resistance, displayed a collateral sensitivity to STZ and BCNU . Verapamil was included in the selection protocol because it has been shown to inhibit the P-glycoprotein-mediated multidrug resistance phenotype and is now in clinical trials as a chemosensitizing agent . The collateral sensitivity to these nitrosoureas seen in the DOX plus verapamil-selected cell lines is due to the functional loss of a DNA repair molecule, O6-Methylguanine DNA methyltransferase (MGMT) . The functional loss of MGMT is secondary to the loss of MGMT gene expression . The loss of MGMT gene expression is not due to loss or gross rearrangement of the MGMT-coding region . If this selection pressure applied in vitro reflects the in vivo situation, then new chemotherapeutic strategies may be devised to exploit this phenomenon . These cell lines will serve as useful models for delineating mechanisms which govern MGMT expression.

Blood, 1992 Sep 15, 80(6), 1528 - 36
Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype; Arceci RJ et al.; The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents . This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P-glycoprotein . Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents . In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents . FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells . The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil . The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive . This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P-glycoprotein . The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8472 - 6
Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis; Ambudkar SV et al.; Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump . In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis . Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B . The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method . P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes . ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein . The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation . Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine . The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases . These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).

Cancer, 1992 Sep 15, 70(6 Suppl), 1799 - 809
Genetic aspects of multidrug resistance; Biedler JL; Mammalian cells exposed to a single cytotoxic natural product drug, such as vincristine or dactinomycin, can develop resistance to the selective agent and cross-resistance to a broad spectrum of structurally and functionally distinct antibiotics and alkaloids . This phenomenon, termed multidrug resistance (MDR), has been widely studied experimentally . The most consistent feature of cells with high-level MDR is amplification and overexpression of genes encoding an integral plasma membrane protein known as P-glycoprotein . The MDR genes belong to a small family (two members in humans and three members in mouse and Chinese hamster) . Based on several lines of evidence, P-glycoprotein is thought to act as an adenosine triphosphate-dependent efflux pump that decreases accumulation of drugs and increases resistance to their effects . The normal function of P-glycoprotein, apart from its role in MDR, is not known . Proposed roles in detoxification and steroid transport systems are speculative but suggest that the membrane protein may have distinct functions in normal tissues and in tumor cells with acquired MDR . Although possible endogenous substrates for P-glycoprotein have not been identified, insight into normal function may be gained from tissue distribution studies . For example, studies using molecular probes to P-glycoprotein messenger RNA and monoclonal antibodies to different epitopes of the molecule have shown that P-glycoprotein is expressed at high levels in the more differentiated or specialized cells of the colon or kidney . Amplification of MDR genes in vivo has not been observed . Whether intrinsic or acquired MDR plays a causal and potentially modifiable role in clinical nonresponsiveness to cancer chemotherapeutic agents is a topic of current interest . Prospective studies and serial determinations during the course of disease are needed to clarify the importance of this membrane protein in clinical drug resistance.

Cancer Lett, 1992 Sep 14, 66(1), 83 - 9
Abnormal chloride conductance in multidrug resistant HL60/AR cells; Gollapudi S et al.; Chloride channel currents were measured in drug sensitive parental HL60 and multidrug resistant (MDR) subline HL60/AR cells, using a whole cell patch-clamp technique . In addition, the in vitro effects of 4,4' diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, on intracellular accumulation and sensitivity to daunorubicin and intracellular pH (pH(i)) in HL60 cells were examined . Baseline DIDS blockable Cl- currents were consistently lower in HL60/AR cells (0.9 pA/pF) as compared to HL60 cells (7.0 pA/pF) . Similarly cAMP-activated Cl- currents were minimal in HL60/AR cells (0.2 pA/pF) as compared to HL60 cells (8 pA/pF) . In vitro treatment of drug sensitive HL60 cells with DIDS resulted in concentration-dependent decreased accumulation and increased resistance to daunorubicin and decreased pH(i) . These data show that altered Cl- permeability is associated with MDR and suggest that Cl- channels may play a role in MDR.

Mol Cell Biol, 1992 Sep, 12(9), 3689 - 98
Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines; Zhen W et al.; We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug . The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008 . Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation . This quantitation was done for total genomic lesions and at the level of individual genes . In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines . Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells . These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level . Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells . There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners . However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links . DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines . Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.

J Med Chem, 1992 Sep 4, 35(18), 3358 - 64
Synthesis and chemical characterization of N-substituted phenoxazines directed toward reversing vinca alkaloid resistance in multidrug-resistant cancer cells; Thimmaiah KN et al.; A series of 21 N-substituted phenoxazines has been synthesized in an effort to find more specific and less toxic modulators of multidrug resistance (MDR) in cancer chemotherapy . Thus, N-(omega-chloroalkyl)- and N-(chloroacyl)phenoxazines were found to undergo iodide-catalyzed nucleophilic substitution on reaction with various secondary amines, including N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidine and (beta-hydroxyethyl)piperazine . Products were characterized by UV, IR, 1H-, and 13C-NMR, mass spectral data, and elemental analyses . All of the compounds were examined for cytotoxicity and for their ability to increase the accumulation of the vinca alkaloids, vincristine (VCR) and vinblastine (VLB) in multidrug-resistant GC3/Cl (human colon adenocarcinoma) and KBChR-8-5 (HeLa variant) cell lines . Compounds were compared to the standard modulator verapamil (VRP) . Substitutions on the phenoxazine ring at position 10 were associated with an increase in antiproliferative and anti-MDR activities . Modification of the length of the alkyl bridge and the type of amino side chain also influenced the potency of these effects . From among the compounds examined, 10 derivatives were found to increase the accumulation of VCR and VLB in GC3/Cl and KBChR-8-5 cells relative to the effect of VRP, suggesting that with the exception of pyrrolidinyl, the tertiary amine attachments to the phenoxazine nucleus linked through a three- or four-carbon alkyl chain resulted in enhanced anti-MDR activity . On the basis of their 50% growth inhibitory (IC50) values, five of the ten compounds, namely, 10-(3'-chloropropyl)phenoxazine, 10-{3'-{N-bis(hydroxyethyl)- amino}propyl}phenoxazine, 10-(3'-N-morpholinopropyl)phenoxazine, 10-(4'-N-morpholinobutyl)phenoxazine and 10-(N-piperidinoacetyl)phenoxazine were selected as relatively nontoxic chemosensitizers . These modulators, at nontoxic concentrations, potentiated the cytotoxicity of VCR and VLB in GC3/Cl and KBChR-8-5 cells . Further, two compounds 10-(3'-N-morpholinopropyl)phenoxazine, and the butyl derivative, enhanced accumulation of VLB in GC3/Cl, KBChR8-5 and highly resistant KB-V1 cells to a level significantly greater than the maximal level achieved with VRP . Additional experiments to understand the mechanism of action of these agents in modulating MDR are in progress.

Am J Trop Med Hyg, 1992 Sep, 47(3), 305 - 9
The effectiveness of permethrin-impregnated bed nets against malaria for migrant workers in eastern Thailand; Kamol-Ratanakul P et al.; A randomized, double-blind, field trial was carried out to compare the effectiveness of permethrin-treated bed nets with that of untreated nets as a method of malaria control for migrant workers in eastern Thailand . The study was conducted using 261 subjects in eastern rural areas that are known to be highly endemic for multidrug-resistant Plasmodium falciparum infection . One hundred twenty-six subjects used treated nets, while 135 used untreated nets . During the 35 weeks of observation, 23 subjects using treated nets and 33 workers using untreated nets developed 28 and 51 episodes of malaria, respectively (P = 0.029) . The reduction in risk per subject due to treated nets was 0.06 . The residual effects of permethrin were tested using a World Health Organization standard bioassay . Anti-mosquito activity was found to be present in the nets for more than 16 months . We conclude that because of the failure of the development of safe, effective, long-lasting prophylactic agents, integrating the use of impregnated nets with large-scale primary health care programs may be a partially effective method for controlling malaria in eastern Thailand.

Chest, 1992 Sep, 102(3), 797 - 801
Multidrug resistant Mycobacterium tuberculosis in patients with human immunodeficiency virus infection; Busillo CP et al.; Multidrug resistant Mycobacterium tuberculosis (MDR-MTB) infection has not been recognized as a serious problem in patients with human immunodeficiency virus (HIV) infection . Multidrug resistance (MDR) has appeared in our medical center in 24 out of 72 patients between January 1990 and May 1991 compared to 8 out of 132 patients within the period from 1982 to 1987 (relative risk 5.50 with 95 percent confidence interval 2.61 to 11.61) . We describe 19 patients with MDR in MTB (isoniazid and at least one additional first line drug), who had serologic evidence of HIV infection, 13 of whom were diagnosed with acquired immunodeficiency syndrome (AIDS) . The MTB cultures from 10 out of 19 patients with MDR were resistant to three or more drugs . Fifteen patients died although 9 out of these 15 had received at least a four-drug regimen for a mean time of seven weeks (range 2 to 12) . This increase in MDR was seen in ten homosexuals and nine intravenous drug users . This rapid appearance of MDR-MTB strains is worrisome . New strategies for empiric therapy of such patients while awaiting sensitivity data are needed.

Arzneimittelforschung, 1992 Sep, 42(9), 1163 - 8
Resistance mechanisms in murine tumors with acquired multidrug resistance; Volm M et al.; Mechanisms of multidrug resistance were studied in murine leukemia (L 1210) and sarcoma (Sa 180) tumors after pretreatment with anthracyclines in vivo . Despite identical pretreatment protocols, a considerable difference in the level of resistance between L 1210 and Sa 180 tumors was noted (for doxorubicin: 45-fold versus 340-fold; for daunorubicin: 51-fold versus 275-fold) . However, no difference in mdr 1 gene-amplification and the overexpression of mdr 1-RNA or P-glycoprotein was demonstrated . None of these parameters did increase by further treatment with a higher concentration of anthracyclines . Resistant sublines of Sa 180 revealed an overexpression of glutathione S-transferase-pi (GST-pi) in comparison to the parental line, whereas in sensitive and resistant sublines of L 1210 tumors the expression of GST-pi was similar . In order to study whether trifluoperazine can reverse the P-glycoprotein mediated component of multidrug resistance, trifluoperazine and doxorubicin were tested in vitro in L 1210 and Sa 180 cells . In contrast to the complete reversal of resistance in L 1210 tumors, resistance in Sa 180 was only partly circumvented . However, by buthionine sulfoximine treatment, the toxicity of multidrug resistant Sa 180 tumors could be increased . It was possible to reverse the resistance of Sa 180 tumors completely by trifluoperazine plus buthionine sulfoximine . Thus, multidrug-resistant Sa 180 tumors express different defense mechanisms whereas L 1210 tumors express only one defense mechanism (P-glycoprotein).

Anticancer Res, 1992 Sep-Oct, 12(5), 1581 - 5
Cytotoxic action of cyclosporins on human tumor cell lines is not dependent on immunosuppressive activity; Larssson R et al.; The cytotoxic activity of cyclosporin A (CsA) and the three non-immuno-suppressive CsA analogues B3-243, WO-039 and B3-665 were studied in tumor cell lines representing both classical and atypical forms of multidrug resistance (MDR): T-ALL GM3639 L100 cells selected for vincristine (vcr) resistance and displaying characteristics of classical MDR, including P-glycoprotein (pgp) expression and increased drug efflux which can be inhibited by pgp blockers (e.g . verapamil), and U-1285/ADR, a small cell lung cancer (SCLC) cell line selected for doxorubicin resistance which lacks pgp, is insensitive to pgp-blockers and shows cross resistance to cis-platinum . At 1 micrograms/ml CsA was the most active agent in reversing Vcr resistance in L100 cells followed by B3-243 and WO-039, with no effect of B3-665 . Parental LO cells were only marginally sensitized to Vcr by these agents . No reversing effect of any cyclosporin was observed in the U-1285/ADR or its parental cell line . Compared to LO cells, L100 cells showed a marked hypersensitivity to CsA > B3-243 > WO-039 with B3-665 being inactive . No collateral sensitivity was observed for cyclosporins in U-1285/ADR cells . Although of different magnitude, the pattern of cytotoxic activity for the different cyclosporins alone closely parallelled that of L100 cells for U-1285, U1285/ADR and LO cells . The results indicate that not only the collateral sensitivity in classical MDR but also the cytotoxic actions of cyclosporins per se on tumor cells alone are independent of immunosuppressive activity . The results also suggest a structure-activity relationship for cyclosporin-induced cytotoxicity similar to, but independent of, MDR reversing activity.

Yeast, 1992 Sep, 8(9), 761 - 8
Sequence of a 12.7 kb segment of yeast chromosome II identifies a PDR-like gene and several new open reading frames; Delaveau T et al.; A 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome . This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5' codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al . (1987) on chromosome VII.

Carcinogenesis, 1992 Sep, 13(9), 1675 - 7
Drug resistance in cultured rat liver epithelial cells spontaneously and chemically transformed; Woo A et al.; Cultured rat liver epithelial cells (RLE) transformed with repeated treatments of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) demonstrate many features of the common biochemical phenotype of multidrug resistance (MDR) seen in vivo in 'resistant hepatocytes' . The cells have increased glutathione-S-transferase placental subunit (GST-Yp), gamma-glutamyltranspeptidase (GGT), glutathione (GSH) and glutathione peroxidase and are resistant to MNNG . Phenotypically identical RLE cells spontaneously transformed by selective culture conditions showed low levels of GGT and GST and were not resistant to MNNG . Both chemical and spontaneous transformants are cross resistant to doxorubicin although resistance is consistently greater in chemical transformants . No direct correlation was found between the degree of resistance to doxorubicin and MDR gene expression in either of the chemically or spontaneously transformed RLE cells . These observations suggest that in chemical carcinogenesis, other mechanisms of drug detoxification are involved and that MDR expression is not a consistent feature.

Southeast Asian J Trop Med Public Health, 1992 Sep, 23 Suppl 4, 39 - 42
The malaria situation and antimalaria program in Laos; Pholsena K; Malaria is endemic in all 17 provinces of Laos . Transmission is perennial with a "seasonal peak" coinciding with the rainy season . The vectors Anopheles minimus and An . balabacensis (=dirus) remain susceptible to DDT . Chloroquine-resistance of Plasmodium falciparum is at the RI-RII level . Multidrug resistance is not yet a problem . Major constraints of the antimalaria program involve logistics and operational problems solutions to which are specifically addressed in the recommendations.

Southeast Asian J Trop Med Public Health, 1992 Sep, 23 Suppl 4, 143 - 8
In vitro sensitivity of Plasmodium falciparum to chloroquine and other antimalarials in east Timor and east Kalimantan, Indonesia; Pribadi W; In Indonesia resistance of Plasmodium falciparum to chloroquine has spread to all provinces except Yogyakarta since the first report in 1974 . The proportion of resistant cases is relatively low except in Irian Jaya and East Timor . The results of in vitro tests performed in East Timor showed 81.0% resistance to chloroquine, 87.5% to amodiaquine, 20.0% to sulfadoxine-pyrimethamine (S-P), 4.8% to mefloquine, and 100% sensitivity to quinine . The percentage of failures was between 11.1% and 71.4% and highest with the S-P combination . Multidrug resistance was observed in 9 cases (or 11.1%) . The results of in vitro tests in East Kalimantan were: 62.8% resistance to chloroquine, 100% to amodiaquine, 85.7% to S-P combination, 3.2% to quinine, and 2.5% to mefloquine . Failures rates ranged between 21.2% and 37.1% the highest being with mefloquine . Multidrug resistance was more prominent and observed in 59 cases (84.3%) of which one case was resistant to 5 antimalarials, while 4 cases (5.7%) were resistant to chloroquine only, compared to 72 cases (88.9%) in East Timor.

Cell Mol Biol, 1992 Sep, 38(6), 561 - 70
Down-regulation of ras and myc expression associated with mdr-1 overexpression in adriamycin-resistant tumor cells; Banerjee S et al.; The murine melanoma tumor cells, B16-BL6, are a recognized model for experimental and spontaneous metastasis . B16-BL6 cells express a lower metastatic phenotype upon acquisition of resistance to adriamycin . Using this novel system, the role of ras, c-myc, and multidrug-resistant gene (mdr1) expression in the metastatic and drug-resistant phenotype was examined . The metastatic cells expressed a high level of c-Ki-ras and c-myc, whereas down-regulation of both proto-oncogenes was observed in the adriamycin-resistant cells . The mdr1 gene, which encodes P-glycoprotein of the drug-resistant superfamily gene, was overexpressed in drug-resistant melanoma cells . These results suggest that altered expression of genes that regulate cellular proliferation and growth may be a determinant of metastasis and drug sensitivity of tumor cells.

J Neurooncol, 1992 Sep, 14(1), 37 - 43
P-glycoprotein expression in brain tumors; Henson JW et al.; Overexpression of P-glycoprotein (P-gp) in cancer cells can result in resistance to several chemotherapy agents (multidrug resistance) including doxorubicin and vincristine . The drugs to which resistance develops also penetrate the blood brain barrier poorly . P-gp expression in brain capillary endothelial cells suggests that P-gp may restrict drug entry into brain tumors and thus be another mechanism of drug resistance . To seek evidence for either of these roles in the drug resistance of brain tumors, we examined the location of expression of P-gp in 49 brain tumors, using an anti-P-gp mouse monoclonal antibody and immunohistochemistry . P-gp expression was observed in tumor cells of two glioblastomas and a meningeal sarcoma but not in low-grade primary or metastatic tumors . In low-grade primary tumors, P-gp was present in all vascular endothelial cells . In the vascular endothelial cells of anaplastic primary brain tumors and brain metastases, P-gp expression was heterogeneous or absent . These findings are consistent with a role for P-gp in the resistance of some brain tumors to chemotherapy agents.

Br J Haematol, 1992 Sep, 82(1), 161 - 8
Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123; Ludescher C et al.; Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells . Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation . Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples . Using two-colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed . In three cases the blast cells showed a rapid and marked Rh123 efflux . In the presence of MDR inhibitors these cells retained Rh123 . To determine whether the efflux of Rh123 was associated with P-gp expression, the leukaemic cells were stained with the monoclonal antibody MRK-16 . In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA . In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P-gp . In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening . Additionally, two-colour flow cytometry was much easier to perform with Rh123 than with daunorubicin . Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.

Ann Hematol, 1992 Sep, 65(3), 124 - 30
Flow cytometric analysis of P-glycoprotein in normal and leukemic cells; Tiirikainen MI et al.; Classical multidrug resistance is characterized by overexpression of a membrane protein, P-glycoprotein, which acts like a drug-extruding pump, reducing accumulation of cytotoxic drugs inside malignant cells . We have developed a simple method for detecting an intracellular epitope of P-glycoprotein in normal and leukemic cells by the monoclonal antibody JSB-1 and fluorescence-activated flow cytometry . Permeabilization of blood and bone marrow cells in unprocessed samples is achieved by a commercially available red blood cell lysing solution which excellently preserves the light scatter properties of leukocytes . The method is suitable for analyzing samples in clinical routine . Lower than 1% reactivity was seen in the lymphoid gate of normal peripheral blood and bone marrow samples as compared with over 60% of reacting cells in some leukemic samples . Twelve patients with acute de novo leukemia were studied at presentation, 13 patients at a refractory stage, and 28 in remission . There was a positive correlation between the P-glycoprotein and the CD34 expression in acute myelogenous leukemia and an association between the P-glycoprotein expression and the blast count in both acute myelogenous and lymphatic leukemias.

Cancer Res, 1992 Sep 1, 52(17), 4735 - 40
Reversal of multidrug resistance by two novel indole derivatives; Kadam S et al.; Two new fused indoles were found to overcome multidrug resistance in P388/Adr cells in vitro . These agents potentiated the cytotoxicity of the antitumor drugs Adriamycin, vinblastine, and vincristine in multidrug-resistant cells with no effect on drug-sensitive parent P388 cells . They significantly increased the ATP-dependent accumulation of {3H}-vinblastine and inhibited efflux of the labeled drug from resistant cells . These compounds also inhibited photoaffinity labeling of P-glycoprotein by {3H}azidopine in P388/Adr cells and membranes isolated from these cells . In addition, the calcium antagonist activity of these compounds was very weak compared with that of verapamil . These data suggest that the compounds reported here may specifically overcome multidrug resistance without the serious hypotensive effects associated with calcium antagonists and that this activity may be independent of their ability to block calcium transport.

Leuk Lymphoma, 1992 Sep, 8(1-2), 9 - 14
Multidrug resistance in acute leukemia: a conserved physiologic function; List AF et al.; Native resistance to conventional chemotherapy remains an important cause of treatment failure in the adult acute leukemias . Delineation of cellular mechanisms of drug resistance therefore represents a prerequisite to the development of more effective treatment strategies . The multidrug resistance (MDR) phenotype represents one such mechanism of resistance with direct clinical relevance . This phenotype occurs normally in certain mammalian tissues, and is detectable in tumor cell lines selected for resistance to naturally occurring antineoplastics . The mdr1 gene or its glycoprotein product, P-glycoprotein, is detected with high frequency in secondary acute myeloid leukemia (AML) and poor-risk subsets of acute lymphoblastic leukemia . In prospective studies in AML, MDR overexpression is an independent determinant of response to treatment and overall survival with conventional-dose induction regimens . Investigations of mdr1 regulation in normal hematopoietic elements has shown a pattern which corresponds to its regulation in acute leukemia, explaining the linkage of mdr1 to specific cellular phenotypes . Therapeutic trials are now in progress to test the ability of various MDR-reversal agents to restore chemotherapy sensitivity in high-risk acute leukemias.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 164 - 70
Effects of Taxotere on murine and human tumor cell lines; Riou JF et al.; Taxotere (RP 56976, NSC 628503), an analog of taxol, is an inhibitor of depolymerisation of microtubules and is currently in Phase I clinical trials . Comparisons of the cytotoxicities of Taxotere and taxol have been studied on several murine (P388, SVras) and human cell lines (Calc18, HCT116, T24, N417, KB) . Taxotere was found more potent than taxol (1.3-12 fold), a result which could be explained by its higher affinity than taxol for microtubules . In agreement with its postulated mechanism of action, Taxotere is more cytotoxic on proliferating than on non proliferating N417 cells and does not inhibit cellular DNA, RNA and protein synthesis . Taxotere gives partial cross resistance on P-glycoprotein resistant P388/DOX cell line, in contrast to taxol which gives a complete cross resistance . On the other hand, no cross resistances were observed on Calc18/AM and P388/CPT5 cell lines, bearing modified activities of topoisomerase II and topoisomerase I, respectively . These results underline the higher cytotoxic activity of Taxotere compared to taxol, and the lack of cross resistance of that class of agent with the topoisomerase I and II-related multidrug resistance phenotypes.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 488 - 92
Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells; Sakurada K et al.; Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells . Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles . In all cases, calyculin A was more potent than okadaic acid . Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively . It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.

Biochim Biophys Acta, 1992 Aug 24, 1109(2), 161 - 71
Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells; Doige CA et al.; Multidrug resistant (MDR) cells overexpress a 170-180 kDa membrane glycoprotein, the P-glycoprotein, which is believed to export drugs in an ATP-dependent manner . Plasma membrane vesicles from the MDR CHRC5 cell line, but not the AuxB1 drug-sensitive parent, showed uptake of {3H}colchicine and {3H}vinblastine that was stimulated by the presence of ATP and an ATP-regenerating system . Steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min . Non-hydrolysable ATP analogues were unable to support drug uptake, indicating that ATP hydrolysis is essential for transport . ATP-stimulated drug uptake appeared to result from drug transport into inside-out vesicles, since uptake was osmotically sensitive and could be prevented by detergent permeabilization . Steady-state uptake was half-maximal at 100 microM colchicine and 200 nM vinblastine and was inhibited by a 10-100-fold excess of MDR drugs and chemosensitizers, in the order vinblastine greater than verapamil greater than daunomycin greater than colchicine . In addition to being vanadate-sensitive, drug uptake was inhibited by 10-200 microM concentrations of several sulfhydryl-modifying reagents, suggesting that cysteine residues play an important role in drug transport . Vesicular colchicine was rapidly exchanged by an excess of unlabelled drug, demonstrating that drug association is the net result of opposing colchicine fluxes across the membrane.

Biochim Biophys Acta, 1992 Aug 24, 1109(2), 149 - 60
ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells; Doige CA et al.; In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs . The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity . As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form . Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS . The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells . The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp . This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein . Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM . P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate . Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.

FEBS Lett, 1992 Aug 17, 308(2), 175 - 8
Modulation of expression of multidrug resistance gene (mdr-1) by adriamycin; Kato S et al.; The acquired resistance to various drugs in cancer is mediated by P-glycoprotein (P-gp) which is encoded by the mdr-1 gene . An increased level of mdr-1/P-gp was demonstrated after chemotherapy administered to treat cancer in humans . To clarify the direct effect of anticancer drugs on mdr-1/P-gp expression, we investigated the change in transport of adriamycin (ADR), and the expression of the mdr-1 gene and P-gp in an ADR-treated, multidrug-resistant leukemic cell line (K562/ADR500) . The addition of ADR induced the over-expression of mdr-1/P-gp, which led to a transient decrease in the intracellular accumulation of ADR although the difference was not statistically significant . A maximal effect was observed after 4 h incubation, returning to the baseline level after further incubation for 12-24 h . The phosphorylation of P-gp was inversely correlated with the levels of P-gp . These observations suggest that ADR itself modulates both the expression and function of P-gp . Determination of the optimal schedule for administering adriamycin is essential to achieving the optimal effect in treating cancer.

Cancer Res, 1992 Aug 15, 52(16), 4427 - 32
Monoclonal antibody MRK16 reverses the multidrug resistance of multidrug-resistant transgenic mice; Mickisch GH et al.; Using multidrug-resistant (MDR)-transgenic mice, whose bone marrow cells express the human MDR1 gene at a level approximately equal to that found in many human cancers, we determined the efficacy of human-specific anti-P-glycoprotein monoclonal antibody MRK16 in overcoming multidrug resistance in an intact animal . MRK16 alone (2 mg) did not significantly affect the WBC counts of the MDR-transgenic mice, but MRK16, as well as the F(ab')2 fragments of MRK16, led to a dose-dependent circumvention of bone marrow resistance against daunomycin, doxorubicin, vincristine, vinblastine, etoposide, and taxol . This sensitizing effect could not be enhanced by combining MRK16 with low molecular weight chemosensitizing agents such as verapamil, quinine, quinidine, or cyclosporin A . We also investigated the concept of specifically targeting and killing multidrug-resistant cells by using MRK16 coupled to Pseudomonas exotoxin (PE) . MRK16-PE resulted in a dose-dependent killing of bone marrow cells in MDR-transgenic mice, whereas no bone marrow toxicity was observed in normal control mice . Administration of excess MRK16 prior to injection of MRK16-PE successfully blocked the effect of MRK16-PE . MOPC-PE, a non-MDR-related control monoclonal antibody conjugate, did not target and kill multidrug-resistant bone marrow cells in MDR-transgenic mice . Thus, these immunological approaches to reversing multidrug resistance appear to be both specific and effective.

Cancer Res, 1992 Aug 15, 52(16), 4361 - 71
Cytogenetic alterations associated with P-glycoprotein- and non-P-glycoprotein-mediated multidrug resistance in SW-1573 human lung tumor cell lines; Nieuwint AW et al.; Multidrug resistance can be induced in mammalian cells by selection with a single cytotoxic agent . Overproduction of the energy-dependent drug efflux pump P-glycoprotein, encoded by the mdr1 gene, has been identified as the cause of one form of multidrug resistance . The molecular basis of other forms of multidrug resistance is unknown . Doxorubicin selection of the human squamous lung cancer cell line SW-1573 resulted in multidrug-resistant sublines in which a non-P-glycoprotein-mediated form of multidrug resistance precedes mdr1 expression . Here we present a cytogenetic analysis of both non-P-glycoprotein-mediated multidrug-resistant and P-glycoprotein-mediated multidrug-resistant sublines derived from SW-1573 . Three independently derived non-P-glycoprotein-mediated multidrug-resistant sublines showed a heterozygous deletion of the short arm of chromosome 2 (p23-pter), whereas alterations of chromosome 7 were present in the P-glycoprotein-mediated multidrug-resistant cell lines . In one series of clonally derived P-glycoprotein-mediated multidrug-resistant sublines, mdr1 overexpression was accompanied by various markers of chromosome 7 with breakpoints at 7q22, the mdr1 gene being known to be located at 7q21.1 . Our data suggest that in SW-1573 cells acquisition of non-P-glycoprotein-mediated multidrug resistance is accompanied by a specific deletion or a translocation involving the short arm of chromosome 2, whereas in the emergence of P-glycoprotein-mediated multidrug resistance a rearrangement of the long arm of chromosome 7 is a critical event.

Nature, 1992 Aug 13, 358(6387), 591 - 3
The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis; Zhang Y et al.; Tuberculosis is responsible for one in four of all avoidable adult deaths in developing countries . Increased frequency and accelerated fatality of the disease among individuals infected with human immunodeficiency virus has raised worldwide concern that control programmes may be inadequate, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in several recent fatal outbreaks in the United States . Isonicotinic acid hydrazide (isoniazid, INH) forms the core of antituberculosis regimens; however, clinical isolates that are resistant to INH show reduced catalase activity and a relative lack of virulence in guinea-pigs . Here we use mycobacterial genetics to study the molecular basis of INH resistance . A single M . tuberculosis gene, katG, encoding both catalase and peroxidase, restored sensitivity to INH in a resistant mutant of Mycobacterium smegmatis, and conferred INH susceptibility in some strains of Escherichia coli . Deletion of katG from the chromosome was associated with INH resistance in two patient isolates of M . tuberculosis.

J La State Med Soc, 1992 Aug, 144(8), 379 - 82
New developments in mycobacteria identification: public health laboratory modernization; Dupree WG et al.; Clinical laboratory mycobacteriology has traditionally involved long delays for results, primarily due to the slow growth rate of most members of the genus Mycobacterium . Several new methodologies are now available that enable dramatic reductions in turn-around times . These methodologies include the Bactec TB System for isolation and drug susceptibility testing of Mycobacterium tuberculosis, the use of DNA probes, and high performance liquid chromatography (HPLC) for the identification of mycobacterial isolates . The spread of multidrug-resistant tuberculosis has made it imperative that laboratories take advantage of these new methodologies whenever possible . The Mycobacteriology Unit of the Louisiana Office of Public Health's Division of Laboratories has pioneered the use of these methodologies in a public health laboratory setting . Great reductions in turn-around times have been achieved and further reductions are expected as the methods are refined and adapted to the needs of a high-volume public health laboratory.

Biochem Pharmacol, 1992 Aug 4, 44(3), 509 - 17
Doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance; Cuvier C et al.; We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer . Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line . These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively . Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e . cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox . In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p . injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor . (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF) . In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e . whether free or carried by nanospheres . Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox . Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively . Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS . Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance . LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.

Cancer Res, 1992 Aug 1, 52(15), 4121 - 9
Structure-activity study and design of multidrug-resistant reversal compounds by a computer automated structure evaluation methodology; Klopman G et al.; We have studied the relation between the structure and the multidrug resistance-reversal activity of a set of diverse chemicals with the MULTICASE structure-activity program . A number of key structural features were identified as being related to multidrug resistance reversal activity . Using these key features, we identified seven new compounds predicted to have substantial activity . These were obtained and tested experimentally on a CHO/CHRC5 cell line derived from the AB1 Chinese hamster ovary line in the presence of vincristine and vinblastine . Of the seven compounds tested so far, four showed substantial reversal activity, the most potent of them exhibiting activity at par with verapamil.

Ann Intern Med, 1992 Aug 1, 117(3), 251 - 3
A leap of faith . What can we do to curtail intrainstitutional transmission of tuberculosis?
Iseman MD.
Large-scale, epidemic transmission of tuberculosis to patients, nonprofessional staff, nurses, and physicians has been documented recently in hospitals, clinics, acquired immunodeficiency syndrome (AIDS) residencies, and correctional facilities . Prominent factors in these outbreaks have included human immunodeficiency virus (HIV) infection and AIDS, delayed diagnosis of tuberculosis, multidrug-resistant strains of tuberculosis that resulted in protracted shedding of mycobacteria, and ventilation patterns in buildings that resulted in the accumulation of infectious particles . Multiple deaths from tuberculosis have resulted . Various strategies, including vaccines, masks, augmented ventilation, air filters, and ultraviolet irradiation have been proposed to control this situation . Although no well-controlled studies exist to document the utility of any of these modalities, ultraviolet germicidal irradiation seems both the best theoretical model and the most practical tactic . Ultraviolet systems should be widely deployed throughout high-risk institutions.

J Trop Med Hyg, 1992 Aug, 95(4), 284 - 7
Multidrug resistant enteric fever; Chandra R et al.; Multidrug resistant typhoid fever (MDRT) is becoming an alarming public health problem in and around Pondicherry, South India . A retrospective review of the multidrug resistant typhoid fever cases admitted to the paediatrics ward of JIPMER Hospital, Pondicherry (India) during 1990 is presented . Prolonged pyrexia, chills and rigors, toxaemia, and tender hepatomegaly often more than 3 cm below the costal margin (often without splenomegaly) were striking features of MDRT cases . The incidence of complications was also greater . Positive blood cultures were observed even after weeks of antibiotic therapy, indicating persistent bacteraemia; resistance was almost always observed for multiple drugs (two or more) . The fluoroquinolone group of drugs such as ciprofloxacin have been found to be the best for MDRT in terms of rapid response and cost effectiveness . Cefotaxime has moderate efficacy.

J La State Med Soc, 1992 Aug, 144(8), 371 - 3
Attacking today's tuberculosis problem: a multifaceted, coordinated effort; Degraw C et al.; Tuberculosis is making a comeback in communities across the nation . Increased rates of the disease, particularly with those having HIV/AIDS, have sounded the alarm that quick and decisive action is needed to halt the spread of TB . Multidrug-resistant TB is becoming a primary concern with public health officials . Specific plans and efforts, instigated by the Centers for Disease Control, have outlined the appropriate steps local public health workers, the medical community, and civic and community organizations should take in order to eliminate TB by the year 2010 . With the creation of the Task Force on Drug Resistant Tuberculosis, Louisiana has a vehicle with which to combat its growing TB problem.

J La State Med Soc, 1992 Aug, 144(8), 357 - 61
AIDS and multidrug-resistant tuberculosis: an epidemic transforms an old disease; Farley TA; Since 1985, tuberculosis case counts in the United States have increased, primarily because of the influence of the HIV epidemic . In addition, during this time outbreaks of multidrug-resistant tuberculosis among patients with AIDS or HIV infection have been reported in New York City and Florida . These outbreaks have occurred in hospitals and prisons and have been characterized by high case fatality rates, disease transmission within the institutions, and high infection rates in health care workers . The increase in tuberculosis rates and the outbreaks have raised concern that multidrug-resistant tuberculosis could become a widespread problem in the United States . Dealing with tuberculosis in the 1990s will require reconsideration of our current methods of tuberculosis prevention, diagnosis, treatment, and control.

Pathol Res Pract, 1992 Aug, 188(6), 804 - 7
The genetic basis of multidrug resistance; Pauly M et al.; Cellular multidrug resistance, a common side-effect of anticancer chemotherapy frequently leading to failure of the treatment, has been characterized as an acquired resistance to several antimitotic drugs simultaneously . Multidrug resistance could mainly be attributed to the overexpression of the P-170 glycoprotein, considered as a drug-efflux pump encoded by the mdr 1 gene . Overexpression of this protein can be induced either by an accidental amplification or activation or both of the mdr 1 gene . Recent investigations focused on these mechanisms, aiming at a better understanding of the appearance of multidrug resistance during a chemotherapy . P-glycoprotein mediated drug resistance, however, is only one, albeit quite an important detoxification pathway, and some observations revealed genetic interactions with other systems . On the basis of this new knowledge, the development of novel therapeutic strategies to circumvent this clinical side-effect of cancer treatment has already begun.

Invest New Drugs, 1992 Aug, 10(3), 137 - 48
In vitro and in vivo circumvention of multidrug resistance by Servier 9788, a novel triazinoaminopiperidine derivative; Pierre A et al.; S 9788 is a novel triazinoaminopiperidine derivative which does not belong to any of the classes of compounds known to reverse multidrug resistance (MDR) . S 9788 was far more potent than verapamil (VRP) in reversing resistance to adriamycin (ADR) in the ADR-selected murine leukaemia cell lines P388/ADR-1 and P388/ADR-10, and the human chronic myelogenous leukaemia K562/R . Fold reversion with S 9788 (5 microM) was, respectively, 3.5, 5.4 and 11.3 times greater than that with VRP (5 microM) . S 9788 was also a more potent reversant of ADR resistance in the intrinsically resistant human colon adenocarcinoma COLO 320DM (2.3 fold), and of vincristine (VCR) resistance in the human MDR1 gene-transfected squamous lung carcinoma line S1/tMDR1 (5.6 fold) . The activity of S 9788 depended on both the MDR cell line and the cytotoxic agent . S 9788 (50-100 mg/kg/d) administered IP once a day on days 1-4 resulted in a dose-dependent increase in the chemotherapeutic effect of VCR (0.25 mg/kg/d) in P388/VCR - bearing mice and ADR (4 mg/kg/d) in P388/ADR - bearing mice . Increases in antitumor activity were (% T/C) of +20-34% in the P388/ADR model and + 50-78% in the P388/VCR model with respect to cytotoxic agent treatment alone . S 9788 appeared to be devoid of toxicity at its effective doses . The mechanism of action of S 9788 is unknown but S 9788 (0.5-10 microM) induced a dose-dependent increase in ADR accumulation in KB-Al cells and compared to verapamil its effect was twice as active and approximately seven times more potent . We conclude that S 9788 is a novel agent capable of reversing MDR in vitro and in vivo, and whose pharmacological profile warrants its selection as a candidate drug for eventual assessment in the clinic.

Chem Pharm Bull (Tokyo), 1992 Aug, 40(8), 2182 - 4
Sensitivity to antitumor drugs and vinblastine binding to membrane in rat ascites hepatoma AH66 cells; Wakusawa S et al.; Rat ascites hepatoma AH66 cells have lower sensitivity to Vinca alkaloids and anthracycline antibiotics than AH66F cells, a subline of AH66 cells . AH66 cells expressed P-glycoprotein, while the protein was not detectable in AH66F cells . There are two affinity sites for {3H}vinblastine binding in the AH66 cell membrane, while AH66F cells have only one affinity site . The high affinity {3H}vinblastine binding in AH66 cells was inhibited by Adriamycin, verapamil, nicardipine, and reserpine . The high affinity site of the binding may be the multidrug transporter, P-glycoprotein . {3H}Vinblastine binding was not influenced by adenosine 3'-5'-monophosphate (AMP), adenosine triphosphate (ATP), or guanosine triphosphate (GTP) . The multidrug resistance in AH66 cells may depend on P-glycoprotein which is not modulated by nucleotide.

Anticancer Drugs, 1992 Aug, 3(4), 419 - 25
SDZ PSC 833 and SDZ 280-446 are the most active of various resistance-modifying agents in restoring rhodamine-123 retention within multidrug resistant P388 cells; Pourtier-Manzanedo A et al.; Multidrug resistance (MDR) of tumor cells may result from overexpression of P-glycoprotein (Pgp) but may be down-modulated by resistance-modifying agents (RMAs) . The cyclosporin SDZ PSC 833 and the cyclopeptolide SDZ 280-446 were found to be the strongest RMAs known to date for restoring the sensitivity of MDR cells to anticancer drugs, as well as for restoring their retention of daunomycin, a fluorescent anthracycline . Using rhodamine-123 (Rhod-123), another fluorescent probe of Pgp function which also differentiates sensitive and MDR cells, several RMAs were compared for their capacity to inhibit Pgp function . At variance with the data obtained with the daunomycin probe, a series of RMAs did not detectably restore Rhod-123 retention by the MDR cells . With the remaining RMAs, achieving the same levels of Rhod-123 retention required 3 times lower RMA concentrations when the RMA was added to the MDR cells for both the initial uptake and the efflux of Rhod-123 rather than for its uptake only . Nevertheless, the data emphasized the large superiority of SDZ PSC 833 and SDZ 280-446 over all other RMAs.

Cell Growth Differ, 1992 Aug, 3(8), 531 - 40
Activation of multidrug resistance (P-glycoprotein) mdr3/mdr1a gene during the development of hepatocellular carcinoma in hepatitis B virus transgenic mice; Kuo MT et al.; The expression of multidrug resistance (mdr) genes was investigated in the livers of transgenic mice that express the human hepatitis B virus large envelope polypeptide under the transcriptional control of a liver-specific promoter . These mice develop a storage disease due to the accumulation of a nonsecretable form of hepatitis B surface antigen in the hepatocyte . Liver cell injury is followed by a hepatocellular proliferative response, dysplasia, microscopic nodular hyperplasia, and finally hepatocellular carcinoma . The expression of mdr1, mdr2, and mdr3 genes was analyzed in livers at different stages of the disease by RNase protection assay, Western blot, and immunohistochemistry . RNase protection assay revealed that mdr3 mRNA expression was moderately increased in tissue with microscopic nodular hyperplasia and significantly overexpressed in hepatocellular carcinoma but undetectable in earlier stages of the disease . Western blot using isoform-specific anti-mdr3 antibody demonstrated that the expression of mdr3 protein reflected the steady-state level of mdr3 mRNA . Immunohistochemical analyses using anti-mdr3 isoform-specific antibody and monoclonal antibody C219, which recognizes all the three mdr isoforms, demonstrated selective overexpression in preneoplastic foci during the stage of microscopic nodular hyperplasia as well as in neoplastic hepatocytes in hepatocellular carcinoma . No consistent activation of mdr1 and mdr2 (but occasional coactivation with mdr1) genes during hepatocarcinogenesis was observed . Our results suggest that the hepatocellular mdr3-specific activation mechanism is associated with the late events of hepatocarcinogenesis in this model . The predictable kinetics of mdr gene expression in this transgenic tumor model suggest that it is suitable for future studies of the mechanism of mdr gene activation and the possible pharmacological consequences for mdr3 gene expression of hepatocellular carcinoma.

Lancet, 1992 Aug 1, 340(8814), 255 - 9
Modulation of multidrug-resistant multiple myeloma by cyclosporin . The Leukaemia Group of the EORTC and the HOVON; Sonneveld P et al.; Resistance to chemotherapy in refractory multiple myeloma is frequently associated with expression of multidrug resistance (MDR) . In resistant cells, intracellular accumulation of doxorubicin and vincristine does not occur because the MDR-1 gene product, a membrane glycoprotein (PgP), is an energy-dependent efflux pump . Cyclosporin is one of several non-cytotoxic drugs that can block the function of PgP . In a prospective study, we assessed the possibility that cyclosporin could be used clinically to modulate MDR . We studied 21 patients with multiple myeloma; disease had progressed during primary chemotherapy in 6 and was resistant to VAD (vincristine, doxorubicin, dexamethasone) in 15 . The patients received cyclosporin by continuous infusion during VAD treatment; there were three cyclosporin dosage groups (5, 7.5, 10 mg/kg daily) . Serum cyclosporin concentrations adequate for MDR modulation were reached in all patients receiving 7.5 or 10 mg/kg daily . 47% (7) of the VAD-refractory patients and 48% (10) of the whole group responded to VAD . Before treatment, MDR-1 expression was present in 12 patients . After VAD plus cyclosporin, no MDR-1-positive plasma cells were present in 6 of 8 patients tested . The response rate in MDR-1-positive patients was 58% compared with 33% in all our patients . Toxic effects were mild and reversible and did not include nephrotoxic or serious cardiovascular side-effects . 12 months after the start of treatment, survival was 85%, and disease-free survival at a median of 9 months after the response was 65% . Thus, in multiple myeloma clinical resistance to VAD can be circumvented by cyclosporin, which enables the cytotoxic drugs to eliminate resistant myeloma cells.

J Urol, 1992 Aug, 148(2 Pt 1), 441 - 5
Establishment and characterization of doxorubicin-resistant human bladder cancer cell line, KK47/ADM; Kimiya K et al.; A human bladder cancer cell line resistant to doxorubicin, KK47/ADM has been established in vitro by exposing KK47 parent cells to progressively higher concentrations of the drug over a period of 16 months . The KK47/ADM was 271 times more resistant to doxorubicin than the KK47 parent . The KK47/ADM exhibited cross-resistance to doxorubicin derivatives (pirarubicin, epirubicin), vinca alkaloids (vinblastine, vincristine) and etoposide, but not to cisplatin, carboplatin, mitomycin C, peplomycin and methotrexate . Unlike the KK47 parent, about 70% of the KK47/ADM cells showed a positive reaction with monoclonal antibody against P-glycoprotein, MRK16 . Uptake and efflux studies with {14C}doxorubicin indicated that the resistance exhibited by the KK47/ADM line was mainly due to a lower accumulation of the drug caused by an increased active efflux, and these were reversed in the presence of verapamil . Although verapamil enhanced doxorubicin sensitivity of KK47/ADM, a complete overcoming of the resistance could not be obtained . These two lines with different chemosensitivity are thus considered to be a useful model for developing new chemotherapeutic strategies against multidrug resistant bladder cancer.

Anticancer Drugs, 1992 Aug, 3(4), 359 - 66
Multidrug resistance in a small cell lung cancer line: rapid selection with etoposide and differential chemosensitization with cyclosporin A; Glisson BS et al.; We developed a multidrug resistant small cell lung cancer line, VPR-2, by exposing H69 parent cells to etoposide (20 microM) for 1 h daily for 3 days every 21-28 days, a schedule similar to that used in the clinic . Resistance (20-fold) to the cytostatic and DNA cleavage activities of etoposide emerged after the third treatment, and this phenotype was stable in the absence of drug exposure for 2.5 years . VPR-2 cells exhibited cross resistance to intercalating agents and vinca alkaloids, but remained sensitive to X-radiation, cisplatin and 5-fluorouracil . The human mdr1 gene was overexpressed in the resistant line, but steady-state concentrations of etoposide were reduced only 1.5-fold . Topoisomerase II catalytic and etoposide stimulated DNA cleavage activity in nuclear extracts from both lines were identical despite retention of a 3-fold level of resistance to etoposide-induced strand breaks in isolated nuclei from VPR-2 cells . Cyclosporin A and verapamil, both of which bind to P-glycoprotein, enhanced accumulation of etoposide in VPR-2 cells, and H69 cells to a lesser extent . Yet only cyclosporin A was effective in differentially enhancing etoposide cytostasis in VPR-2 relative to H69 . In VPR-2 whole cells, cyclosporin A enhanced etoposide-induced DNA single-strand break frequency 9-fold but had no effect in isolated nuclei . Rapid selection of this line with a clinically relevant drug exposure schema and stability of the resistant phenotype suggest these cells may have been a steady subpopulation of the parent line through years of serial passage in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Jul 21, 31(28), 6366 - 72
Modulation of P-glycoprotein phosphorylation and drug transport by sodium butyrate; Bates SE et al.; P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents . Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased . In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux . Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function . This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D . Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp . Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment . Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation . These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.

MMWR Morb Mortal Wkly Rep, 1992 Jul 17, 41(28), 507 - 9
Transmission of multidrug-resistant tuberculosis among immunocompromised persons in a correctional system--New York, 1991; Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage; MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, United KingdomFlow cytometry and laser scanning confocal imaging have been used to analyze the uptake of the anticancer topoisomerase II poison mitoxantrone by intact mammalian cells and the results correlated with the induction of DNA damage . Unlike Adriamycin, mitoxantrone displays only minimal levels of red fluorescence when excited at 514 wavelength . However, using these excitation and emission conditions, flow cytometry could detect low levels of fluorescence in human transformed fibroblasts exposed to high concentrations (5-20 microM) of mitoxantrone for 1 h . Over this dose range whole cell fluorescence was a function of cell size and increased with drug concentration while drug-induced DNA-protein cross-linking showed saturation . Confocal microscopy revealed the time- and dose-dependent appearance of fluorescence, interpreted here as reflecting the disposition of drug molecules, preferentially within the cytoplasm, nuclear membrane, and nucleoli . This pattern contrasted with the intense intranuclear fluorescence observed in Adriamycin-treated human cells . Loss of the nuclear membrane during mitosis resulted in an apparent increase in chromatin-associated fluorescence . Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosomal, location for fluorescence from human cells exposed for 1 h to a low but cytotoxic concentration (0.1 microM, yielding approximately 90% cell kill) of mitoxantrone . At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNA-protein cross-linking . Murine cells, displaying mitoxantrone resistance as part of the P-glycoprotein-mediated multidrug resistance phenotype, showed specific extinction of mitoxantrone-associated fluorescence from inside nuclei but not from within extranuclear compartments . The study demonstrates the feasibility of high resolution studies on the intracellular distribution of mitoxantrone in intact living cells . We suggest a mechanism by which cytoplasmic sequestration of mitoxantrone may be important in determining the response of normal and multidrug-resistant cells as they attempt to progress through mitosis.

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