Atypical Multiple Drug Resistance in a Human Leukemic Cell Line Selected for Resistance to Teniposide (VM-26)1

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[CANCER RESEARCH 47, 1297-1301, March 1, 1987] Atypical Multiple Drug Resistance in a Human Leukemic Cell Line Selected for Resistance to Teniposide (VM-26)1

Mary K. Danks, Jack C. Yalowich2, and William T. Beck3

Department of Biochemical and Clinical Pharmacology [M. K. D.. W. T. B.) and Pharmacokinetics and Pharmacodynamics Section, Pharmaceutical Division [J. C. Y], St. Jude Children's Research Hospital. Memphis, Tennessee 38101

ABSTRACT and an mAMSA-resistant P388 line (12) and HL-60 line (20) that are cross-resistant to the epipodophyllotoxins and sensitive Resistance to the cytotoxic effects of many natural product drugs after to the Vinca alkaloids. exposure to a single agent is a common observation. The classes of drugs included in the "classic" multiple drug resistance phenotype are Vinca We report here the development and characterization of a teniposide-resistant human leukemic lymphoblastic cell line alkaloids, anthracyclines, epipodophyllotoxins, and antibiotics. We re that expresses "atypical" MDR. This line (CEM/VM-1) has a port here the characterization of a human leukemic cell line (CEM/VM- 1) with "atypical" multiple drug resistance: despite resistance and cross- pattern of cross-resistance similar to cells having the "classic" resistance to etoposide, anthracyclines, mitoxantrone, and 4'-((9-acridi- MDR phenotype with the exception that it is completely sen nyl)amino)methanesulphon-in-anisidide (mA.MSA), these cells retain sitive to the Vinca alkaloids. Also, in contrast to the "classic" sensitivity to the Vinca alkaloids. Further, even though this cell line is MDR phenotype, the CEM/VM-1 cells accumulate nearly as Â»40-foldcross-resistant to the cytotoxic effect of etoposide (VP-16), it much drug as do drug-sensitive CEM cells, suggesting a mech is similar to drug-sensitive CEM cells in the cellular pharmacology of [3H)VP-16 as determined by zero time binding, initial influx rate, steady anism of resistance other than decreased drug retention. Com parison of the CEM/VM-1 line and our well-characterized state drug concentration, and unidirectional efflux. Our studies suggest "classic" MDR cell line CEM/VLBIOO is also reported. A pre that the resistance of CEM/VM-1 cells to epipodophyllotoxins is due to an altered interaction between drug and its cellular target(s) by a mech liminary account of this work has been presented (21). anism unrelated to the decreased cellular concentration of drug associated with the "classic" multiple drug resistance phenotype. MATERIALS AND METHODS Chemicals and Supplies. Eagle's minimal essential medium, with INTRODUCTION Earle's spinner salts, was purchased from Grand Island Biological Co. The simultaneous resistance to several classes of "natural (Grand Island, NY) and fetal bovine serum was from Hyclone Labora product" drugs in vivo and in vitro after exposure to a chemically tories (Sterile-Systems, Inc., Logan, UT). VCR and VLB were generous dissimilar agent is termed M DR.4 This is most frequently seen gifts of Eli Lilly and Co. (Indianapolis, IN). DOX was from Adria Laboratories (Wilmington, DE), and bleomycin, VM-26, and VP-16 with cells selected for resistance to alkaloids or to anthracy were provided by Bristol-Myers Laboratories (Syracuse, NY); dimethyl clines. Compared to drug-sensitive cells, MDR cells usually sulfoxide was from Fisher Scientific Co. (Fair Lawn, NJ). The drug have a decreased steady state drug level (1-5), decreased reten solutions were prepared as reported previously (22); mAMSA, obtained tion of drug (1,6, 7), and specific changes in plasma membrane from the Drug Synthesis and Chemistry Branch, DTP, DCT, National glycoproteins (8-11). The relationship between the pharmaco Cancer Institute, was dissolved in 100% dimethyl sulfoxide and then logical and biochemical changes is unclear. The mechanism by diluted with 0.9% sodium chloride to a concentration of 1 HIMbefore which MDR cells accumulate and/or retain less drug than its dilution to final concentration with 0.9% sodium chloride. Methanol and acetonitrile were purchased from Burdick and Jackson Laboratories sensitive cells is also unknown. (Muskegon, MI). | 'II|VI'-I6 was purchased from Moravek Biochemi- Resistance or cross-resistance to the epipodophyllotoxins (semisynthetic derivatives of the "natural product" podophyl- cals. Brea, CA; and [carÂ¿ary/-uC]inulin was from New England Nuclear Products, Boston, MA. Propidium iodide was obtained from Calbi- lotoxin) and its relationship to MDR has been studied in cell ochem (San Diego, CA). AHother chemicals and supplies were obtained lines developed in several laboratories (12-20). While most of from commercial sources. these lines showed decreased drug accumulation and cross- Cells and Culture Conditions. Teniposide-resistant cells were selected resistance to the Vinca alkaloids as is typical of the MDR from the original parental CCRF-CEM cell line by growth in the phenotype, there are reports of a teniposide-resistant Chinese intermittent presence of sublethal concentrations of drug as described hamster ovary line with minimal cross-resistance to VLB (17), for our VCR-resistant cells (22). The CEM/VM-1 line was cloned by limiting dilution (23) and retained its resistance to VM-26 for at least Received 8/15/86; revised 11/20/86; accepted 12/4/86. 2 months in the absence of drug. All cells used for experiments were The costs of publication of this article were defrayed in part by the payment grown in drug-free media for at least 1 week. We have described the of page charges. This article must therefore be hereby marked advertisement in selection and maintenance conditions for the (TAI/VI,HH>), cell line accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by Research Grant CA30103 (to W. T. B.) and Cancer elsewhere (11). Center Support (CORE) Grant CA21765, from the National Cancer Institute, Growth Inhibition Assay. The concentration of drug required to Bethesda, MD; by National Research Service Award CA06795 (to M. K. D.) inhibit growth of cells by 50% (ICjo) in 48 h was determined for each from the National Cancer Institute; and by American Lebanese Syrian Associated cell line, as described previously (22). Fold resistance is calculated by Charities. dividing the K '<â€žofthe resistant line by the K \(>of that drug in the 2Leukemia Society of America, Special Fellow; Recipient of Biomedicai Research Support Grant RR05S84-21 from NIH. CEM parent line. 3To whom requests for reprints should be addressed, at St. Jude Children's DNA Content. CEM, CEM/VLB100 and CEM/VM-1 cells were Research Hospital, Department of Biochemical and Clinical Pharmacology, 332 analyzed by now cytometry for DNA content and cell cycle distribution North Lauderdale, P.O. Box 318, Memphis, TN 38101. 4The abbreviations used are: MDR, multiple drug resistance; CEM, parent as described previously (24). CCRF-CEM human lymphoblast cell line; CEM/VLB100, CEM subline selected Cellular Pharmacology of VP-16. Influx, efflux, accumulation, and for resistance to vinblastine; VLB, vinblastine; CEM/VM-1, cloned CEM subline retention of ['HJVP-16 by the cell lines were determined by the cold selected for resistance to VM-26; VM-26, teniposide [4'-demethylepipodophyllotoxin 9-(4,6-O-2-thenylidene-0-r>glucopyranoside)]; VP-16-213, VP-16, [eto buffer wash method, as previously described (25). Intracellular water poside; 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-/3-r>glucopyrano- volume was determined from the difference between the wet and dry side)]; niAMSA. 4'-{(9-acridinyl)amino)methanesuIphon-m-anisidide; DOX,dox- weights of cell pellets, taking into account the extracellular volume, as orubicin; VCR, vincristine; HPLC, high pressure liquid chromatography. determined with [14C]inulin. Intracellular water (ml/g dry weight) was 1297 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. DRUG RESISTANCE TO TENIPOSIDE IN A HUMAN LEUKEMIC CELL LINE determined to be 4.3, 4.5, and 3.9 for CEM, CEM/VM-1, and CEM/ Table 2 Doubling times and cell cycle distribution of drug-sensitive and -resistant VLBioo cells, respectively. Data are expressed either as nmol VP-16/g cell lines dry weight or /imol VP-16/liter cell water as previously reported (25). of cells Some drug accumulation studies were also done using a standard in phases of cycle SDfG,44Â±4(% Â± silicone oil technique (26) for separating the cells from the extracellular time(h)"31 medium. At indicated times, aliquots of cell suspension were layered CelllineCCRF-CEMCEM/VM-1 +M11 over 0.5 ml Nyosil 50 oil (William F. Nye, Inc., New Bedford, MA) in Â±533 Â±5 Â±3' a microcentrifuge tube and centrifuged for 5 min at room temperature. Â±6 53 Â±8 38 Â±8 10Â±3 The [3H]VP-16 used in the experiments was 93-97% pure by HPLC CEM/VLB.ooDoubling32 Â±7Distribution50 Â±7S45 37 Â±8Gj 14 Â±4 " Determined graphically and defined as the number of hours required for cells (27). HPLC Analysis for Possible Cellular Metabolism of |3H)VP-16. Cell in log phase growth to double in number. Values are the mean Â±SD of four to five experiments. pellets were prepared for drug metabolism studies after a 60-min * Determined by DNA content, as indicated in "Materials and Methods." incubation at 37*C using an initial extracellular VP-16 concentration ' Percentages do not equal 100 when added, since these are mean values of of 170 pM. After incubation with drug, cell pellets containing [3H]VP- three separate experiments. 16 were treated twice with 100 M' of 100% methanol; this treatment extracted 100% of the radiolabel. Analysis for potential metabolites of [3H]VP-16 was by the HPLC method of Sinkule and Evans (27). Statistics. Comparison of rates of influx of VP-16 in the three cell lines was done with a Wald \- analysis (28). /' values for simple comparisons of drug accumulation, retention, and r were obtained by Student's r test (28).

RESULTS Drug Cytotoxicity. Forty-eight-h IC50 values of several drugs for the parent drug-sensitive (CEM), VLB-resistant (CEM/ VLB,oo), and VM-resistant (CEM/VM-1) cell lines are shown in Table 1. Each of the resistant lines is equally resistant or cross-resistant to VM-26 and VP-16, permitting direct com parison of degrees of cross-resistance and cellular pharmacol ogy data. They are also each =20-fold cross-resistant to mitoxantrone and 80- to 150-fold cross-resistant to DOX. The CEM/ VM-1 line is resistant to mAMSA, but sensitive to the Finca alkaloids VLB and VCR. By contrast, the CEM/VLBIOO cells are resistant to Vinca alkaloids. Both resistant lines are sensitive Fig. 1. Initial uptake of I3H]VP-16 by CEM, CEM/VM-1, and CEM/VLB,Â«, to bleomycin. cells. External concentration of [3H]VP-16 was 5 Â»IM.Eachpoint is the mean of triplicate determinations from three experiments run on separate days. liur\. Flow Cytometric Findings. Since similarities or differences in mean â€¢SE.See -Materials and Methods" for experimental details. degrees of resistance to the drugs in Table 1 could be due to differences in growth rates or cell cycle distribution, we deter mined doubling times for each line and analyzed, with cells in 10 s to determine initial influx rates in the three cell lines (Fig. log phase growth, the DNA content of each line. Results of 1). Using the first three time points (0.5-2.0 s), comparison of these experiments are shown in Table 2. The similarity of both the rates of drug influx (slopes) showed only a 2-fold difference these measurements in all three lines suggests that growth between CEM and CEM/VM-1 cells, but a greater than 10- kinetics do not account for contrasting cross-resistance patterns fold difference between the CEM and CEM/VLB100 cells. These in these lines, and lends further support to the comparability of initial influx rates are given in Table 3. There is no significant the other data presented in this paper. difference (P = 0.91) in zero-time binding (Y intercept value) Influx, Efflux, Steady State Accumulation, and Retention of among the cell lines (Fig. 1). |3H|VP-16. The uptake of [3H]VP-16 was measured from 0.5 to Despite the fact that the two drug-resistant lines are equally cross-resistant to VP-16, they accumulated different amounts Table 1 Cross-resistance ofCCRF-CEMsublines selected for resistance to of drug at steady state, as shown in Fig. 2. CEM/VM-1 cells VM-26 or VLB accumulated 70% as much [3H]VP-16 as the CEM cells, but Degree of resistance* CCRF-CEM the CEM/VLBioo cells accumulated less than 10% as much DrugsVM-26VP-16VLBVCRDOXMitoxantronemAMSABleomycinICwdiM)Â«48.0 [3H]VP-16 as the drug-sensitive controls. These steady state Â±39.0416 values are listed in Table 3. Â±2002.76 Removal of [3H]VP-16 from the extracellular compartment Â±2.502.40 Â±2.1029.0 results in the rapid loss of drug from all cell lines to a plateau Â±24.012.2 (Fig. 2). Statistically, these levels of nonexchangeable drug Â±14.756 (Table 3) were significantly different only when comparing the Â±71. 26 Â±0.84Â°CEM/VM-I47413'0.3"841630*3.2GEM/VLB,Â«324418620231522161.6two resistant cell lines. " 50% inhibitory concentration in a 48-h growth inhibition assay Â±SD. Values Unidirectional efflux of [3H]VP-16 was determined in these shown for CEM cells are means of 14 to 18 separate experiments. For CEM/ cell lines after "loading" for 30 min with 5 /IM [3H]VP-16 to VM-1 and CEM/VLB,oo cells, values are means of 3 lo 12 separate experiments. * 1C,,,of the resistant cell line divided by that of the parent CCRF-CEM line. achieve steady state drug levels (Fig. 2). As observed for influx, ' Units/liter. there was a similar rate of efflux of VP-16 from the parental d Degree of resistance significantly different (P < 0.001) from CEM/VLB,Â«, CEM and CEM/VM-1 cells (Table 3). (Because of the small cells. ' Degree of resistance significantly different (/> < 0.02) from CEM/VLB,,Â» standard error, these rates are statistically different; but the cells. difference is of doubtful biological significance.) However, ac- 1298 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. DRUG RESISTANCE TO TENIPOSIDE IN A HUMAN LEUKEMIC CELL LINE

Table 3 Analysis of VP-16 influx, efflux, accumulation, and retention in conditions intracellular accumulation was approximately 3.6 CEM, CEMjVM-l, and CEM/VLBIM cells MMin each cell line. concentration VP-16 Metabolism in CEM/VM-1 Cells. Since the CEM/ (nmol/g dry weight)Nonex- Influxrate(nmol/g VLBioo cells and the CEM/VM-1 cells were equally resistant to VP-16, but the cellular drug concentration in the CEM/VM- Cell line dry weight/s)Â°Efflux(iÂ»,s)'VP-16 Total changeable 1 cells was Â»8-foldhigher than in the CEM/VLB100 cells, the CEM 0.230 Â±0.084 102 Â±1 21.39 + 0.80 0.73 + 0.21 CEM/VM-1 0.123 Â±0.026 94 Â±Ie 14.63 Â±0.94 0.41 Â±0.04 question of drug metabolism was addressed. If the parent drug CEM/VLB.oo 0.021 Â±0.046'' 256Â±33C'' 1.79 Â±0.1S*' 0.20 Â±0.03' were metabolized to an inactive or less active form in the CEM/ " Includes first three points from 0.5-2.0 s. Data are presented as mean Â±SE VM-1 cells, the effective drug concentration in the two lines from three separate experiments run on separate days. Three to four measure might be similar. We therefore examined our CEM/VM-1 cells ments at each of five different times were taken as previously described (25). * Cells were incubated with 5 /IM [3H]VP-16 for 30 min, after which they were for metabolites of VP-16. No metabolites were found by HPLC washed two times in ice cold buffer. Total VP-16 was measured in triplicate and analysis (data not shown), suggesting that the mechanism of the cells were resuspended in two to three times the original volume of VP-16â€¢ free buffer at 37'C. Over the next 300 s, at least 12 measurements were made of resistance in the CEM/VM-1 cells is not due to increased cellular VP-16 concentration. The log of the fraction of exchangeable VP-16 metabolism of the parent compound. (determined from the difference between total and nonexchangeable VP-16) remaining in the cell as a function of time after resuspension was plotted (not shown) and the resulting linear efflux rate was used to calculate the i, values DISCUSSION shown. Nonexchangeable VP-16 was determined from measurement of VP-16 concentration taken 30-45 min after resuspension of cells in VP-16-free buffer. The two CEM sublines in this report are resistant to at least Data are presented as the mean Â±SE from three experiments run on different five of the eight drugs studied; therefore, each line could be said days. ' Significantly different from CEM (P < 0.01). to be "multiply drug resistant." All the drugs are natural prod ' Significantly different from CEM (P < 0.05). ucts or their semisynthetic derivatives, and all except bleomycin ' Significantly different from CEM/VM-1 (p < o.OI). f Significantly different from CEM/VM-1 (P < 0.001). have been reported to be involved in MDR (2, 8, 12, 14). The proposed mechanisms of cytotoxicity of the drugs in Table 1 include DNA damage (29-32) (DOX, mitoxantrone, bleomy cin, and mAMSA), tubulin binding (33) (VLB and VCR), and 16 r alteration of topoisomerase activity (34-37) (mAMSA, DOX, VM-26, and VP-16). However, the CEM/VLB100 line is resist ant to the Vinca alkaloids, while the reverse is true of the CEM/ VM-1 line. These contrasting cross-resistance data suggest that even though the lines are equally resistant to the epipodophyllotoxins, the mechanisms of resistance differ. In support of this conclusion, the resistance of the CEM/VLBioo line to VP-16 was likely due to the characteristic decrease in steady state drug concentration associated with the "classic" MDR phenotype. This was probably not the case for the CEM/VM-1 line. There fore, despite resistance to several classes of natural product drugs, the MDR of the CEM/VM-1 cells is "atypical" because the cellular pharmacology of VP-16 in these cells is similar to that in the CEM cells and also because the CEM/VM-1 cells Minutes are sensitive to the Vinca alkaloids. Fig. 2. Accumulation and retention of VP-16 in CEM, CEM/VM-1, and CEM/VLB,oo cells. Cells were incubated with 3.6 MM|'111V]' 16 and intracellular Our observations with the CEM/VLBioo line (Table 1) are VP-16 was determined at the times indicated. After 25 min, cells were harvested, consistent with those in the literature in that in each MDR line washed twice in ice-cold 0.85% sodium chloride, and resuspended at 37Â°Cintwo showing decreased accumulation of natural drug products, there to three times the original volume of drug-free incubation buffer. Over the next is cross-resistance to both the Vinca alkaloids and anthracy- 20 min the cell-associated epipodophyllotoxin concentration was measured. clines (4-6, 38-40). Comparable studies with lines similar to the CEM/VM-1 line are limited: of the three lines reported cumulated [3H]VP-16 leaves the CEM/VLB100 cells more slowly, the fy, being 2.5-fold greater than that observed for the other two lines (Table 3). CEM The accumulation of [3H]VP-16 by the three cell lines was = 50 - also studied using the silicon oil method (Fig. 3). By this technique, both CEM and CEM/VM-1 lines accumulated VP- 16 to a similar steady state concentration. This contrasts with the results using the cold buffer wash method where VP-16 concentrations were 30% lower in CEM/VM-1 cells than in CEM (Fig. 2). The steady state concentration of VP-16 in the CEM/VLBioo cells was about 25% that of the parent line [versus 10% by the buffer wash method (Fig. 2)]. 200 400 600 At 5 MMextracellular [3H]VP-16, CEM cells accumulated Seconds Fig. 3. Determination of |3H)VP-16 accumulation by CEM, CEM/VM-1, similar concentrations of epipodophyllotoxin regardless of the CEM/VLBiao cells using the silicone oil rapid separation method. Cells were technique used to quantitate drug uptake (5.0 MMversus 5.9 MM incubated in buffer containing 5 MM[3H]VP-16. At the indicated times, portions of the cell suspension (Â«Sx to' cells) were centrifuged through silicon oil as for wash method and silicon oil method, respectively). Accu described in "Materials and Methods." The cell pellet was analyzed for associated mulation experiments were also done by each method with an radioactivity after correction for small amounts of contaminating buffer carried extracellular concentration of [3H]VP-16 3.6 MM.Under these with cells through the silicon oil (25).

1299 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. DRUG RESISTANCE TO TEN1POSIDE IN A HUMAN LEUKEMIC CELL LINE with an "atypical M DR" phenotype (12, 17, 20), single point C., and Ozols, R. F. Adriamycin accumulation and metabolism in adriamycin-sensitive and -resistant human ovarian cancer cell lines. Biochem. I'har- drug accumulation studies have been done on one line (12,41). macol., 35:467-472, 1986. Consistent with our results, Kessel Ã©tal.(41) showed no change 4. Skovsgaard, T. Mechanism of cross-resistance between vincristine and dauno rubicin in Ehrlich ascites tumor cells. Cancer Res., 38:4722-4727, 1978. in accumulation of mAMSA or daunorubicin in the P388/ 5. Kartner, N., Shales, M., Riordan, J. R., and Ling, V. Daunorubicin-resistant AMSA line. Chinese hamster ovary cells expressing multidrug resistance and a cell-surface Comparison of results shown in Figs. 2 and 3 suggest that /"-glycoprotein. Cancer Res., Â«.-4413-4419, 1983. there is a fraction of accumulated VP-16 in both resistant lines 6. Yanovich, S., and Taub, R. N. Differences in daunomycin retention in sensitive and resistant P388 leukemic cells as determined by digitized video (but not in the parent line) that is lost from the cell by washing fluorescence microscopy. Cancer Res., 43:4167-4171, 1983. at 0Â°C;this fraction may be intracellular free drug (i.e., osmot- 7. Inaba, M., Fujikura, R., and Sakurai, Y. Active efflux common to vincristine and daunorubicin in vincristine-resistant P388 leukemia. Biochem. Pharma- ically active) or drug loosely bound in or on the cell. These col., 30: 1863-1865, 1981. results are consistent with another study reporting loss of VM- 8. Peterson, R. H. F., Meyers, M. B., Spengler, B. A., and Biedler, J. L. 26 during cold buffer washing of LI210 cells resistant to VM- Alteration of plasma membrane glycopeptides and gangliosides of Chinese hamster cells accompanying development of resistance to daunorubicin and 26 (40). vincristine. Cancer Res., 43: 222-228, 1983. Our findings are unique in that the data suggest a mecha 9. Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug perme nism^) of epipodophyllotoxin resistance in CEM/VM-1 cells ability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455: 152-162, 1976. different from that in the CEM/VLBIOO cells. Resistance to VP- 10. Garman, I)., and Center, M. S. Alterations in cell surface membranes in 16 in the CEM/VLB100 cells probably involves a "classic" MDR Chinese hamster lung cells resistant to adriamycin. Biochem. Biophys. Res. Commun., 105:157-163, 1982. mechanism with decreased drug accumulation and retention 11. Beck, W. T., Mueller, T. J., and Tanzer, L. R. Altered surface membrane associated with characteristic membrane changes (1). The glycoproteins in Vinca alkaloid-resistant human leukemic lymphoblasts. Can CEM/VM-1 cells, in contrast, show only a slight decrease from cer Res., 39: 2070-2076,1979. 12. Johnson, R. K., and Howard, R. S. Development and cross-resistance char control in cell-associated drug and no detectable metabolism of acteristics of a subline of P388 leukemia resistant to 4'-(9-acridinylamino)- parent compound that might result in a lower effective drug methanesulfon-m-anisidide. Fur. J. Clin. OncoL, Â¡8:479-487, 1982. concentration. Other data5 indicate that these cells do not 13. Hill, B. T., and Whelan, R. D. H. Establishment of vincristine-resistant and vindesine resistant lines of murine lymphoblasts in vitro and characterization overexpress the MDR "marker" glycoprotein (gplSO), or its of their patterns of cross-resistance and drug sensitivities. Cancer Chemother. mRNA. Pharmacol., 8: 163-169, 1982. 14. Hill, B. T., and Bellamy, A. S. Establishment of an etoposide-resistant human Kinetic data (Table 3) in this paper present the first evidence epithelial tumour cell line in vitro: characterization of patterns of cross- that, in contrast to cells with the "classic" MDR phenotype resistance and drug sensitivities. Int. J. Cancer, 33: 599-608, 1984. 15. Wilkoff, L. J., and Dulmadge, E. A. Partial cross-resistance of cultured (CEM/VLBioo), alterations in transport and accumulation of murine leukemia vincristine-resistant P388 cells to 4'-demethylepipodophyl- VP-16 most likely do not account for epipodophyllotoxin re lotoxin ethylidene-/3-r>glycoside (40823). Proc. Soc. Exp. Biol. Med., 164: sistance in our human leukemic cell line (CEM/VM-1). Prelim 51-56, 1980. inary experiments with VM-26 (data not shown) and VCR (21) 16. Glisson, B., Gupta, R., Smallwood-Kentro, S., and Ross, W. Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell produced similar results. Further, we showed that resistance to line: loss of drug-stimulated DNA cleavage activity. Cancer Res., 46: 1934- VP-16 in the CEM/VM-1 cell line is likely not due to drug 1938, 1986. 17. Gupta, R. S. Genetic, biochemical, and cross-resistance studies with mutants metabolism. We suggest, therefore, that the mechanism of of Chinese hamster ovary cells resistant to the anticancer drugs, VM-26 and epipodophyllotoxin resistance in the CEM/VM-1 line is alter VP-16-213. Cancer Res., 43: 1568-1574, 1983. 18. Lee, T. and Roberts, D. Selection and characterization of L1210 sublines ation of a cellular drug target(s). The compounds to which this resistant to teniposide (VM-26). Cancer Res., 44:2981-2985, 1984. line is cross-resistant (epipodophyllotoxins, anthracyclines, 19. Pommier, Y., Schwartz, R. E., Zwelling, L. A., Kerrigan, D., Mattern, M. mAMSA) implicates changes in topoisomerase II (34-37) or in R., Charcosset, J. Y., Jacquemin-Sablon, A., and Kohn, K. W. Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells other factors involved in topoisomerase II activity (42, 43); but resistant to topoisomerase II inhibitors. Cancer Res., 46:611-616, 1986. this remains to be determined. 20. Odaimi, M., Andersson, B. S., McCredie, K. B., and Beran, M. Drug We conclude that the CEM/VM-1 line is resistant to the sensitivity and cross-resistance of the 4'-(9-acridinylamino)methanesulfon- m-anisidide-resistant subline of HIM) human leukemia. Cancer Res., 46: cytotoxic action of multiple natural drug products, but because 3330-3333, 1986. of its sensitivity to the Vinca alkaloids and minimal alteration 21. Danks, M. K., and Beck, W. T. Studies of resistance and cross-resistance to in drug accumulation, this line expresses an "atypical" MDR vincristine (VCR), VM-26 and VP-16-213 in human leukemic cells (CEM). Proc. Am. Assoc. Cancer Res., 24: 1113, 1983. phenotype. Studies are currently in progress in our laboratories 22. Conter, V., and Beck, W. T. Acquisition of multiple drug resistance by to define the mechanism of this "atypical" MDR. CCRF<:EM cells selected for different degrees of resistance to vincristine. Cancer Treat. Rep., 68: 831-839,1984. 23. Norman, M. R., and Thompson, E. B. Characterization of a glucocorticoid- ACKNOWLEDGMENTS sensitive human lymphoid cell line. Cancer Res., 37: 3785-3791, 1977. 24. Beck, W. T., Cirtain, M. C., Look, A. T., and Ashmun, R. A. Reversal of Vinca alkaloid resistance but not multiple drug resistance in human leukemic The authors gratefully acknowledge and thank Margaret Cirtain, cells by verapamil. Cancer Res., 46: 778-784, 1986. 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1301 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. Atypical Multiple Drug Resistance in a Human Leukemic Cell Line Selected for Resistance to Teniposide (VM-26)

Mary K. Danks, Jack C. Yalowich and William T. Beck

Cancer Res 1987;47:1297-1301.

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