Alkylating Agents: in Vitro Studies of Cross-Resistance Patterns in Human Cell Lines1

Home , Nitrogen mustard

[CANCER RESEARCH 46, 4379-4383, September 1986] Alkylating Agents: In Vitro Studies of Cross-Resistance Patterns in Human Cell Lines1

Beverly A. Teicher,2 Carol A. Cucchi, Jonathan B. Lee, Jennifer L. Flatow, Andre Rosowsky, and Emil Frei III

Dana-Farber Cancer Institute, Boston, Massachusetts 02115

ABSTRACT there is compelling evidence that the cytotoxicity of many alkylating agents is the result of DNA monoadduct formation The alkylating agents represent one of the most important classes of followed by inter- or intrastrand cross-links in DNA (4-7). The antitumor agents and play a major role in combination with other agents kinetics of monoadduct and cross-link formation, the nature in the curative chemotherapy of selected human cancers. By repeatedly exposing cells to escalating doses of an alkylating agent, we have devel and sites of binding to nucleic acid bases, and the efficiency and oped four human tumor cell lines which are relatively stably resistant to kinetics of repair vary substantially among the alkylating agents the drug with which the culture was treated. The response of these cell (4-7). Similarly, other determinants of drug action, such as lines to a variety of alkylating agents was compared to the response of plasma membrane transport and intracellular biotransforma- the parent cell lines to the same drug. The Kaji/IIN2 line was 7-fold tion (activation or inactivation), vary substantially among al resistant to nitrogen mustard and about 3-fold resistant to 4-hydroxype- kylating agents (8-11). Nevertheless, it has been widely as roxycyclophosphamide, but it was not resistant to .Y,.\"-bis(2-chloro- sumed that there is cross-resistance among the alkylating ethylKV-nitrosourea (BCNU), melphalan (MEL), busulfan, trimethyl- agents. If this were true, all alkylating agents would be essen eneiminethiophosphoramide, 4-hydroperoxyifosfamide, or cisplatin \cis- diamminedichloroplatinum(II)| (CDDP). The Raji/BCNU line was 5.3- tially equivalent except for differences based on differing routes fold resistant to BCNU and 4-fold resistant to both MEL and CDDP. of administration and some differences, generally slight, in side The Raji/CP line was 7-fold resistant to CDDP and 3-fold resistant to effects and antitumor spectrum. On the other hand, absence of both nitrogen mustard and BCNU, but it was not resistant to busulfan, cross-resistance among the alkylating agents would have sub trimethyleneiminethiophosphoramide, or 4-hydroperoxyifosfamide. The stantial therapeutic implications, among which the most im SCC-25/CP line, which was 12-fold resistant to CDDP, was S-fold portant would be the possibility of using these drugs in se resistant to MEL and 3-fold resistant to 4-hydroxyperoxycyclophos- quence, in combination, or in high-dose combination in the phamide. The SCC-25/CP line was almost 24-fold resistant to metho- bone marrow transplant setting. trexate after 30-min treatment and about 7-fold resistant to methotrexate Recently developed information concerning the mechanisms after continuous treatment. None of the other cell lines was resistant to of action and resistance of alkylating agents indicates substan methotrexate. The survival of SCC-25 and SCC-25/CP cells exposed to tial heterogeneity among these agents (12). Resistance to indi several antineoplastic agents was examined over several logs of survival. The SCC-25/CP cells are highly resistant to CDDP, the ratio of the vidual alkylating agents in 1.1210 and P388 mouse leukemia slopes of the survival curves (SCC-25/CP to SCC-25) of the two lines by selection pressure in vivo was described in 1978 by Schabel was 43. At survivals of 1%, resistance to MEL and BCNU became and coworkers (13). In these systems, in general, there was no evident in the SCC-25/CP line. At survivals of 0.1%, resistance to cross-resistance among the alkylating agents. More recently, mitomycin C and, to a lesser degree, to Adriamycin and vincristine was we have used selection pressure in culture to develop alkylating evident. It is more difficult to produce resistance to alkylating agents, agent resistance in two human tumor cell lines (12). In this even with extended selection pressure, than to other antineoplastic drugs paper, the cross-resistance patterns of four of the resistant cell such as antimetabolites and natural products. We found no evidence of lines to antineoplastic agents from the various classes of drugs pleiotropic resistance in any alkylating agent-resistant cell line. Our are examined. The response of a CDDP-resistant head and neck results suggest that a judicious choice of alkylating agents given in squamous cell carcinoma cell line (SCC-25/CP) to several sequential or concurrent combination may be a rational treatment strategy anticancer drugs is also examined in detail. with potential applications in the clinic.

INTRODUCTION MATERIALS AND METHODS

The alkylating agents represent one of the most important Drugs. HN2, BCNU, MEL, ADR, FU, VCR, and thiotEPA were classes of antitumor agents (1) and play a major role in com obtained from the Dana-Farber Cancer Institute pharmacy. HN2 as the bination with other agents in the curative chemotherapy of hydrochloride salt was resuspended in 0.1 M HC1. In this form it selected human cancers (2). The introduction of CDDP,3 a remains stable for up to 1 yr at -20"( ' (12). Aliquots were thawed and used immediately. BCNU lyophilized powder was resuspended in 95% nonclassical alkylating agent, has further extended the efficacy ethanol and stored, protected from light, at 4"( '. This preparation of alkylating agents in the clinic (3). results in 10% degradation in 78 days (14). MEL was dissolved in IK I The clinically effective alkylating agents are bifunctional; that acidified ethanol and diluted in serum-free DME just before use. FU is, they have the capacity to form two covalent linkages. While and VCR were diluted with DME just before use. CDDP pure powder they may combine with many nucleophilic sites within the cell, was a gift from Johnson-Matthey, Malvern, PA. HO2-CPA was kindly Received 3/14/86; revised 6/9/86; accepted 6/10/86. provided in the powder form by M. Colvin of Johns Hopkins University The costs of publication of this article were defrayed in part by the payment and was prepared in DME just before use. MitoC was purchased from of page charges. This article must therefore be hereby marked advertisement in Sigma Chemical Co., St. Louis, MO. Both ADR and MitoC were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work is supported by National Cancer Institute Grant 1P01-CA38493. resuspended in H2O and diluted with DME just before use. MTX was 2To whom requests for reprints should be addressed. a gift from the Pharmaceutical Resource Branch, National Cancer 'The abbreviations used are: CDDP, cisplatin [rÃ¡-diamminedichloropla- Institute, Bethesda, MD. MTX and thiotEPA were dissolved in IM) tinum(II)]; HN2, nitrogen mustard; BCNU, yv,W-bis(2-chloroethyl)-/V-nitrosou- and diluted with DME just before use. Busulfan, obtained from the rea; MEL, melphalan; HO2-CPA, 4-hydroxyperoxycyclophosphamide; MTX, Burroughs Wellcome Co., Research Triangle Park, NC, was dissolved methotrexate; FU, 5-fluorouracil; HOj-IFA, 4-hydroperoxyifosfamide; MitoC, mitomycin C; ADR, Adriamycin; thiotEPA, trimethyleneiminethiophos- in ethanol and diluted with DME just prior to use. IK>, 11A was phoramide; VCR, vincristine; DME, Dulbecco's modified Eagle's medium; KM,. obtained from M. Peukert of Asta-Werke AG, Federal Republic of 50% inhibitory concentration. West Germany, and prepared in DME just prior to use. 4379

Cells. The Raji cell line, derived from a human Burkitt lymphoma, of alkylating agents was compared to the response of the parent was obtained from H. Lazarus of the Miami Comprehensive Cancer cell lines to the same drug. The ratios of the drug concentrations Center. Cells were grown in suspension in DME supplemented with needed to reduce the survival of the resistant lines and the 10% fetal bovine serum, L-glutamine (292 pg/ml), penicillin (10 Â¿ig/ ml), and streptomycin (100 ^g/ml) in 8% CO2/92% air at 37'C. The parent lines to 50% are shown in Table 1. The Raji/BCNU cell line which was 5.3-fold resistant to BCNU was appreciably SCC-25 cell line was derived from biopsy of a human squamous cell cross-resistant (4-fold) to both MEL and CDDP. The Raji/ carcinoma of the tongue and was established and characterized initially by J. G. Rheinwald at this Institute (IS). Monolayers were maintained HN2 cells were almost 7-fold resistant to HN2. These cells in the DME supplemented with 20% fetal bovine serum and antibiotics. were about 3-fold resistant to HO2-CPA. Almost no resistance For the SCC-25 line, hydrocortisone (0.4 Mg/ml) was included in the was observed with BCNU, MEL, busulfan, thiotEPA, HO2- medium ( 12). IFA, or CDDP. The Raji/CP cell line was about 7-fold resistant Production of Resistance. Two drug schedules were used to develop to CDDP; it was also about 3-fold resistant to both HN2 and resistant cell lines: intermittent and continuous (daily). For the inter BCNU. This cell line was not resistant to busulfan, thiotEPA, mittent schedule, the SCC-25 cells (2 x IO5cells per 100-mm dish) and or HO2-IFA. The SCC-25/CP cell line which was 12-fold Raji cells (5 x 10s cells per ml) were treated for 30 min with the 1CÂ«, resistant to CDDP was also 5-fold resistant to MEL and almost concentration of the drug. The cultures were observed daily and allowed 3-fold resistant to HO2-CPA. Most often cross-resistance de to grow until they reached the initial density or greater as determined by trypan blue exclusion (Raji) or microscopic observation (SCC-25). veloped to MEL and HO2-CPA. Resistance to HO2-CPA did For the Raji cells, the concentration was increased primarily on the not correlate with resistance to HO2-IFA. Cross-resistance with basis of recovery time. For short times (1 to 2 wk) a 1.5- to 2-fold busulfan, thiotEPA, or HO2-IFA was not observed in any of increase in concentration was made; for recoveries of 2 to 4 wk, the the four cell lines. increase was smaller (10 to 50%); and for recoveries exceeding 4 wk, Very little cross-resistance (pleiotropic resistance) was ob the concentration was either kept the same or decreased. For the SCC- served with any of the four alkylating agent-resistant cell lines 25 cells, concentration changes were based on the reduction and recov to nonalkylating agent drugs (Table 2). All of the alkyiating ery of colony number and size. For continuous (daily) exposure of Raji agent-resistant lines were more sensitive (collaterally sensitive) and SCC-25 cells, daily "pulsing" for 30 min with selected alkylating agents in serum-free medium was used. The resistant lines are treated than the parent lines to ADR after short (30 min) or continuous exposure to the drug. To a lesser degree, all of the alkylating weekly to maintain the resistance. agent-resistant cell lines were also more sensitive to VCR than Cloning. The cloning of resistant populations of Raji cells was accomplished by the limiting dilution technique (16) and was performed the parent cell lines. On the other hand, the SCC-25/CP cell at least twice to ensure clonal derivation. Daily microscopic observation line was almost 24-fold resistant to MTX (30-min treatment) confirmed that the clone was derived from a single cell. Cloning of and about 7-fold resistant to MTX (continuous treatment). SCC-25 monolayers was accomplished by isolation of a single large None of the other cell lines including the Raji/CP line was colony in a cloning chamber, followed by removal and trypsinization. Measurement of the I(.'â€¢,â€žwasmade periodically with each clone to resistant to MTX. We have examined in greater detail the survival of SCC-25 determine the degree of resistance. and SCC-25/CP cells exposed for 1 h to a range of concentra Cytotoxicity. The drug concentration that reduced the number of tions of drugs from various classes of antineoplastic agents. viable cells to 50% of control (ICso) was determined as follows. For the SCC-25 line, 1 x IO3 cells were plated on a 60-mm dish and allowed From the data in Fig. 1, it is evident that the SCC-25/CP cells to attach overnight; 24 h later the medium was replaced with serum- are highly resistant to CDDP. At 100 MMof drug there was a 4-log kill in the SCC-25 cell line and only 50% kill in the SCC- free DME containing the drug. Treatment was terminated after 30 min by washing with DME and replacing with fresh medium. After 2 wk, 25/CP cell line. Twenty % of the SCC-25/CP cells survived the plates were fixed with phosphate-buffered 10% formalin, and the treatment with 500 MMCDDP. The ratio of the slopes of the colonies were stained with 0.2% mÃ©thylÃ¨neblue.An appropriate con survival curves (SCC-25/CP to SCC-25) of the two cell lines centration range was used. Raji cells were diluted to 5 x 10* cells per was 43. Thus the degree of resistance varies depending upon ml in serum-free DME. Drugs were added, and the cells were incubated the assay method used. The curvilinear nature of the dose- (30 min at 37Â°C),washed, and resuspended in fresh medium. The response curve for the resistant line in Fig. 1, as compared to number of viable cells was determined on Day 3, initially by trypan the sensitive line, leads to a determination of 12-fold resistance blue exclusion, then confirmed with a limiting dilution technique as a at the K \,i. but substantially greater resistance when slopes or clonogenic assay for cytotoxicity (16). Survival Curves. SCC-25 and SCC-25/CP cells in exponential growth survivals are compared at concentrations greater than the IC50. were treated with various doses of the drugs described above. After The response of these cells to treatment with other alkylating exposure to the agent or vehicle for 1 h, the cells were washed 3 times agents is shown in Fig. 2. After 1-h exposure to 250 MMMEL, with phosphate-buffered 0.9% saline solution and suspended by treat there was a 2-log kill of SCC-25 cells, and there was a 1-log ment with 0.25% trypsin/0.1% EDTA. The cells were plated in dupli kill of SCC-25/CP ceils. This 1-log differential persisted at 500 cate at 3 dilutions for colony formation. After 2 wk the colonies were MMMEL. The ratio of the survival curve initial slopes for this visualized by staining with crystal violet, and colonies of 50 cells or drug was 3, and the ratio of the terminal slopes was 1.1, greater were counted. The results were expressed as surviving fraction indicating that, at high drug concentrations, the remaining of treated cells compared to vehicle-treated control cells. populations of cells in both cell lines are equally sensitive to Data Analysis. Quantitative analysis of survival curves was performed using the log-probit iterative least-squares method of Litchfield and MEL. Although at low drug concentrations there was no dif ference in the survival of SCC-25 and SCC-25/CP cells exposed Wilcoxon ( 17) as revised by Tallarida (18). Calculations were performed on an Apple 11+ microcomputer. to BCNU, at higher drug concentrations the survival curves diverged, so that at 500 MMBCNU there was >l-log differential in cell kill between the SCC-25 and SCC-25/CP cells. The ratio RESULTS of the terminal slopes of these survival curves was 3.6. Busulfan, By repeatedly exposing cells to escalating doses of an alkyl after 1-h exposure, was not a very effective cytotoxic agent in ating agent, we have developed four human tumor cell lines these cell lines. There was no difference in cell survival between which are relatively stably resistant to the drug with which the the SCC-25 and SCC-25/CP cells after treatment with busulfan. culture was treated. The response of these cell lines to a variety At low concentrations of MitoC, the ratio of the slopes of the 4380

Table 1 Resistance ratios to various alkylating agents The resistance ratio equals the ICÂ»of the resistant line divided by the K â€¢â€žofthe parental line. The ICsoS II/M) for the Raji parental line were: HN2, 2.6; MEL, 20; BCNU, 30; HO2-CPA, 18; busulfan, 6.6; thiotEPA, 2.4; CDDP, 30; HO2-IFA, 23.3; MitoC,* 10.2; and MitoC,* 0.042. The ICÂ»sGIM)for the SCC-25 parent line were: HN2, II; MEL, 8; BCNU, 200; HO2-CPA, 25; busulfan, 15.5; thiotEPA, 2.4; CDDP, 17; HOrlFA, 25; MitoC,* 3.3; and MitoC,' 0.004.

CelllineRaji/BCNU

Raji/HN2 6.6 1.4 1.9 2.8 1.4 Raji/CPSCC-25/CPHN2'1.93.21.8MEL"4.01.35.0BCNU'5.32.62.0H02-CPA"1.61.02.8Busulfan''1.20.91.0ThiotEPA'1.61.51.7CDDP"4.02.37.312.0HOrlFA"0.91.21.6MitoC01.00.81.0MitoC*2.01.51.2

" Drug exposure for 30 min. * Drug exposure continuous. ' Drug exposure for 24 h.

Table 2 Resistance ratios to various antineoplastic agents 1.0 1.0 The resistance ratio equals the l( '.â€žofthe resistant line divided by the H',,, of MELPHALAN BCNU the parental line. The IC** (,/M) for the Raji parental line were: ADR,* 0.66; ADR,*0.04; VCR*0.11; FU,'44; MTX,"41; and MTX,*0.022. The lCâ€žs(^M) for the SCC-25 parental line were: ADR,' 2.8; ADR,* 0.013; VCR," 0.21; FU,C 52; MTX,Â°34; and MTX,* 0.006. 0.1 CelllineRaji/BCNU 0.1

Raji/HN2 0.3 0.70.20.9FV1.03.41.90.6MTX*1.41.2 Raji/CPSCC-25/CPADR*0.30.20.6ADR*0.50.50.4VCR*0.7 1.623.8MTX*2.51.57.3

0.01 0.01 Â°Drugexposure for 30 min. * Drug exposure continuous. ' Drug exposure for 24 h.

1.0 CDDP

0.1 0.1 I BUSULFAN u 0.01 Â£

I 0.001 0.01 0.001 CO

m 0.0001

0.0001 O.OOO01 50 100 250 600 100 250 500 Drug Concentration uMolar 0.00001 Fig. 2. Survival of SCC-25 (â€¢)and SCC-25/CP (O) cells exposed for 1 h to 50 100 250 500 various concentrations of MEL, BCNU, MitoC, or busulfan. Survival was meas Drug Concentration pMoiar ured by colony formation. Points, mean for three independent experiments; bars, SE. Fig. 1. Survival of SCC-25 (â€¢)and SCC-25/CP (O) cells exposed for 1 h to various concentrations of CDDP. Survival was measured by colony formation. Points, mean for three independent experiments; bars, SE. the VCR-treated SCC-25 and SCC-25/CP cells was identical. In the terminal phase of these dose-response curves, the SCC- survival curves for the SCC-25/CP and SCC-25 cell lines was 25/CP cells showed less sensitivity to this drug than did the 8. At a concentration of 50 UMthere was a 2.5-log greater kill SCC-25 cells. The ratio of the terminal slopes was 2.3. As is of the SCC-25 cells than of the SCC-25/CP cells. The ratio of evident from Fig. 4, there was no difference in the survival of the terminal slopes of the curves was 11.3, indicating continued SCC-25 and SCC-25/CP cells treated with FU. The SCC-25/ resistance in the SCC-25/CP cells even at very high drug levels. CP cell line was highly resistant to MTX. At a dose of 150 Â¿IM, From the 50% inhibitory concentrations shown in Table 2, there was a 2-log kill in the SCC-25 cell line, and there was a it appears that all of the alkylating agent-resistant lines are 50% kill in the SCC-25/CP cell line. At 500 n\Â¡MTX, there somewhat more sensitive to ADR than are the parent Raji and SCC-25 lines. When the survival of SCC-25/CP and SCC-25 was a nearly 3-log kill of SCC-25 cells while 30% of the SCC- cells treated with ADR was examined over several logs, the 25/CP cells survived. The ratio of the slopes of the survival results shown in Fig. 3 were observed. Only after a 2-log kill curves at low drug concentrations was 14.4, and the ratio of the did the survival curves begin to diverge, and then only slightly. slopes of the terminal portions of the curve was 3.7. We have The ratio of the terminal slopes of the survival curves (SCC- shown that a cell line which is highly resistant to CDDP (>10% 25/CP to SCC-25) is 1.5. As was indicated in Table 2 from the of the cells are not killed by 500 ^M CDDP) contains a popu 50% inhibitory concentrations, the resistant cell lines are lation (about 3% of the cells) which is highly resistant to MEL, slightly more sensitive to VCR than are the parent cell lines. a population (about 10% of the cells) which is highly resistant Through the first two logs of cell kill on Fig. 3 the survival of to BCNU, a population (about 0.04% of the cells) which is 4381

1.0 ship between dose and log cell kill through some 4 to 5 logs is ADRIAMYCIN VINCRISTINE generally maintained for the alkylating agents in the drug- sensitive lines (compare Figs. 1 and 2), whereas it is generally 0.1 curvilinear with the nonalkylating agents (note particularly MTX, FU, and VCR). Indeed, with MTX and VCR, for ex ample, at 4-log and 2-log kill, respectively, further increase in u 0.01 dose would not be expected to provide greater cytoreduction. The lack of cross-resistance between HO2-CPA and HO2-IFA indicates that even small change in molecular structure can lead I 0.001 to alkylating agents with differing resistance mechanisms. The observation that resistance to a given antitumor agent may impart pleiotropic resistance to a number of seemingly 0.0001 unrelated agents has major therapeutic implications (20). We examined the alkylating agent-resistant lines for cross-resist

0.00001 ance to several nonalkylating agent antitumor agents including 50100 250 500 100 250 500 ADR, MTX, VCR, and FU. We found no evidence of pleio Drug Concentration, uMolar tropic resistance in any alkylating agent-resistant cell line ex Fig. 3. Survival of SCC-25 (â€¢)and SCC-25/CP (O) cells exposed for l h to various concentrations of ADR or VCR. Survival was measured by colony cept for the cross-resistance of the SCC-25/CP line to MTX. formation. Points, mean for three independent experiments; bars, SE. The fact that most of the alkylating agent-resistant lines are collaterally sensitive to other antitumor agents was reassuring, 1.0 METHOTREXATE 5-FLUOROURACIL since the latter are commonly used in combination with alkyl ating agents, either concurrently or sequentially (21). Finally, the relationship of cell kill carried out over 3 to 4 logs with increasing concentration of drug may provide infor mation not evident when 50% inhibitory concentrations deter 0.1 mined over a 1-log range are used. For the SCC-25 line, there was generally a log linear relationship between cell kill and increasing concentration for the alkylating agents CDDP, BCNU, MEL, and MitoC. This is important, since for nonal 0.01 kylating agents there is a tendency, at increasing doses, for a reduced fractional cell kill that may occur as early as 1 or 2 logs. The data for the parent SCC-25 cell line follow the classical steep dose-response relationship for alkylating agents and thus support the idea of dose intensification therapeutic 0.001 strategies with these drugs. However, there was a consistent 50100 250 500 50100 250 500 difference between the SCC-25 and SCC-25/CP cell lines. For Drug Concentration. jjMolar the CDDP-resistant line, whether treated with CDDP or the Fig. 4. Survival of SCC-25 (â€¢)and SCC-25/CP (O) cells exposed for l h to various concentrations of MTX or FU. Survival was measured by colony forma other alkylating agents, there was a loss of linearity and, there tion. Points, mean for three independent experiments; bars, SE. fore, lower fractional kill with increasing concentrations of drug. For example, with CDDP, resistance was 20-fold at the highly resistant to MitoC, and a population (about 30% of the 50% survival level, 40-fold at the 20% survival level, and much cells) which is highly resistant to MTX. greater at lower levels in comparison with the parent cell line. The differences, while not nearly so great, were nevertheless present for the other alkylating agents, adding a measure of DISCUSSION complexity to the interpretation of cross-resistance patterns It is more difficult to produce resistance to alkylating agents and suggesting greater heterogeneity among tumor cell lines than to other antineoplastic drugs, such as antimetabolites and not only specifically with respect to alkylating agent resistance, natural products, under conditions of extended selection pres but perhaps also more generally with respect to cytokinetic, sure. We have been able to produce only relatively low resistance cytogenetic, and biochemical properties not directly linked to (< 12-fold when measured by ICso) to alkylating agents as com alkylating agent resistance. Since alkylating agents are muta- pared to the many hundredfold resistance which can be pro genie, cell lines made resistant to an alkylating agent might be duced to other antitumor agents (12). This has major therapeu expected to exhibit more heterogeneity than the parent lines tic implications. It is well known experimentally and clinically (22). that the dose-response curves for alkylating agents are steep Clinical cancer chemotherapy is now in a position to aim for (13). In the autologous marrow transplant setting, a 5- to 15- the curative treatment of solid tumors. To achieve "cure" it is fold increase in alkylating agent dosage is possible (19). Thus, necessary to eradicate essentially all the neoplastic stem cells intensive treatment with alkylating agents (for example, a 10- in the body. Multidrug regimens using combinations of alkyl fold increase in dose) ought to produce major, or perhaps total, ating agents with a low degree of cross-resistance offer consid cell kill, since drug-resistant clones in a heterogeneous popu erable theoretical promise in this regard, but there has been, lation should rarely exceed 10-fold resistance. On the other until recently, a notable lack of experimentally based guidelines hand, for nonalkylating agents, much higher levels of resistance for the design of such regimens. The present in vitro studies are possible, suggesting that dose intensification designed for indicate that cross-resistance among some of the alkylating cytoeradication may be a less realistic goal than with the alkyl agents is very low, while for others partial cross-resistance may ating agents. This is borne out by the fact that a linear relation- occur. In Raji cells, for example, CDDP resistance and HN2 4382

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. ALKYLATING AGENT CROSS-RESISTANCE resistance are associated with minimal cross-resistance to any 5. Lindahl, T., Karran, P., Demple, B., Sedgwick, B.. and Harris, A. Inducible DNA-repair enzymes involved in the adaptive response to alkylating agents. of six other alkylating agents, while BCNU resistance is asso Biochimie (Paris), 64: 581-583, 1982. ciated with cross-resistance to both CDDP and MEL. More 6. Sedgwick, B., and Lindahl, T. A common mechanism for the repair of ()''â€¢â€¢ methylguanine and O'-ethylguanine in DNA. J. Mol. Biol., 154: 169-175, over, the pattern of cross-resistance may not be the same in 1982. every type of tumor cell, as illustrated by the finding of cross- 7. Meyn, R. E., Jenkins, S. F., and Thompson, L. H. Defective removal of resistance between CDDP and MEL in SCC-25 cells but not in DNA cross-link in a repair-deficient mutant of Chinese hamster cells. Cancer Res., Â«.-3106-3110, 1982. Raji cells. Taken altogether, however, our results do suggest 8. Goldenberg, G. .1..and Begleiter, A. Membrane transport of alkylating agents. that a judicious choice of alkylating agents given in sequential Pharmacol. Ther., 8: 237-274, 1979. 9. Goldenberg, G. .1.. Vanstone, C. L., Israels, L. G., Ilse, D., and Bihler, I. or concurrent combination should be a rational treatment strat Evidence for a transport carrier of nitrogen mustard in nitrogen mustard- egy with potential applications in the clinic. A further conclu sensitive and -resistant LSI78Y lymphoblasts. Cancer Res., 30: 2285-2291, sion which emerges from this work is that alkylating agent 1970. 10. Vistica, D. T., Toal, J. N., and Rabinowitz, M. Amino acid conferred resistance may be associated with surprising patterns of altered protection against melphalan. Biochem. Pharmacol., 27: 2865-2870, 1978. sensitivity to antineoplastic drugs of the nonalkylating agent 11. Redwood, W. R., and Colvin, M. Transport of melphalan by sensitive and type. For example, there is a suggestion from our data that resistant LI210 cells. Cancer Res., 40:1144-1149, 1980. 12. Frei, E., Ill, Cucchi, C. A., Rosowsky, A., Tantravahi, R., Bernal, S., Ervin, BCNU, HN2, and CDDP resistance in Raji cells is associated T. J., Ruprecht, R. M., and Haseltine, W. A. Alkylating agent resistance: in with enhanced sensitivity to ADR. On the other hand, CDDP vitro studies with human cell lines. Proc. Nati. Acad. Sci. USA, 82: 2158- resistance in SCC-25 cells seems to be associated with a de 2162, 1985. 13. Schabel, F. M., Jr., Trader, M. W., Laster, W. R., Jr., Wheeler, G. P., and crease in sensitivity to MTX that would argue against a sequen Witt, M. H. Patterns of resistance and therapeutic synergism among alkyl tial use of these two agents with CDDP preceding MTX. ating agents. Antiobiot. Chemother., 23: 200-215, 1978. 14. Dorr, R. T., and Fritz, W.L. Cancer Chemotherapy Handbook, p. 297. New Studies are currently in progress in our laboratory to elucidate York: Elsevier, 1980. the basis of these patterns of altered nonalkylating agent sen 15. Rheinwald, J. G., and Beckett, M. A. Tumorigenic keratinocyte lines requir sitivity in alkylating agent-resistant cell lines. In vitro studies ing anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res., 41: 1657-1663, 1981. of this type should make it possible to ultimately design new 16. Bast, R. C., Jr., DeFabritis, P., Lipton, J., Gelber, R., Marer, R., Nadler, L., rational combinations of alkylating and nonalkylating drugs for Sallan, S., and Ritz, J. Elimination of malignant clonogenic cells from human bone marrow using multiple monoclonal antibodies and complement. Cancer the treatment of solid tumors in patients. Res., 45: 499-503, 1985. 17. Litchfield, J. T., and Wilcoxon, F. A simplified method of evaluating dose- effect experiments. J. Pharmacol. Exp. Ther., 96:99-113, 1949. REFERENCES 18. Tallarida, R. J., and Murray, R. B. Manual of Pharmacologie Calculations with Computer Programs. New York: Springer-Verlag, 1981. 1. Colvin, M. The alkylating agents. In: B. Chabner (Ã©d.).Pharmacologie 19. Dicke, K. A., Spitzer, G., Zander, A. R., and ( .orÃn.N. C. Autologous Bone Principles of Cancer Treatment, pp. 276-306. Philadelphia: Saunders, 1982. Marrow Transplantation. Houston: The University of Texas, M.D . Ander 2. Frei, E., III. Curative cancer chemotherapy. Cancer Res., 45: 6523-6537, son Hospital and Tumor Institute, 1985. 1985. 20. Kartner, N., Charles, M., Riordan, J. R., and Ling, V. Daunorubicin-resistant 3. Zwelling, L. 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Beverly A. Teicher, Carol A. Cucchi, Jonathan B. Lee, et al.

Cancer Res 1986;46:4379-4383.

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