Mechanism of Melphalan Resistance Developed in Vitro in Human Melanoma Cells1

Home , Melphalan

[CANCER RESEARCH 41, 1525- 534, April! 981] 0008-5472/81 /0041-OOOOS02.00 Mechanism of Melphalan Resistance Developed in Vitro in Human Melanoma Cells1

P. G. Parsons,2 F. B. Carter, L. Morrison, and Sr. Regius Mary

Queens/and Institute of Medical Research, Herston [P. G. P., F. B. C., L. M.], and Cytogenetics Clinic, Mater Hospital. South Brisbane Â¡P.M.]. Queensland. Australia 4006

ABSTRACT and repair (9, 16, 24, 50). However, the relevance of these processes to chemotherapy in humans is not clear, partly Melphalan resistance developed previously in a human mel because of the wide variety of target material within cells, the anoma cell line (MM253) could not be further increased. Cross- lack of sensitive and resistant human cells suitable for experi resistance was found to nitrogen mustard but not to ultraviolet mental studies, difficulties in studying drug damage at minimum light radiation. A clone of MM253 had the same drug sensitivity lethal doses, and the requirement for metabolic activation of and heterogeneous chromos orne complement as did the parent some drugs. culture. The melphalan-resiutant cells (MM253-12M) had 2.6- Melphalan (phenylalanine nitrogen mustard) is a bifunctional fold the Do, 1.5-fold the size, 1.3-fold the RNA content, 1.4- agent which has been used extensively in the treatment of fold the protein content, and 2.6-fold the DMA content of the breast cancer (15), malignant melanoma (31), myeloma (2), sensitive parent line. There was no evidence for activation or and ovarian cancer (44). Melphalan undergoes hydrolysis un detoxification of melphalan by intact melanoma cells or by der physiological conditions with a half-life of 60 to 90 min in mouse liver microsomes competent for the activation of other human plasma (1) or 0.1 M phosphate buffer (53); no metabo drugs. Melphalan transport was similar in both cell lines, reach lites other than the mono- and dihydroxy hydrolysis products ing a steady-state level 3 timas the concentration in the medium and alkylated proteins have been identified in plasma or urine after 2.5 min. Both lines covalently bound the same total (1, 34). The transport of melphalan into murine and human amount of [3H]melphalan per cell, but in MM253-12M a 50% cells is carrier mediated and can be inhibited by L-leucine (6, decrease in binding to DNA was almost sufficient to account 21, 38, 48). Melphalan-resistant L1210 cells had impaired for the increase in resistance. The level of melphalan-induced melphalan transport compared with the sensitive parent cells, DMA interstrand cross-links, which were heat labile but not but this alteration alone was not sufficient to fully account for alkali labile, reached a maximum during the 4-hr treatment the degree of resistance (38). DNA cross-links induced by period and then declined slowly. The degree of cross-linking in melphalan in mouse leukemia L1210 cells were formed and MM253-12M was 50% less than that in MM253. Unlike ultra were removed more slowly than were HN2-induced cross-links violet light, methyl methane sulfonate, and nitrogen mustard, (41); in an earlier study (34), covalent binding of melphalan to melphalan at equitoxic doses did not damage the DNA suffi DNA could not be detected. DNA-protein cross-linking was ciently to immediately inhibit DNA synthesis. Although both found in a human lymphoblastoid cell line (6) but not in Ehrlich- lines were proficient for repair of ultraviolet light and methyl LettrÃ©mouse tumor cells (25). A melphalan-resistant Yoshida methane sulfonate damage, melphalan did not induce signifi sarcoma had a higher glutathione content than did the sensitive cant levels of DNA repair synthesis and had little effect on the parent tumor but showed no difference in growth rate, chro rate of DNA chain elongation. In MM253 cells, strand breaks mosome number, or drug uptake (3). A melphalan-sensitive were detected only at high melphalan doses; MM253-12M rodent tumor became resistant after transfection with DNA formed breaks more readily. This evidence suggests that the extracted from melphalan-resistant cells (37). toxicity of melphalan results from comparatively rare DNA A melphalan-resistant human melanoma cell line was derived cross-linking events and that developed resistance arises from which had an increased chromosome number and decreased decreased susceptibility of DNA to this damage. frequency of melphalan-induced chromosome aberrations compared with the sensitive parent line (36). The former line INTRODUCTION showed cross-resistance to chlorambucil and hyperthermia but not to the monofunctional agents MMS, A/-methyl-/V'-nitro-/V- Extensive studies of rodent tumors resistant to bifunctional nitrosoguanidine, and DTIC (35). We have now compared these alkylating agents have derronstrated a variety of resistance lines with respect to melphalan uptake and metabolism and mechanisms based mainly on differences in drug transport (9, 25, 38), SH3 content (3, 19), and DNA cross-linking damage DNA damage and repair, with the aim of defining the lesion(s) responsible for melpahalan toxicity and the mechanism of resistance. Other agents were included in this study to dem onstrate known patterns of DNA damage and repair: UV, which ' This work was assisted by a giant from the National Health and Medical induces thymidine dimers and subsequent long-patch excision Research Council. Canberra, Australia. repair (39) and postreplication repair (29); MMS, which alkyl- 2 To whom requests for reprints siould be addressed. 3 The abbreviations used are: SH, sulfhydryl; HN2. nitrogen mustard; MMS, ates phosphodiester groups and the N-7 of guanine with sub methyl methanesulfonate; DTIC, 4-dimethyltriazenoimidazole-5-carboxamide; sequent short-patch repair (39) and postreplication repair (7); TNE, 0.1 M NaCI, 10 mw Tris, and 2 mm EDTA, pH 8; PBS, 0.15 M NaCI, 10 mw and HN2 (a bifunctional agent), which forms inter- and intra- Na2HPO4. and 3 rriM KH2PO4, pH 7.2; SDS. sodium dodecyl sulfate; TCA, strand DNA cross-links (27), forms DNA-protein cross-links trichloroacetic acid; HU. hydroxyurej. Received May 16. 1980: accepted December 29. 1980. (25), and induces excision repair (11, 16, 32).

APRIL 1981 1525

MATERIALS AND METHODS 30 min at 37Â°, the reaction mixtures were precipitated with cold 10% TCA and filtered onto glass fiber discs prior to Biological Material. The MM253 and MM253-12M human solubilization with Soluene 350 (Packard Instruments) and melanoma cell lines have been described (36). The MM253- counting. The effectiveness of these treatments (>95% diges 14M line was derived from MM253-12M by 2 cycles of a tion) was demonstrated in each experiment using cells prela- treatment involving UV exposure (254-nm radiation, 5 J/sq m) beled with [3H]thymidine, [3H]leucine, and [14C]uridine. followed 3 days later by melphalan (15 jug/ml). MM253-15M To determine the uptake of unbound melphalan, a suspen was derived from MM253-14M following a further treatment sion of 3 x 107 cells in 5 ml of medium containing melphalan with melphalan alone (20 /Â¿g/ml). (20 fig/ml) was incubated at 37Â°for 15 min. After centrifugation Cells were cultured in Roswell Park Memorial Institute Me (100 x g for 10 min), the cell pack was dispersed in 1 ml water dium 1640 (Commonwealth Serum Laboratories, Melbourne, and lysed with SDS (0.2%). The viscous solution was sheared Australia) containing 10% fetal calf serum, penicillin (100 IU/ by 5 passes through a 26-gauge needle and then heated with ml), streptomycin (100 jug/ml), and 10 mM A/-2-hydroxyethyl- 50 /Â¿I4-(p-nitrobenzyl)pyridine (5% in acetone) at 100Â°for 20 piperazine-W-2-ethanesulfonic acid. Periodic assays for My- min. After cooling, 50 /il 50% TCA and 0.5 ml acetone were coplasma (36) were negative. All comparative studies of agents added, the precipitate was removed by centrifugation, and the and cell lines were performed simultaneously using the same supernatant was mixed with 200 /Â¿11NNaOH. The absorbance reagents; cell lines were used 0 to 24 hr after passage. Cell at 600 nm was read immediately. survival was determined by the clonal assay described previ Cell Size and Composition. Mean cell diameter was deter ously (23). For preparation of the microsome fraction, the livers mined using a particle data counter (Particle Data, Elmhurst, from 8 mice, given 0.1% phÃ©nobarbital in the drinking water III.). For determination of total SH, 107 cells were frozen and during the previous 7 days, were collected in cold 0.15 M KCI; thawed 3 times in 200 fil 0.02 M EDTA (pH 4.7). A 50-/J aliquot homogenized in 10 ml of 0.1 M KCI, 5 mM MgCI2, 5% sucrose, was mixed with 150 /tl 0.2 M Tris (pH 8.2) and 10 /il 0.1 M 5,5'- and 18.6 mM KH2PO4 (pH 7.2); and centrifuged (9000 x gmax) dithiobis(2-nitrobenzoic acid), the volume was made up to 1 ml for 10 min. The supernatant was stored at â€”70Â°in 1-ml with methanol, and the absorbance at 420 nm was determined aliquots. With the addition of NADP (5 mM) and glucose 6- after 30 min at room temperature. Dithiothreitol was used as a phosphate (10 mM), this mixture enhanced the toxicity for standard. Soluble SH was estimated after precipitation of pro MM253 of DTIC and cyclophosphamide 30-fold and 100-fold, tein with 10% TCA (4Â°for 10 min). Protein was determined respectively. using the method of Lowry ef al. (30), DNA was determined by Metaphase cells were prepared for chromosome analysis as the method of Gilles and Myers (18), and RNA was determined described (36) except that potassium chloride (0.075 M for 8 by the method of Ceriotti (8). min) was used. Higher chromosome counts were obtained for Sedimentation of DMA in Alkaline Sucrose. Approximately the MM253 lines than reported previously (36), possibly be 2.4 x 10" cells prelabeled for 24 hr with [14C]thymidine (0.02 cause of the improved quality of the preparations. C-banding /iCi/ml; control) or [3H]thymidine (0.5 /tCi/ml; treated) were was carried out according to the method of Sumner (46). mixed and lysed for 1 hr in 200 /il 1 N NaOH containing 1 mM Cell Adherence. Cells were labeled for 3 days with [methyl- EDTA and 0.5% Sarkosyl, followed by cosedimentation on 5 to 3H]thymidine (0.1 /iCi/ml; 48 Ci/mmol; Radiochemical Centre, 20% alkaline sucrose gradients as described previously (22). Amersham, Buckinghamshire, U. K.) before seeding in Linbro Weight-average molecular weights were calculated according trays (5 x 10" cells/16-mm well). Treatments were carried out to the method of Lehmann (29). For DNA elongation studies, on the following day, the medium being replaced after 4 hr. cells prelabeled with [14C]thymidine were treated with the drug Adherent cells from duplicate cultures (cells remaining after for 1 hr, pulsed with [3H]thymidine (10 /iCi/ml), and incubated removal of medium and 1 wash with TNE) were harvested in unlabeled medium for the times indicated. (trypsin) onto glass fiber discs at daily intervals, followed by Sedimentation of Nucleoids. The method of Cook and Bra- liquid scintillation counting. zell (13) was modified as follows. Cells (5 x 105/60-mm dish) [3H]Melphalan. An ethanolic solution of [G-3H]melphalan (8 were suspended in 130 /il PBS, diluted with 400 /il lysis solution Ci/mmol; Radiochemical Centre) was evaporated to dryness (2.6 M NaCI, 0.13 M Tris, 2.7 mM EDTA, and 0.67% Triton X- and dissolved in 20 /il of 1 N MCI. Complete medium (100 /il) 100, pH 8.0) and layered on duplicate 12-ml (90-mm) gradients was added, adjusted to neutral pH, diluted to 2 fig/ml (50 /iCi/ of 10 to 20% sucrose containing 1.95 M NaCI, 1 mM EDTA, ml), and used immediately. The hydrolysis products were sep and 10 mM Tris, pH 8. After 15 min, the gradients were arated by descending paper chromatography (n-butanyl alco centrifuged at 4200 rpm (4850 x gmax)for 1 hr in an IEC 981 hol saturated with water) of a 40-/il aliquot. Chromatograms rotor using adaptors which allowed all gradients for each time were cut into strips, and the radioactivity was eluted with 0.5 point to be run simultaneously. The nucleoid band was located ml water before counting in Instagel (Packard Instruments, by siphoning gradients through an A26omonitor. DNA Interstrand Cross-Linking. Cultures of 5 x 10'' cells Zurich, Switzerland). Cells treated with [3H]melphalan were washed twice in me were labeled overnight with [3H]thymidine (0.2 juCi/ml), washed dium at room temperature and twice in PBS and were either with medium, and treated as described. The cells were then fixed and washed in water for autoradiography or harvested suspended in 50 /il 15 mM NaCI:15 mM sodium citrate (pH 7.0) using trypsin. After resuspension in 200 fi\ TNE, the cells were and added to 0.5 ml 6.8 M sodium perchlorate (pH 7) containing lysed with 0.2% SDS and sheared by 10 passes through a 26- 1 mM EDTA and 0.2% Sarkosyl. This mixture was heated at gauge needle; 20-/il duplicates were used for digestion with 75Â°for 5 min to denature the DNA and then cooled in ice water Pronase, RNase (each 100 fig in 1 ml TNE), or DNase (50 jug for 1 min. To estimate the extent of renaturation, duplicate in 1 ml 50 mM Tris:5 mM magnesium chloride, pH 7.5). After aliquots (20 /il) were added to 1 ml of buffer [native calf thymus

1526 CANCER RESEARCH VOL. 41

DNA(100fÂ¿g/ml), heat-denatured DNA (10 jig/ml), 0.1 M KCI, RESULTS 0.1 mM ZnSO4, and 25 mw sodium acetate, pH 4.5] containing 200 units Si nuclease (Sigma Chemical Co., St. Louis, Mo.). Properties of MM253 Sublines. Table 1 shows that the After 30 min at 37Â°,samples were precipitated with cold 10% MM253-12M subline retained the increased melphalan resist TCA, washed, and counted. Renaturation was expressed as ance and chromosome number reported previously (36). At the percentage of acid-insoluble readioactivity remaining after tempts to derive further resistance resulted in subÃ®mes Si nuclease treatment, typically 20 to 25% of control DMA. (MM253-14M and MM253-15M) which had properties similar Less than 5% of native DNA (control or melphalan treated) was to those of MM253-12M. The melphalan-resistant lines were rendered acid soluble under these conditions. For size analy resistant to HN2 but not UV. A clone of MM253 (MM253cl) was sis, the DNA solutions wero dialyzed overnight at 4Â°against aneuploid and had the same drug sensitivity as did the un- 15 mM NaCI:15 HIM sodium citrate (pH 7.0) before lysis and cloned line. Cell-doubling times were determined 24 to 72 hr sedimentation in alkaline sucrose as described above. after seeding because very little growth occurred during the To purify DNA for A26o determination of cross-linking, a first 24 hr, but melphalan toxicity was the same whether treat nucleoid pellet was prepared from 2 x 108 cells by lysis with ment was carried out 2 or 24 hr after seeding. The presence of Triton X-100 as above and sedimentation (35,000 x gmaxfor caffeine (10 to 100 fig/ml), L-leucine (100 jug/ml), or L-arginine 20 min) through 10% sucrose containing 1.95 M NaCI, 1 mM (200 fig/ml) 1 hr before and during treatment did not affect the EDTA, and 10 mM Tris (pH 8). Further deproteinization was survival of MM253 or MM253-12M cells. carried out for 2 hr at 50Â°in 20 ml TNE containing SDS (0.5%) The results from chromosome banding studies were difficult and proteinase K (50 Â¿ig/ml), followed by extractions with to analyze in detail because of the large and variable number phenol and chloroform. of chromosomes and absence of a clearly defined modal num DNA-Protein Cross-Linking. Cells were prelabeled for 24 hr ber. However, C-banding showed that all of the MM253 sub- with [2-14C]thymidine (0.02 ,iCi/ml; 50 mCi/mmol; Radiochem- lines had human karyotypes of the same sex as that of the ical Centre) and L-[4,5-3H]leucine (5 /Â¿Ci/ml; 60 Ci/mmol; donor (male). Chromosomes 1, 2, 7, and 20 contributed most Radiochemical Centre). After the drug treatments, cells were to the hyperploidy of MM253 and to the increased chromosome either lysed directly (5 x IO5 in 50 fi\ TNE containing 0.2% number of MM253-12M. The latter line was not tetraploid. G- Sarkosyl) or isolated as a nucleoid pellet before lysis. The banding revealed a number of possible markers, but charac lysate was added to 1 ml 10 mM Tris (pH 8) and submitted to terization of consistent banding differences between MM253 manual countercurrent distribution using 0.5-ml volumes of and the other sublines is not yet feasible. phenol (preequilibrated with buffer) as the lower phase. The The volume and macromolecular contents of MM253 sub- phases were diluted separately with 2 ml 10% TCA and filtered lines were also compared (Table 2), since melphalan resistance for counting. could conceivably result from the presence of an excess of

Table 1 Properties of MM253 subÃ®mes Chromosome no.Cell

(jÂ«i lineMM253 levels100-110 time(hr)20 ml)0.33 Oig/ml)0.095 (J/sqm)1 Â±0.03Â°(4)" Â±0.03 (2) .9 Â±0.3(5)2.1 MM253C1 10-15 65 51-121 22 0.35 0.09 Â±0.01 (4) MM253-12M 120-130 9498 78-102 25 0.85 Â±0.1 (5) 0.45 Â±0.15(2) Â±0.1 (5) MM253-14M 30-35 57-99 27 1.1 Â±0.2 (6) 0.47 Â±0.15(2) 1.9 Â±0.2(5) MM253-15MPassage40-50Mode6284Range51-7166-147Doubling210*Melphalan0.94 Â±0.2 (4)HN20.51 Â±0.1 (2)UV1.9 Â±0.2(4) 0 Dose r squired to reduce survival by 1/e. Drug treatments were 4 hr. " Determined 24 to 72 hr after seeding. c Mean t S.E. d Numbers in parentheses, number of experiments.

Table 2 Size and composition of MM253 cells

Dilible:solu-ble4.94.8 (cu/jm)3080s3610"4590a52906DNA Ce IlineMM2Ãœ3MM2Ãœ3-12MVolume (pg)1436RNA(pg)95120Protein(ng)0.480.68Soluble(pmol)0.470.64SHInsoluble(pmol)2.33.1In:,

1.49" Ratio 2.6 1.3 1.4 1.4 1.3 1.0 1.46* a "wo-day culture, "hree-day culture.

APRIL 1981 1527

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. P. G. Parsons et al. alternative target material. As expected from microscopic ob servation, MM253-12M cells were 50% larger than MM253 cells, and the size distribution showed less heterogeneity (Ta ble 2; Chart 1). MM253-12M cells had 2.6-fold more DNA but only 1.3- to 1.4-fold more RNA, protein, and SH. The ratio of insoluble to soluble SH was not changed. The DNA content of both lines was higher than that expected from the average chromosome number [5 to 8 pg/46 chromosomes (45)] but is consistent with the excess of large chromosomes. Normal human leukocytes used in these experiments as a control contained 7.3 pg DNA per cell, within the reported range of 6.9 to 11 pg (45). HeLa cells, with a mode of approximately 80 chromosomes, contain 14 to 17 pg DNA (45). Selective loss of cell subpopulations by detachment and disintegration soon after treatment could affect the results of temporal responses to drugs. To test this possibility, cultures were extensively labeled with [3H]thymidine, and the adherent cells were harvested at daily intervals after treatment. Un treated cells showed 50% loss of original label after 3 days. Melphalan (0.5 and 10 /Â¿g/ml)had little effect, except that the higher dose level led to an additional 25% loss of MM253 label after 2 days. UV (2 J/sq m) and HN2 (1 jug/ml) treatments led 1Ãœ 20 to a 50% loss over 3 days compared with controls harvested FRACTION at the same time; MMS (20 /Â¿g/ml)had little effect. Chart 2. Chromatographie separation (left to right) of medium containing [3H]melphalan (2 ^g/ml) after incubation with MM253 (â€¢)or MM253-12M (â€¢) Decomposition of Melphalan. Paper chromatography of cells at 36Â°for 1 hr. Arrow, Rr of melphalan. medium containing [3H]melphalan separated 2 products of decreased mobility (Chart 2) which were assumed to be the mono- and dihydroxy derivatives. Most of the radioactivity was found on the base line, and this was not entirely due to binding with protein since it was produced in PBS and in medium lacking calf serum (Chart 3) and was resistant to digestion with proteases. The half-life of melphalan, determined by chroma tography of aliquots sampled after 1, 2, and 4 hr at 37Â°,was approximately 1 hr. Neither MM253 nor MM253-12M cells had any detectable effect on the rate or products of decomposition (Charts 2 and 3). Only 50% of the original activity was re covered after 4 hr, possibly because of loss of tritium from the melphalan molecule during decomposition. Cultures treated with [3H]melphalan (1 to 4 hr) and washed 3 times did not secrete any 3H products detectable by chromatography when the medium was analyzed 2 to 18 hr later. As a biological test for cell-specific activation or detoxifica tion of melphalan, MM253 and MM253-12M cells were mixed TIME(hr) in 1:2 and 2:1 ratios in the survival assay. The survival curve Chart 3. Decomposition of [3H]melphalan in PBS (O, â€¢),medium without serum (O. â€¢),and complete medium (A, A), assayed by Chromatographie deter mination of loss of melphalan (â€¢â€¢,A) or formation of material at the origin (D, 100 O, A).

of the 1:2 mixture, determined largely by the excess of MM253- 12M cells, should have been steeper than expected if MM253 2T cells released an activated metabolite of melphalan, whereas the converse effect would have been expected in the 2:1 50 mixture if MM253-12M cells detoxified melphalan to any sig nificant extent. In fact, the survival curves in each case were the same as if the treatments had been carried out separately. In a further test for drug metabolism, a mouse liver microsomal system added to the medium during melphalan treatment was found to have no effect on survival.

15 20 25 Transport and Incorporation of Melphalan into Cells. Col CELL DIAMETER orimetrie assay of cells after brief treatment in suspension with Chart 1. Size distribution of MM253 (â€¢)and MM253-1 2M (â€¢)cells. melphalan (20 /Â¿g/ml)revealed no difference in the amount of

1528 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. Mechanism of Melphalan Resistance drug taken up by each cell line in complete medium at 37Â°. Use of [3H]melphalan (2 fig/ ml) gave a similar result when the cells were still attached to the dish (Chart 4); melphalan uptake at 0Â°was very low. In repeated experiments, MM253-12M cells took up slightly more drug than did MM253 cells presum ably because of their larger volume. The intracellular accumu lation of melphalan reached a level 3-fold higher than the concentration in the mediurr, similar to the 5-fold enhancement found for murine cells (38). Although the melanoma cells were 10-fold larger, the melphalan content was similar to that of the mouse cells (38), possibly because the latter experiments were carried out in PBS in the absence of amino acids known to inhibit melphalan transport (6, 38, 48). The specific activity of [;H]melphalan was sufficient to ex Chart 5. Binding of [3H]melphalan (2 /ig/ml) to cells (â€¢).protein (O), RNA (D). DNA (â€¢),and cells in alkali-stable form (A). A, MM253; B. MM253-12M. amine its covalent incorporation into cells. Autoradiography of Points, means Â±S.E. of 4 experiments (mean Â±S.D. of 1 experiment for alkali- cells 1, 4, and 24 hr after treatment with [3H]melphalan (2 jug/ stable activity). ml) showed that 90% of cells were labeled. Grains were evenly distributed over the cell witn 30% being found in the nucleus. No significant differences were found between MM253 and MM253-12M with respect :o grain count in the cytoplasm or 100 nucleus. To quantitate the incorporation of [3H]melphalan into DNA, RNA, and protein, lyscites of washed cells were submitted to enzymic digestion. The same amount of melphalan was incorporated by each line, with respect to the total per cell and to the amount bound to RNA (Chart 5). The DMA of MM253 however, bound nearly twice as much drug (1 molecule/ 250,000 bases or 7.5 X 107 daltons) as MM253-12M DNA, at the expense of protein. The amount of alkali-digestible label was also greater in MM253 cells. The time response showed a more rapid loss of label from the protein and DNA of MM253 compared with MM253-12VI but is complicated by the possi bility of selective loss of heavily labeled cells over the extended period. DNA Synthesis. The timo and dose responses of DNA syn thesis following melphalan treatment were similar to those

24 2 TIME(hr) Chart 6. Inhibition of DNA synthesis in MM253 (A, ÃŸ)and MM253-12M (C, D). Cells seeded the previous day in microtiter wells (2 x 10" cells/6-mm well) were treated with the appropriate drug or exposed to 254 nm UV in the absence of medium, pulsed with [3H]thymidine (10 /iCi/ml) for 45 min at the times indicated, and harvested (trypsin) onto glass fiber discs for liquid scintillation counting. After 4 hr, the medium of remaining cultures was changed. A, C: O, 0.3 ^g/ml;Â«, 1 fig/ml; O, melphalan (10 ng/ml). 8, D:G, UV, 1 J/sq m;Â«, MMS(10 /ig/ml); O, HN2, (0.1 /ig/ml). Points, mean of quadruplicates. All standard deviations are s10%.

reported previously (36), notable features being the lack of 10 TIME (mm ) effect of low doses (near D0) and, with a high dose, no effect until after 2 hr of treatment (Chart 6). The occasional enhance Chart 4. Uptake of melphalan Dy melanoma cells. Cultures seeded 24 hr previously (5 x 105 cells/60-mrr dish) were treated with medium containing ment of DNA synthesis at 1 hr (36) also occurred following [3H]melphalan (2 ng/ml; 0.21 Ci/Timol). At the indicated times, the cells were MMS or HN2 treatment and was independent of the specific washed rapidly with 4 x 5 ml ice-cold PBS and harvested with 0.5 ml trypsin. activity of the label used and the pH of the medium. Low doses and the dish was rinsed with 0.5 ml water. The mixture of cells, trypsin, and rinse liquid was treated with Sarkosyl (0 2%) and then solubilized for liquid scintillation of UV, MMS, and HN2 were sufficient to inhibit DNA synthesis counting. Correction was made, using a dish without cells, for incomplete removal of the medium but not for surface binding of [3H]melphalan to cells. A, MM253 within 2 hr. cells treated at 37Â°;A, MM253-12M cells treated at 37Â°;Â»,MM253 cells treated DNA Repair Synthesis. Autoradiographic determination of at 0Â°;O, MM253-12M cells treated at 0Â°. unscheduled DNA synthesis showed that, although MM253

APRIL 1981 1529

UV Urn'2) arid MM253-12M cells exhibited repair synthesis when treated HNjfjug/ml ) with UV or MMS, melphalan had little effect (Table 3). As 10 20 05 expected from previous studies of human melanoma cells (22) and other cells (12), HU had little qualitative effect on these 300 â€¢ results; the overall increase in unscheduled synthesis found in all cultures presumably results from a reduction in thymidine pool size (26) or enhancement of repair synthesis (10). DMA repair synthesis determined directly by thymidine incorporation during suppression of semiconservative synthesis was not greatly affected by the HU concentration (Chart 7). Dose-re sponse studies carried out using 20 rriM HU gave results similar to those with autoradiography; UV and MMS induced signifi cant levels of repair synthesis, even at doses close to the D0, whereas the response to melphalan and HN2 was very low (Chart 8). 10 20 50 100 200 MELPHALAN (ug/ml) MMS(>Jg^nl) DNA Strand Breaks. Sedimentation of DMA in alkaline su Chart 8. DNA repair synthesis in MM253 (O, â€¢)andMM253-12M (C. â€¢)cells crose readily detected DNA breaks induced by high levels of as measured by incorporation of [3H]thymidine in the presence of 20 mM HU. UV and MMS 4 hr after treatment (Table 4); insufficient cells O. D, melphalan or HN2; â€¢.â€¢UV or MMS. All standard deviations (duplicates) remained attached at 24 hr for analysis. Melphalan doses of are <10%. similar toxicity, however, did not change the sedimentation profiles of DNA from either cell line. No breaks were detected Table 4 Alkaline sucrose determination of DNA breaks in MM253 cells by Ross et al. (41) in mouse L1210 cells following treatment Wt av. M.W. {% of control)* with melphalan or HN2. It should be noted that interpretation of Treatment"Melphalan hr103 Table 3 (1 0 fig/ml) Unscheduled DNA synthesis in MM253 sublines MMSdOOfig/ml) 7698 74 NT0NT Cells seeded on the previous day on coverslips were treated as described and HN2 (1 fig/ml) incubated at 36Â°with [3H]thymidine (10 piCi/ml) for 3 hr. After autoradiography UV(10 J/sqm)4hr95 8724 (22), the percentage of cells undergoing unscheduled DNA synthesis (2 to 100 Medium changed after 4 hr. grains/nucleus) was determined by counting 300 cells. 6 8.1 X 10'dallons. c NT, not tested. % of sparsely labeled nuclei

MM253TreatmentControlUV such results may be ambiguous in that the reduction in DNA mWHUa14453011MM253-12MNoHU41614520rnwHUa17302919size as a result of enzymically or alkali-induced breaks may be offset by interstrand cross-linking. The nucleoid sedimentation (5 J/sqm)MMS method, which depends on loss of supercoiled DNA structure (100(ig/ml)Melphalan (13) at neutral pH, demonstrated a marked response to low (10ng/ml)NoHU5713520 doses of UV and MMS (Chart 9). Equitoxic melphalan (0.5 /xg/ ml) had only a limited effect on MM253-12M cells and no effect Added 1 hr before treatment. on MM253 cells unless a much higher dose was used (10 fig/ ml). The level of DNA breaks induced in both lines by HN2 was similar to that induced by melphalan. The time course of repair was similar in all cases, with breaks increasing to 2 hr followed by some recovery. The cross-linking potential of melphalan is unlikely to have prevented loss of supercoils because melphalan-treated samples sedimented in the presence of ethidium bromide or irradiated with UV gave the same reduced sedimen tation rate as did controls. In addition, breaks induced in HeLa cell DNA by the cross-linking agent mitomycin C were detected using this method (13). It should be noted, however, that although the nucleoid method gives results equivalent to alka line sucrose sedimentation for detection of pH-independent breaks induced by UV and ionizing radiation (13), confirmation

20 50 of the results using alkylating agents has not yet been possible HYDROXYUREA(mM) because of the lack of an independent method for detecting Chart 7. Effect of HU concentration on [3H]thymidine incorporation by MM253 breaks with sufficient sensitivity at neutral pH. cells following UV (2 J/sq m) (â€¢)or during treatment with melphalan (5 fig/ml) DNA Chain Elongation. Under conditions of equal incorpo (â€¢).Cells were treated with HU 2 hr after seeding in Lmbro plates (3 x 10s cells/ ration of [3H]thymidine, UV and MMS treatments led to a 16-mm well). After 1 hr, the cells were exposed to UV where indicated, and fresh medium was added which contained [3H]thymidine (10 jiCi/ml) and the required reduction in size of newly synthesized DNA and inhibition of level of drug and HU. After a further 3 hr at 36Â°,cells were harvested onto glass the elongation of this DNA during the following 2 hr (Table 5). fiber discs, lysed, washed extensively with water, and then solubilized for counting. The results were expressed as a percentage of controls treated with Melphalan and nitrogen mustard had less effect, with no differ HU alone. Points, means of 3 experiments; Ã¶ars,S.E. ence evident between MM253 and MM253-12M cells.

1530 CANCER RESEARCH VOL. 41

that of purified DNA and to that of crude DNA denatured in NaOH by the method of Harrap et al. (24). With the perchlorate- Si nuclease method, MM253 cells were found to undergo more melphalan-induced cross-linking than did MM253-12M, with a maximum at the end of the 4-hr treatment period (Chart 10-4). Other results (not shown) indicated that in both lines most of this cross-linking occurred within 2 hr. During the 16 hr follow ing treatment, the level of cross-linking was considerably re duced in MM253 but not in MM253-12M cells. Ross ef al. (41 ) found that interstrand cross-linking induced by melphalan in L1210 cells reached a maximum 4 to 12 hr after a 0.5-hr treatment, followed by a gradual decline. Dose-response stud ies (Chart 10ÃŸ)showed that the extent of cross-linking differed in the 2 lines at melphalan doses up to 20 /Â¿g/mlbut reached the same maximum level at 40 jiig/ml. Since differences in the size of the melted DNA could affect the degree of rapid renaturation (17), it was possible that the lower renaturation level found for MM253-12M was not due to reduced cross-linking but to reduced size of the DNA. However, at the 2 melphalan doses giving maximum differences in rena turation, MM253-12M DNA was either slightly larger (10 jug/ ml) or the same size (20 Â¿ig/ml)as was MM253 DNA (Chart

Chart 9. DMA strand breaks in MM253 (O, â€¢)and MM253-12M (D. â€¢}cells, as detected by reduction in the di stance sedimented by nucleoids, compared with controls. A, melphalan (0.5 /Â¿g/ml),B, melphalan (10 /ig/ml); C, MMS (20 fig/ml) (O, D) and HN2 (1 /ig/ml) (Â«l.â€¢);D, UV (2 J/sq m). The drug treatments were continuous. Points, means of duplicates; bars, S.D.

Table 5 Elongation of DNA in MM253 and MM253-12M cells MM2Ã•.3Treatment

ChaseMin (1 hr)ControlUV M,3 M. Hr TO 20 TIME (hr) MELPHALAN (pg/rnl) 3*2077 4.84.34.04.51.52221.521.5M.7.37.45.16.26.78.03 5.1 1.5 Chart 10. DNA size and interstrand cross-linking in MM253 (O, â€¢)and MM253-12M (D, â€¢)cells. A, temporal response of cross-linking with melphalan (5 J/sqm)MMSdOOfig/ (1 0 fig/ml) present for the first 4 hr, as measured by the increase in renaturable DNA; B, dose response of cross-linking determined at 4 hr (â€¢,â€¢)and DNA size ml)Melphalan after renaturation (O, D). Mâ€ž,weight-average molecular weight.

(10 'A Â»ig/ml)HN2 37 5.15.5 6.67.88.6MM253-12MPulse7.53 5.2 1.5

(1 fig/ml)PulseMin7b 3M."5.34.7ChaseHr2 4.8 1.5 8.0 40 Mw, weight-average molÃ©cula weight x 10 7. 6 Separate experiments. ?20

DNA Interstrand Cross-Linking. Initial studies with purified MM253 DNA melted (70Â°) and renatured (0Â°)in sodium per-

chlorate showed that the level of renaturation in DNA from g 60 control cells (15 to 20%) was increased to 40% by a 4-hr D treatment with melphalan (10 /ug/ml), presumably because of 0140 interstrand cross-linking (1 7). This result was obtained using either A26omonitoring of hyperchromicity or the single-strand- 20 specific Si nuclease. Sinco labeling studies showed that 50 to 90% of the original DNA was lost during purification, a new A tgttt method was devised whersby cells were lysed in perchlorate 10 5 10 10 solution, followed by meltirg and rapid renaturation of the total, FRACTION unpurified DNA and direct analysis using Si nuclease. The 7"m Chart 11. Detection of DNA-protein cross-linking by countercurrent distribu tion of nucleoids isolated from MM253 cells labeled with [14C]thymidine (O) and of this DNA, determined using S, nuclease, was the same as that of purified DNA (48Â°) The extent of renaturation of DNA [3H]leucine (â€¢).Top, radioactivity recovered from the aqueous phase; bottom, phenolic phase; transfer of the upper phase was carried out from left to right. A, from control and melphalan-treated cells was also similar to untreated cells; B, melphalan (10 /ig/ml) for 4 hr; C, HN2 (1 fig/ml) for 4 hr.

APRIL 1981 1531

10S). Prolonged heating in perchlorate (75Â° for 30 min) re which can be expected to react with the same nucleophilic moved the cross-links. groups as do melphalan and HN2, did not show cross-resist DNA-Protein Cross-Linking. After countercurrent distribu ance (35) and because the increase in DNA content exceeded tion of lysates prepared from untreated cells, the DNA that of the alternative targets. The similar rates of melphalan ([3H]thymidine labeled) was found in the leading fractions of influx and degree of covalent binding to cell protein and RNA the upper aqueous phase and most of the protein ([14C]leucine indicate that melphalan transport is not impaired in MM253- labeled) remaining in the lower phenol layer of the initial frac 12M cells. tions. To enhance the sensitivity of the method for nuclear As expected from previous studies (1, 34, 53), melphalan protein, nucleoids were isolated prior to lysis and countercur decomposed rapidly under physiological conditions, and there rent distribution. This procedure yielded partition profiles which was no evidence for activation or detoxification by liver micro- showed that HN2 bound sufficient protein to DNA to retain the somes or by intact melanoma cells. The equal labeling by product almost completely in the initial fractions of the phenolic [3H]melphalan of most cells present suggests that most phases phase (Chart 11C). Equitoxic melphalan had no effect upon the of the cell cycle were involved. Assuming that melphalan up partitioning of DNA but increased the amount of nuclear protein take, like that of other mustards (5, 28), is directly proportional associated with the DNA in the phenolic phase (Chart 11S), to the dose, extrapolation to D0 (the dose which on average suggesting some degree of DNA-protein cross-linking. will kill a cell) provides the estimate that 2 x 104 molecules bind to the DNA of each cell (for MM253, 1 molecule per 5 x 10s daltons or 1.5 x 106 bases). This is similar to D0 alkylation DISCUSSION levels of 2.5 x 104 per cell with HN2 in ascites tumor cells (9), Although melphalan itself is mutagenic (33) and UV pretreat 2.3 x 10" per HeLa cell using sulfur mustard (14), 1 alkylation ment was also used, the melphalan resistance and chromo per 5 x 105 bases in mouse L-cells using sulfur mustard (49), some complement of MM253-12M could not be greatly in and 1 alkylation per 106 bases in solid tumors under therapeutic creased by repeated cycles of treatment with melphalan. If conditions (52). resistance in MM253-12M was related to increased chromo More information concerning the nature of melphalan dam some number, the former may be limited by the ability of the age can be obtained by extrapolation of interstrand cross- cells to replicate at even higher ploidy levels. Although hyper- linking to the Do, providing an estimate of 1 cross-link per 2 ploidy was predicted as a means whereby mammalian cells X 109 daltons or 25% of total alkylation products. The heat could develop resistance to alkylating agents (43), only 3 (19, sensitivity of these lesions correlates with that of alkylated 20, 37) of 13 pairs (3, 9, 20, 42, 47, 50, 51) of sensitive and purines (4). This level of interstrand cross-linking was similar resistant rodent cell lines exhibited this association. As part of to that of total cross-linking found in previous studies (9, 51, a separate study of the scope of the effect in human cells, we 54), but the latter consisted mainly of intrastrand links. The found that resistance developed in MM253 to DTIC was also limited supply of [3H]melphalan has thus far prevented estima accompanied by an increase in chromosome complement.4 tion of intrastrand cross-linking in this study and of the extent The basis for such a relationship could be that additional gene of unhooking repair processes of the type reported for other copies increase the probability that a cell will retain its repro cross-linking agents (54). The slow removal of the interstrand ductive capacity (43). However, this makes the assumption links found here may represent spontaneous depurination that the additional target material will not also be lethally rather than an active repair process. damaged; furthermore, it cannot account for the reduced chro The biochemical effects of melphalan damage differed from mosome damage (36), lack of cross-resistance to monofunc- those of UV and MMS in that no rapid inhibition of semicon- tional agents (35), and reduced DNA alkylation found in servative DNA synthesis or DNA chain elongation occurred, MM253-12M cells. Elucidation of these problems may first and the levels of repair synthesis and strand breaks were very require an understanding of the chromosomal heterogeneity low. The insensitivity to caffeine of melphalan toxicity provided and selective increase in the number of large chromosomes further evidence that chain elongation (40) was not a relevant found in the parent line and in other human aneuploid lines factor. The only difference between the sensitive and resistant such as HeLa. Chromosome counts of MM253cl suggest that lines was that the latter showed DNA strand breaks at low the wide range of chromosome numbers found in the uncloned doses; a similar difference was noted with monofunctional line originates not from particular sublines but from every agents (35). Since the decreased level of interstrand cross- proliferating cell, but this apparent instability does not affect linking in MM253-12M was associated with a similar decrease the modal (or average) number even during long periods in in DNA alkylation, the strand breaks probably represent inci culture (36). From the difficulties encountered in the banding sions induced by monoalkylation products rather than unhook studies, it seems likely that chromosomal markers will not be ing of one arm of interstrand-linked melphalan molecules. useful for prediction of drug resistance. However, a third possibility cannot yet be excluded, that in In contrast to sulfur mustard-resistant clones of mouse cells trastrand unhooking occurs and contributes to the resistance (50), MM253-12M cells were larger than were the parent cells. of MM253-12M. The corresponding increase in protein, SH, and RNA content In contrast to previous studies with human lymphocytes (32), raises the question (3, 19, 51) of whether more alternative HN2 did not induce significant DNA repair synthesis in the target material could reduce the effective intracellular level of melanoma lines. It is possible that, in some actively proliferating melphalan. This is an unlikely explanation for the resistance of cells, semiconservative DNA synthesis cannot be depressed MM253-12M cells, however, because monofunctional agents, sufficiently by HU to detect repair synthesis resulting from low levels of short-patch repair. DNA breaks were produced by 4 P. G. Parsons and L. Morrison, unpublished observations. HN2, however, as well as DNA-protein cross-links. The latter

1532 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. Mechanism of Melphalan Resistance result agrees with the work cf Klatt ef al. (25), who found that 19. Goldenberg, G. J. Properties of L5178Y lymphoblasts highly resistant to HN2, but not melphalan, produced extensive DNA-protein nitrogen mustard. Ann. N. Y. Acad. Sci., 163: 936-953. 1969. 20. Goldenberg, G. J., and Alexander. P. The effects of nitrogen mustard and cross-linking in Ehrlich ascites tumor cells. The low level of dimethylmyleran on murine leukaemia cell lines of different radiosensitivity protein-DNA cross-links detected in this study using melphalan in vitro. Cancer Res., 25. 1401-1409, 1965. 21. Goldenberg, G. J., Lee. M., Lam, H-Y. P., and Begleiter, A. Evidence for requires further evaluation using the alkaline elution method (6, carrier-mediated transport of melphalan by L5178Y lymphoblasts in vitro. 41), taking into account the effects of DNA breaks and inter- Cancer Res., 37. 755-760, 1977. strand cross-linking on the elution rate. 22. Goss, P. D., and Parsons, P. G. Temperature-sensitive DNA repair of ultraviolet damage in human cell lines. Int. J. Cancer, 17: 296-303, 1976. Overall, this evidence suggests that the killing effect of 23. Goss. P. D., and Parsons, P. G. The effect of hyperthemia and melphalan on melphalan at minimum letha doses results from DNA cross- survival of human fibroblast strains and melanoma cell lines. Cancer Res., 37. 152-156, 1977. linking involving purine base;; at a frequency too low to detect 24. Harrap, K. R.. Riches, P. G., Gascoigne, E. W.. Sellwood, S. M.. and repair. The resistance of MM253-12M can be explained largely Cashman. C. C. The alkylating agent: does a knowledge of its mode of on the basis of reduced accessibility of DNA to the cross- action suggest leads for improving its chemotherapeutic effectiveness? In: Biological Characterization of Human Tumours, pp. 106-121. Amsterdam: linking effect of melphalan and similar agents, presumably as Excerpta Medica, 1975. a result of specific changes at the DNA or chromatin level. 25. Klatt, O., Stehlin, J. S., Jr., McBride. C., and Griffin. A. C. The effect of Comparisons of nuclear structure now appear necessary, as nitrogen mustard on the deoxyribonucleic acid of sensitive and resistant Ehrlich tumor cells. Cancer Res., 29. 286-290, 1969. well as studies of the scope of this resistance phenomenom 26. Lampidis, T. J., and Little, J. P. Enhancement of UV-induced unscheduled using melphalan-resistant allogeneic lines, other autologous DNA,synthesis by hydroxyurea. Exp. Cell Res.. 110: 41-46, 1977. 27. Lawley, P. D. Effects of some chemical mutagens and carcinogens on pairs, and other alkylating agents. nucleic acids. Prog. Nucleic Acid Res. Mol. Biol., 5 89-131, 1966. 28. Lawley. P. D.. and Brookes. P. Molecular mechanisms of the cytotoxic action of difunctional alkylating agents and of resistance to this action. Nature ACKNOWLEDGMENTS (Lond.), 208. 480-483. 1965. 29. Lehmann, A. R. Post replication repair of DNA in ultraviolet-irradiated mam We thank Irena Jaskiewicz for excellent technical assistance. We are indebted malian cells. J. Mol. Biol., 66. 319-337, 1972. to R. Simmons and C. Bishop for canying out the cell size analyses. 30. Lowry. O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., )93: 265-275, 1951. REFERENCES 31. Luce, J. K. Chemotherapy of malignant melanoma. Cancer (Phila.), 30. 1604-1615, 1972. 1. Alberts, D., Chang, S., Melnick, .., Himmelstein. K.. Walson. P.. Gross. J.. 32. Maurice, P. A., and Lederrey, C. Increased sensitivity of chronic lymphocytic and Salmon, S. Melphalan disposition in man. Proc. Am. Assoc. Cancer leukemia lymphocytes to alkylating agents due to a deficient DNA repair Res., 18: 128. 1977. mechanism. Eur. J. Cancer, 13: 1033-1039, 1977. 2. Alexander. R., Bergsagel, D. E.. Migliore, P. J., Vaughn, W. K., and Howe, 33. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcino C. D. Melphalan therapy for plasma cell myeloma. Blood, 37. 1-10, 1968. gens as mutagens in the Sa/mone//a/microsome test: assay of 300 chemi 3. Ball. C. R.. Connors. T. A., Double, J. A., Ujhazy, V., and Whisson, M. E. cals. Proc. Nati. Acad. Sci. U.S.A., 72. 5135-5139, 1975. Comparison of nitrogen-mustard-sensitive and -resistant Yoshida sarcomas. 34. Milner, A. N., Klatt. O.. Young. S. E., and Stehlin, J. S. The biochemical Int. J. Cancer, 1: 319-327, 196(i. mechanism of action of u-phenylalanine mustard. I. Distribution of u-phenyl- 4. Bannon. P., and Verly. W. Alkylation of phosphates and stability of phosphate alanine mustard-H3 in tumor-bearing rats. Cancer Res., 25. 259-264, 1965. triestersin DNA. Eur. J. Biocherri., 31: 103-111, 1972. 35. Parsons, P. G., and Brown, S. G. Cytotoxicity studies of human melanoma 5. Brookes, P.. and Lawley. P. D. Effects of alkylating agents on T2 and T4 cells and fibroblasts. Ausi. J. Exp. Biol. Med. Sci., 57: 161-170, 1979. bacteriophages. Biochem. J.. 89 138-144, 1963. 36. Parsons, P. G.. and Morrison, L. E. Melphalan-induced chromosome damage 6. Brox, L. W., Gowans, B., and Beli:h, A. L-Phenylalanine mustard (melphalan) in sensitive and resistant human melanoma cell lines. Int. J. Cancer, 21: uptake and cross-linking in the FPMI 6410 human lymphoblastoid cell line. 438-443, 1978. Cancer Res., 40: 1169-1172, 1 380. 37. Podgajetskaja, D. J., Bresler, V. M., Surikov, I. M., Ignatova, T. N., and 7. Buhl, S. N., and Regan. J. D. D^A replication in human cells treated with Olenov, J. M. The transforming action of deoxyribonucleic acid isolated from methylmethane sulphonate. Mutiit. Res.. 18: 191-197, 1975. sarcolysine-resistant tumors of sarcolysine-sensitive tumors. Biochim. Bio- 8. Ceriotti, G. Determination of nuc eie acids in animal tissues. J. Biol. Chem., phys. Acta, 80. 110-115. 1964. 214.: 59-70. 1955. 38. Redwood, W. R., and Colvin. M. Transport of melphalan by sensitive and 9. Chun, E. H. L., Gonzales, L.. LÃ®wis,F. S., Jones. J., and Rutman, R. J. resistant L1210 cells. Cancer Res., 40: 1144-1149, 1980. DiffÃ©rences in thÃ©m vivo alkylation and cross-linking of nitrogen mustard- 39. Regan, J. D., and Setlow, R. B. Repair of human DNA: radiation and chemical sensitive and -resistant lines of I ettrÃ©-Ehrlich ascites tumors. Cancer Res., damage in normal and xeroderma pigmentosum cells. In: J. M. Yuhas. R. W. 29. 1184-1194, 1969. Tennant, and J. D. Regan, (eds.). Biology of Radiation Carcinogenesis, p. 10. Clarkson, J. M. Enhancement of repair replication in mammalian cells by 103. New York: Raven Press, 1976 hydroxyurea. MutÃ¢t.Res.. 52. 2'3-284. 1978. 40. Roberts, J. J. Possible diagnostic value of caffeine as an inhibitor of 11. Clarkson, J. M.. and Evans, H. J. Unscheduled DNA synthesis in human postreplication repair of chemically modified DNA: enhancement of toxicity lymphocytes after exposure to UV-light. X-rays and chemical mutagens. and chromosome damage and effects of newly synthesized DNA. In: R. MutÃ¢t.Res., 14. 413-430, 197ÃŽ. Montesano, H. Bartsch. and L. Tomatis (eds.). Screening Tests in Chemical 12. Cleaver, J. E. Repair replication of mammalian cell DNA: effects of com Carcinogenesis, IARC Scientific Publication No. 12, pp. 605-612. Lyon. pounds that inhibit DNA synthesis or dark repair. RadiÃ¢t. Res., 37: 334- France: International Agency for Research on Cancer. 1976. 348, 1969. 41. Ross. W. E., Ewig, R. A. G., and Kohn, K. W. Differences between melphalan 13. Cook, P. R., and Brazell, I. A. Detection and repair of single-strand breaks and nitrogen mustard in the formation and removal of DNA cross-links. in nuclear DNA. Nature (Lond.), 263. 679-682. 1976. Cancer Res., 38. 1502-1506, 1978. 14. Crathorn. A. R.. and Roberts, J. J. Mechanism of cytotoxic action of 42. Scott, D., Fox, M., and Fox, B. W. The relationship between chromosomal alkylating agents in mammalian cells and evidence for the removal of aberrations, survival and DNA repair in tumor cell lines of different sensitivity alkylated groups from deoxyribDnucleic acid. Nature (Lond.), 211: 150- to X-rays and sulphur mustard. MutÃ¢t.Res.. 22. 207-221. 1974. 152. 1966 43. Simon, 2. Lethal mutation hypothesis for the mechanism of action of the 15. Fisher, B.. Carbone, P., Economou. S. G., Frelick, R., Glass, A., Lerner, H., cytostatic alkylating agents. J. Theor. Biol., 8. 193-197, 1965. Redmond, D., Zellen, M., Band. 3., Katrych, D. L.. Wolmark, N., and Fisher, 44. Smith, J. P., and Rutledge, F. N. Chemotherapy in advanced ovarian cancer. E. R. L-Phenylalanine mustard in the management of primary breast cancer. Nati. Cancer Inst. Monogr.. 42: 141-143, 1974. N. Engl. J. Med., 292. 117-122,1975. 45. Sober, H. A. (ed.). Handbook of Biochemistry, p. H-113. Cleveland, Ohio: 16. Fox, M.. and Fox, B. W. Repair teplication and unscheduled DNA synthesis Chemical Rubber Company, 1970. in mammalian cell lines showing differential sensitivity to alkylating agents. 46. Sumner. A. T. A simple technique for demonstrating centromeric hetero- MutÃ¢t.Res.. 19: 119-128, 197Ã•I. chromatin. Exp. Cell Res., 75. 304-305, 1972. 17. Geiduschek. E. P. On the factors controlling the reversibility of DNA dena- 47. Tsukagoshi, S., and Hashimoto, Y. Increased immunosensitivity in nitrogen turation. J. Mol. Biol.. 4: 467-4IÃŒ7, 1962. mustard-resistant Yoshida sarcoma. Cancer Res., 33. 1038-1042, 1973. 18. Gilles, K., and Myers. A. An improved diphenylamine method for the esti 48. Vistica, D. T.. Toal, J. N., and Rabinovitz, M. Amino-acid-conferred protec mation of deoxyribonucleic acid Nature (Lond.), 206. 93. 1965. tion against melphalan. Biochem. Pharmacol., 27. 2865-2870, 1978.

APRIL 1981 1533

49. Walker, I. G. Sulphur mustard: reaction with L-cells treated with 5-fluoro- fixation of C" of labelled alkylating agents by bilaterally grown sensitive and deoxyuridine. Science (Wash. D. C.), 151: 99-101, 1976. resistant tumors. Cancer Res.. 24: 1331-1337, 1964. 50. Walker. I. G., and Reid, B. D. Some properties of substrains of L-cells with 53. Williamson, C. E., and Witten. B. Reaction mechanism of some aromatic a decreased sensitivity to bis(2-chloroethyl)sulfide. Cancer Res., 3). 510- nitrogen mustards. Cancer Res., 27. 33-38, 1967. 515. 1971. 54. Yin, L., Chun, E. H. L., and Rutman, R. J. A comparison of the effects of 51. Wheeler, G. P. Studies related to mechanisms of resistance to biological alkylation on the DNA of sensitive and resistant Lettre-Ehrlich cells following alkylating agents. Cancer Res., 23. 1334-1349, 1963. in vivo exposure to nitrogen mustard. Biochim. Biophys. Acta. 324. 472- 52. Wheeler, G. P., and Alexander. J. A. Studies with mustards. V. In vivo 481, 1973.

1534 CANCER RESEARCH VOL. 41

P. G. Parsons, F. B. Carter, L. Morrison, et al.

Cancer Res 1981;41:1525-1534.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/4/1525

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/41/4/1525. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.