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A comparative cytogenetic study of melphalan-sensitive and -resistant murine L1210 leukemia cells
Cancer Letters, 8 (1980) o Elsevier/North-Holland
335-341 Scientific Publishers Ltd.
335
A COMPARATIVE CYTOGENETIC STUDY OF MELPHALAN-SENSITIVE AND -RESISTANT MURINE L1210 LEUKEMIA CELLS
BARBARA
P. SCHUETTE,
MARCO
RABINOVITZ
and DAVID
T. VISTICA
Laboratory of Medicinal Chemistry and Biology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Ml3 20205 (U.S.A.) (Received 11 October 1979) (Accepted 21 November 1979)
SUMMARY
A cytogenetic analysis of melphalan-sensitive and -resistant murine L1210 leukemia cells maintained in vivo indicated that the drug-resistant tumor had a modal number of 40 chromosomes while the sensitive tumor possessed a mode of 41 chromosomes. Two marker chromosomes were present in both the sensitive and the resistant tumors. One was of medium length with secondary constrictions, and the other was a minute chromosome. A single in vitro exposure of the sensitive tumor cells to a cytotoxic concentration of melphalan was not accompanied by a change in modal number but resulted in chromatid exchange. No alterations were found in resistant tumor cells which were exposed to the same drug concentration. However, exposure of resistant tumor cells to higher doses of melphalan resulted in chromatid breaks, chromatid gaps and the formation of acentric chromosomes. The resistant tumor, transplanted once without drug injection, maintained a sharp mode of 40 chromosomes.
INTRODUCTION
Melphalan (L-phenylalanine mustard, L-PAM) is an alkylating agent [ 111 with a broad range of antitumor activity both in experimental animals [ 161 and in human neoplasms where it is used in the treatment of multiple myeloma [ 6,101, ovarian carcinoma [ 4,181, breast cancer [ 51 and malignant melanoma [ 81. Objective clinical response to melphalan often declines after repeated courses of therapy due to the emergence of a drug resistant cell population. In order to determine whether the development of resistance all correspondence to : David T. Vistica, Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, Building 37, Room 6B09, Bethesda, MD 20205, U.S.A.
Address
336
to melphalan is accompanied by a specific change in karyotype, we have undertaken a comparative cytogenetic study of a melphalan-sensitive murine tumor and a melphalan-resistant variant developed from it by repeated in vivo exposure to sub-optimal doses of the drug [ 121. MATERIALS
AND METHODS
RPMI-1630 medium and phosphate buffered saline were obtained from the NIH Media Unit. Gentamicin was purchased from Microbiological Associates, Bethesda, MD and bovine serum albumin was obtained from Miles Laboratories, Elkhart, IN. Fetal bovine serum was obtained from Flow Laboratories, Rockville, MD. Melphalan was purchased from Burroughs Wellcome, Research Triangle Park, NC. Colcemid was obtained from Grand Island Biological Co., Grand Island, NY and gentian violet was purchased from Fisher Scientific Co., Fairlawn, NJ. The murine L1210 leukemia was obtained under contract from the Mason Research Institute, Boston, MA. A melphalan-resistant tumor was developed from this line at the Southern Research Institute, Birmingham, AL and kindly provided by Mrs. Mary Trader. Passage of tumor
Mice were killed by cervical dislocation and tumor cells from male BDF, mice bearing the melphalan-sensitive and -resistant murine L1210 leukemia were removed by flushing the peritoneum with phosphate buffered saline (PBS) containing 0.1 mM bovine serum albumin and 0.25% glucose. Mice were injected intraperitoneally (i.p.) with 10’ sensitive cells or with lo6 resistant cells. Mice receiving the resistant tumor were given an i.p. injection of melphalan (7.5 mg/kg) 48 h after inoculation with tumor. Melphalan was prepared as a 0.1 M stock solution in 90% dimethyl sulfoxide containing equimolar hydrochloric acid, diluted with PBS to the final desired concentration and injected in a final vol. of 0.1 ml.. Preparation analysis
of cultures
for in vitro melphalan
treatment
and chromosome
Cells were harvested 5-6 days after passage from the ascitic fluid of 2-3-month-old male CDFl mice bearing 3 tumor variants: drug-sensitive, drug-resistant with melphalan injection and drug-resistant without melphalan injection. The procedure for harvesting the cells was similar to that described above except that RPMI-1630, supplemented with 20% heat-inactivated fetal bovine serum and 40 I_cg/mlgentamicin, was used. Drug-sensitive and -resistant cells were then washed twice in the same medium by centrifugation at 300 Xg, seeded at 7.5 X lo5 cells/ml and incubated 18-24 h at 37°C. They were then treated at the same concentration in vitro with drug for 1 h at 37°C. Melphalan concentrations were 0.8 PM for the sensitive tumor cells and 0.8 PM and 25 PM for the resistant cells. Melphalan was prepared
337
as a 32 mM stock solution in 75% ethanol containing equimolar hydrochloric acid, diluted with 75% ethanol and used at a concentration of 1 pi/ml of medium. Control cells were exposed for 1 h to an equal vol. of 75% ethanol. Both suspensions were then harvested by centrifugation, washed twice in RPMI-6130, seeded at 7.5 X 10’ cells/ml and incubated at an additional 18-24 h. Chromosome
preparation
Colcemid was added to the cultures at a concentration of 0.1 pg/ml and the cells incubated for 2 h at 37°C. The cells were then centrifuged for 5 min at 300 X g and resuspended in 5 ml 0.075 M KC1 for 15 min at 37°C. Five milliliters of freshly prepared fixative (methanol/glacial acetic acid, 3 : 1 v/v) was added to the hypotonic cell suspension which was then centrifuged for 5 min at 130 X g. The pellets were treated with 1 ml fixative 3 additional times and resuspended in approx. 0.5 ml fixative. Three slides of each treatment group were prepared by placing 3 drops of suspension on ethanol-cleaned slides and air-drying. Slides were stained the following day for 2 min with Turks solution (0.01% gentian violet in 1% ace?.ic acid), rinsed twice, air-dried and mounted. Scoring
modal number
The modal number for each treatment group was determined by slides prepared from at least 2 experiments. No less than 5 different slides were used to obtain the modal number. The first 15-25 intact metaphases were scored/slide. One hundred metaphases for each treatment group were counted and recorded to obtain the modal number. Photomicrographs were taken on a Zeiss microscope equipped with a camera attachment containing Kodak high contrast copy film. RESULTS
The melphalan-sensitive tumor cells exhibited a modal number of 41 chromosomes as compared to a modal number of 40 for the drug-resistant tumor cells (Figs. lA,C). Approximately 15% of the drug-sensitive cells containing 40 chromosomes compared to 72% for the drug-resistant tumor. Examination of metaphase spreads of the sensitive and resistant tumor indicated that 2 marker chromosomes, one of medium length possessing secondary constrictions [ 21 and a minute chromosome [ 2,3] were present in both the drug-sensitive and -resistant tumors (Figs. 2A,B). A subtelocentric marker cited by Biedler et al. [2] and Biesele et al. [3] was found in less than 5% of the sensitive and resistant tumor cells. In vitro exposure of the sensitive cells to melphalan was accompanied by an increase in the percentage of cells with 42 chromosomes, but no change in the modal number (Fig. 1B). A single omission of the in vivo maintenance injection of melphalan had no effect on the modal number of chromosomes in drug-resistant cells (Fig. 1D).
L1210 MELPHALAN-
MELPHALAN-SENSITIVE
SENSITIVE
WITHOUT DRUG
WITH
EXPOSURE
DRUG
EXPOSURE
”
z-
D 70
ti z a
60
50
L1210
L1210
MELPHALAN-RESISTANT
MELPHALAN-RESISTA~
WITH DRUG
0
383
-114243
56
WITHOUT
EXPOSURE
78
80
DRUG
82>120
38394.0414243
EXPOSURE
64
72
80
CHROMOSOME NUMBER Fig. 1. Comparison of modal number counts of melphalansensitive and -resistant murine L1210 leukemia cells. One hundred cells were counted and recorded for each treatment group. Melphalansensitive tumors were harvested 5-6 days after transplantation and exposed in vitro to 0.8 PM melphalan or ethanol as described in Materials and Methods. Mice bearing melphalan-resistant tumors were given either 7.5 mg/kg of melphalan or an equivalent volume of saline 48 h after tumor transplantation. Cells were harvested 5-6 days after transplantation as described in Materials and Methods: (A) melphalansensitive tumor without in vitro drug exposure; (B) melphalansensitive tumor with in vitro drug exposure; (C) melphalan-resistant tumor with in vivo drug exposure; and (D) melphalan-resistant tumor without in vivo drug exposure.
Exposure of the sensitive tumor cells to 0.8 PM melphalan in vitro for 1 h resulted in aberrations of the chromatid interchange type in approx. 20% of the metaphases scored (Fig. 2C). No similar structural alterations were found in the resistant tumor after in vitro exposure to the same drug concentration. Exposure of resistant tumor cells to 25 PM melphalan in vitro resulted in chromatid breaks, chromatid gaps and the formation of acentric chromosomes in approx. 20% of the intact metaphases examined (Fig. 2D).
Fig. 2. Metaphase spreads of melphalansensitive and -resistant murine L1210 leukemia cells. Effect of in vitro melphalan exposure; (A) melphalan-sensitive tumor prossessing 41 chromosomes including 2 marker chromosomes one with secondary constrictions (SC) and a minute chromosome (M), 1232X ; (B) melphalan-resistant tumor possessing 40 chromosomes including the 2 marker chromosomes found in the drug-sensitive tumor, 1296X; (C) metaphase spread of melphalansensitive tumor exposure to 0.8 PM melphslan in vitro for 1 h. Chromatid exchange (CE) is evident at arrows, 1216x; and (D) Metaphase spread of melphalan-resistant tumor exposed to 25 rM meWalan in vitro for 1 h. Chromatid breaks (CB), chromatid gaps (CG) and an acentric chromosome (AC) are present, 1168X.
DISCUSSION
The development of resistance to alkylating agents by tumor cells is often, but not always, accompanied by alterations in karyotype. Hirono and Yokoyama [ 71 using nitrogen mustard N-Oxide and Ball et al. [l] using melphalan indicated that there were no chromosomal differences in drug
340
sensitive and drug resistant variants of the Yoshida sarcoma. Ujhazy [ 131 confirmed this observation and also demonstrated that a large telocentric marker chromosome emerged in the resistant karyotype which was analogous to the large subtelocentric marker in the sensitive tumor. Banding studies failed to reveal a relationship between these 2 markers. Results which parallel those found in the present study were reported by Voitovitskii et al. [15] who observed a decrease in modal number from 48 -49 to 46.-47 chromosomes in rat ovarian cells which had become resistant to sarcolysin, the DL form of melphalan. These authors also found a decrease from 48-49 chromosomes to 46-47 chromosomes in rat ovarian tumors resistant to 6-mercaptopurine and thio-tepa [ 141. A possible relationship between chromosome number and drug resistance to alkylating agents has been reported by Yoshida [ 171. He observed that cells from rat ascites hepatomas which were resistant to nitrogen mustard N-Oxide had higher modal numbers than sensitive tumor cells. A recent report [ 91 indicated that the development of resistance to melphalan by a human melanoma cell line was accompanied by an increase in the average chromosome number from 53 to 72. The results described in the present communication failed to demonstrate a relationship between resistance to melphalan, and the emergence of a karyotype with an altered ploidy level. It is not known whether the net loss of 1 non-marker chromosome is responsible for resistance to melphalan or is merely a manifestation accompanying the development of such resistance. Since all non-marker chromosomes are acrocentric, additional studies utilizing giemsa banding techniques were undertaken to define the exact nature of the decrease in modal number. However, differences in the banding patterns of the sensitive and -resistant tumors have, as yet, not been found. REFERENCES 1 Ball, C.R., Connors, T.A., Double, J.A., Ujhazy, V. and Whisson, M.E. (1966) Comparison of nitrogen mustard-sensitive and -resistant Yoshida sarcomas. Int. J. Cancer, 1,319-32’7. 2 Biedler, J.L., Schrecker, A.W. and Hutchison, D.J. (1963) Selection of chromosomal variant in amethopterin-resistant sublines of leukemia L1210 with increased levels of dihydrofolate reductase. J. Natl. Cancer Inst., 31, 575-601. 3 Biesele, J.J., Biedler, J.L. and Hutchison, D.J. (1959) The chromosomal status of drug resistant sublines of mouse leukemia L1210. In: Genetics and Cancer, pp. 295-307, Univ. Texas Press, Austin. 4 IkPalo, G.M., DeLena, M., DiRe, F., Luciani, L., Valaqussa, P. and Bonadonna, G. (1975) Melphalan versus adriamycin in the treatment of advanced carcinoma of the ovary. Surg. Gynecol. Obstet., 141, 899-902. 5 Greenspan, E.M. (1975) Clinical cancer chemotherapy, pp. 151-172, Raven Press, New York. 6 Hammack, W.J., Huguley, C.M. and Chan, Y.K. (1975) Treatment of myeloma. Comparison of melphalan, chlorambucil and axathioprine. Arch. Int. Med., 135, 157-162. 7 Hirono, I. and Yokoyama, C. (1955) Chromosome features in the original and resistant sublines of Yoshida sarcoma. Cytologia, 20,84-88.
34 8 McBride, CM., Sugarbaker, E.V. and Hickey, R.C. (1975) Prophylactic isolation perfusion as the primary therapy for invasive malignant melanoma of the limbs. Ann. Surg., 182, 316-324. 9 Parsons, P.G. and Morrison, L. (1978) Melphalan-induced chromosome damage in sensitive and resistant human melanoma cell lines. Int. J. Cancer, 21, 438-443. 10 Rosen, B.J. (1975) Multiple myeloma. A clinical review. Med. Clin. North Am., 59,375-386. 11 Ross, W.C.J. (1962) Biological alkylating agents, 232 pp, Butterworths, London. 12 Schabel, F.M., Trader, M.W., Laster, W.R., Wheeler, G.P. and Witt, M.H. (1978) Patterns of resistance and therapeutic synergism among alkylating agents. In: Antibiotics and Chemotherapy, pp. 200-215, S. Karger, Basel. 13 Ujhazy, V. (1974) Identical karyotypes in acquired drug-resistant sublines of Yoshida sarcoma. Neoplasma, 21, 665-669. 14 Voitovitskii, V.K., Edygenova, A.K. and Kabiev, O.K. (1970) Cytogenetic analysis of rat ovarian tumor variants resistant to 6-mercaptopurine and thio-tepa. Vopr. Gnkol., 16, 87-92. 15 Voitovitskii, V.K., Edygenova, A.K. and Kabiev, O.K. (1971) Comparative cytogenetic study of the resistance of tumor cells to sarcolysine. Genetika, 7, 89-93. 16 White, F.R. (1960) Sarcolysin and related compounds. Cancer Chemother. Rep., 6,61-93. 17 Yoshida, T. (1957) Studien uber das ascites-hepatom. Virchows Arch. Pathol. Anat., 330, 85-105. 18 Young, R.C., Canellos, G.P., Chabner, B.A., Schein, P.S., Hubbard, S.P. and DeVita, V.T. (1974) Chemotherapy of advanced ovarian carcinoma: A prospective randomized comparison of phenylalanine mustard and high dose cyclophosphamide. Gynecol. Gncol., 2,489--497.
335-341 Scientific Publishers Ltd.
335
A COMPARATIVE CYTOGENETIC STUDY OF MELPHALAN-SENSITIVE AND -RESISTANT MURINE L1210 LEUKEMIA CELLS
BARBARA
P. SCHUETTE,
MARCO
RABINOVITZ
and DAVID
T. VISTICA
Laboratory of Medicinal Chemistry and Biology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Ml3 20205 (U.S.A.) (Received 11 October 1979) (Accepted 21 November 1979)
SUMMARY
A cytogenetic analysis of melphalan-sensitive and -resistant murine L1210 leukemia cells maintained in vivo indicated that the drug-resistant tumor had a modal number of 40 chromosomes while the sensitive tumor possessed a mode of 41 chromosomes. Two marker chromosomes were present in both the sensitive and the resistant tumors. One was of medium length with secondary constrictions, and the other was a minute chromosome. A single in vitro exposure of the sensitive tumor cells to a cytotoxic concentration of melphalan was not accompanied by a change in modal number but resulted in chromatid exchange. No alterations were found in resistant tumor cells which were exposed to the same drug concentration. However, exposure of resistant tumor cells to higher doses of melphalan resulted in chromatid breaks, chromatid gaps and the formation of acentric chromosomes. The resistant tumor, transplanted once without drug injection, maintained a sharp mode of 40 chromosomes.
INTRODUCTION
Melphalan (L-phenylalanine mustard, L-PAM) is an alkylating agent [ 111 with a broad range of antitumor activity both in experimental animals [ 161 and in human neoplasms where it is used in the treatment of multiple myeloma [ 6,101, ovarian carcinoma [ 4,181, breast cancer [ 51 and malignant melanoma [ 81. Objective clinical response to melphalan often declines after repeated courses of therapy due to the emergence of a drug resistant cell population. In order to determine whether the development of resistance all correspondence to : David T. Vistica, Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, Building 37, Room 6B09, Bethesda, MD 20205, U.S.A.
Address
336
to melphalan is accompanied by a specific change in karyotype, we have undertaken a comparative cytogenetic study of a melphalan-sensitive murine tumor and a melphalan-resistant variant developed from it by repeated in vivo exposure to sub-optimal doses of the drug [ 121. MATERIALS
AND METHODS
RPMI-1630 medium and phosphate buffered saline were obtained from the NIH Media Unit. Gentamicin was purchased from Microbiological Associates, Bethesda, MD and bovine serum albumin was obtained from Miles Laboratories, Elkhart, IN. Fetal bovine serum was obtained from Flow Laboratories, Rockville, MD. Melphalan was purchased from Burroughs Wellcome, Research Triangle Park, NC. Colcemid was obtained from Grand Island Biological Co., Grand Island, NY and gentian violet was purchased from Fisher Scientific Co., Fairlawn, NJ. The murine L1210 leukemia was obtained under contract from the Mason Research Institute, Boston, MA. A melphalan-resistant tumor was developed from this line at the Southern Research Institute, Birmingham, AL and kindly provided by Mrs. Mary Trader. Passage of tumor
Mice were killed by cervical dislocation and tumor cells from male BDF, mice bearing the melphalan-sensitive and -resistant murine L1210 leukemia were removed by flushing the peritoneum with phosphate buffered saline (PBS) containing 0.1 mM bovine serum albumin and 0.25% glucose. Mice were injected intraperitoneally (i.p.) with 10’ sensitive cells or with lo6 resistant cells. Mice receiving the resistant tumor were given an i.p. injection of melphalan (7.5 mg/kg) 48 h after inoculation with tumor. Melphalan was prepared as a 0.1 M stock solution in 90% dimethyl sulfoxide containing equimolar hydrochloric acid, diluted with PBS to the final desired concentration and injected in a final vol. of 0.1 ml.. Preparation analysis
of cultures
for in vitro melphalan
treatment
and chromosome
Cells were harvested 5-6 days after passage from the ascitic fluid of 2-3-month-old male CDFl mice bearing 3 tumor variants: drug-sensitive, drug-resistant with melphalan injection and drug-resistant without melphalan injection. The procedure for harvesting the cells was similar to that described above except that RPMI-1630, supplemented with 20% heat-inactivated fetal bovine serum and 40 I_cg/mlgentamicin, was used. Drug-sensitive and -resistant cells were then washed twice in the same medium by centrifugation at 300 Xg, seeded at 7.5 X lo5 cells/ml and incubated 18-24 h at 37°C. They were then treated at the same concentration in vitro with drug for 1 h at 37°C. Melphalan concentrations were 0.8 PM for the sensitive tumor cells and 0.8 PM and 25 PM for the resistant cells. Melphalan was prepared
337
as a 32 mM stock solution in 75% ethanol containing equimolar hydrochloric acid, diluted with 75% ethanol and used at a concentration of 1 pi/ml of medium. Control cells were exposed for 1 h to an equal vol. of 75% ethanol. Both suspensions were then harvested by centrifugation, washed twice in RPMI-6130, seeded at 7.5 X 10’ cells/ml and incubated at an additional 18-24 h. Chromosome
preparation
Colcemid was added to the cultures at a concentration of 0.1 pg/ml and the cells incubated for 2 h at 37°C. The cells were then centrifuged for 5 min at 300 X g and resuspended in 5 ml 0.075 M KC1 for 15 min at 37°C. Five milliliters of freshly prepared fixative (methanol/glacial acetic acid, 3 : 1 v/v) was added to the hypotonic cell suspension which was then centrifuged for 5 min at 130 X g. The pellets were treated with 1 ml fixative 3 additional times and resuspended in approx. 0.5 ml fixative. Three slides of each treatment group were prepared by placing 3 drops of suspension on ethanol-cleaned slides and air-drying. Slides were stained the following day for 2 min with Turks solution (0.01% gentian violet in 1% ace?.ic acid), rinsed twice, air-dried and mounted. Scoring
modal number
The modal number for each treatment group was determined by slides prepared from at least 2 experiments. No less than 5 different slides were used to obtain the modal number. The first 15-25 intact metaphases were scored/slide. One hundred metaphases for each treatment group were counted and recorded to obtain the modal number. Photomicrographs were taken on a Zeiss microscope equipped with a camera attachment containing Kodak high contrast copy film. RESULTS
The melphalan-sensitive tumor cells exhibited a modal number of 41 chromosomes as compared to a modal number of 40 for the drug-resistant tumor cells (Figs. lA,C). Approximately 15% of the drug-sensitive cells containing 40 chromosomes compared to 72% for the drug-resistant tumor. Examination of metaphase spreads of the sensitive and resistant tumor indicated that 2 marker chromosomes, one of medium length possessing secondary constrictions [ 21 and a minute chromosome [ 2,3] were present in both the drug-sensitive and -resistant tumors (Figs. 2A,B). A subtelocentric marker cited by Biedler et al. [2] and Biesele et al. [3] was found in less than 5% of the sensitive and resistant tumor cells. In vitro exposure of the sensitive cells to melphalan was accompanied by an increase in the percentage of cells with 42 chromosomes, but no change in the modal number (Fig. 1B). A single omission of the in vivo maintenance injection of melphalan had no effect on the modal number of chromosomes in drug-resistant cells (Fig. 1D).
L1210 MELPHALAN-
MELPHALAN-SENSITIVE
SENSITIVE
WITHOUT DRUG
WITH
EXPOSURE
DRUG
EXPOSURE
”
z-
D 70
ti z a
60
50
L1210
L1210
MELPHALAN-RESISTANT
MELPHALAN-RESISTA~
WITH DRUG
0
383
-114243
56
WITHOUT
EXPOSURE
78
80
DRUG
82>120
38394.0414243
EXPOSURE
64
72
80
CHROMOSOME NUMBER Fig. 1. Comparison of modal number counts of melphalansensitive and -resistant murine L1210 leukemia cells. One hundred cells were counted and recorded for each treatment group. Melphalansensitive tumors were harvested 5-6 days after transplantation and exposed in vitro to 0.8 PM melphalan or ethanol as described in Materials and Methods. Mice bearing melphalan-resistant tumors were given either 7.5 mg/kg of melphalan or an equivalent volume of saline 48 h after tumor transplantation. Cells were harvested 5-6 days after transplantation as described in Materials and Methods: (A) melphalansensitive tumor without in vitro drug exposure; (B) melphalansensitive tumor with in vitro drug exposure; (C) melphalan-resistant tumor with in vivo drug exposure; and (D) melphalan-resistant tumor without in vivo drug exposure.
Exposure of the sensitive tumor cells to 0.8 PM melphalan in vitro for 1 h resulted in aberrations of the chromatid interchange type in approx. 20% of the metaphases scored (Fig. 2C). No similar structural alterations were found in the resistant tumor after in vitro exposure to the same drug concentration. Exposure of resistant tumor cells to 25 PM melphalan in vitro resulted in chromatid breaks, chromatid gaps and the formation of acentric chromosomes in approx. 20% of the intact metaphases examined (Fig. 2D).
Fig. 2. Metaphase spreads of melphalansensitive and -resistant murine L1210 leukemia cells. Effect of in vitro melphalan exposure; (A) melphalan-sensitive tumor prossessing 41 chromosomes including 2 marker chromosomes one with secondary constrictions (SC) and a minute chromosome (M), 1232X ; (B) melphalan-resistant tumor possessing 40 chromosomes including the 2 marker chromosomes found in the drug-sensitive tumor, 1296X; (C) metaphase spread of melphalansensitive tumor exposure to 0.8 PM melphslan in vitro for 1 h. Chromatid exchange (CE) is evident at arrows, 1216x; and (D) Metaphase spread of melphalan-resistant tumor exposed to 25 rM meWalan in vitro for 1 h. Chromatid breaks (CB), chromatid gaps (CG) and an acentric chromosome (AC) are present, 1168X.
DISCUSSION
The development of resistance to alkylating agents by tumor cells is often, but not always, accompanied by alterations in karyotype. Hirono and Yokoyama [ 71 using nitrogen mustard N-Oxide and Ball et al. [l] using melphalan indicated that there were no chromosomal differences in drug
340
sensitive and drug resistant variants of the Yoshida sarcoma. Ujhazy [ 131 confirmed this observation and also demonstrated that a large telocentric marker chromosome emerged in the resistant karyotype which was analogous to the large subtelocentric marker in the sensitive tumor. Banding studies failed to reveal a relationship between these 2 markers. Results which parallel those found in the present study were reported by Voitovitskii et al. [15] who observed a decrease in modal number from 48 -49 to 46.-47 chromosomes in rat ovarian cells which had become resistant to sarcolysin, the DL form of melphalan. These authors also found a decrease from 48-49 chromosomes to 46-47 chromosomes in rat ovarian tumors resistant to 6-mercaptopurine and thio-tepa [ 141. A possible relationship between chromosome number and drug resistance to alkylating agents has been reported by Yoshida [ 171. He observed that cells from rat ascites hepatomas which were resistant to nitrogen mustard N-Oxide had higher modal numbers than sensitive tumor cells. A recent report [ 91 indicated that the development of resistance to melphalan by a human melanoma cell line was accompanied by an increase in the average chromosome number from 53 to 72. The results described in the present communication failed to demonstrate a relationship between resistance to melphalan, and the emergence of a karyotype with an altered ploidy level. It is not known whether the net loss of 1 non-marker chromosome is responsible for resistance to melphalan or is merely a manifestation accompanying the development of such resistance. Since all non-marker chromosomes are acrocentric, additional studies utilizing giemsa banding techniques were undertaken to define the exact nature of the decrease in modal number. However, differences in the banding patterns of the sensitive and -resistant tumors have, as yet, not been found. REFERENCES 1 Ball, C.R., Connors, T.A., Double, J.A., Ujhazy, V. and Whisson, M.E. (1966) Comparison of nitrogen mustard-sensitive and -resistant Yoshida sarcomas. Int. J. Cancer, 1,319-32’7. 2 Biedler, J.L., Schrecker, A.W. and Hutchison, D.J. (1963) Selection of chromosomal variant in amethopterin-resistant sublines of leukemia L1210 with increased levels of dihydrofolate reductase. J. Natl. Cancer Inst., 31, 575-601. 3 Biesele, J.J., Biedler, J.L. and Hutchison, D.J. (1959) The chromosomal status of drug resistant sublines of mouse leukemia L1210. In: Genetics and Cancer, pp. 295-307, Univ. Texas Press, Austin. 4 IkPalo, G.M., DeLena, M., DiRe, F., Luciani, L., Valaqussa, P. and Bonadonna, G. (1975) Melphalan versus adriamycin in the treatment of advanced carcinoma of the ovary. Surg. Gynecol. Obstet., 141, 899-902. 5 Greenspan, E.M. (1975) Clinical cancer chemotherapy, pp. 151-172, Raven Press, New York. 6 Hammack, W.J., Huguley, C.M. and Chan, Y.K. (1975) Treatment of myeloma. Comparison of melphalan, chlorambucil and axathioprine. Arch. Int. Med., 135, 157-162. 7 Hirono, I. and Yokoyama, C. (1955) Chromosome features in the original and resistant sublines of Yoshida sarcoma. Cytologia, 20,84-88.
34 8 McBride, CM., Sugarbaker, E.V. and Hickey, R.C. (1975) Prophylactic isolation perfusion as the primary therapy for invasive malignant melanoma of the limbs. Ann. Surg., 182, 316-324. 9 Parsons, P.G. and Morrison, L. (1978) Melphalan-induced chromosome damage in sensitive and resistant human melanoma cell lines. Int. J. Cancer, 21, 438-443. 10 Rosen, B.J. (1975) Multiple myeloma. A clinical review. Med. Clin. North Am., 59,375-386. 11 Ross, W.C.J. (1962) Biological alkylating agents, 232 pp, Butterworths, London. 12 Schabel, F.M., Trader, M.W., Laster, W.R., Wheeler, G.P. and Witt, M.H. (1978) Patterns of resistance and therapeutic synergism among alkylating agents. In: Antibiotics and Chemotherapy, pp. 200-215, S. Karger, Basel. 13 Ujhazy, V. (1974) Identical karyotypes in acquired drug-resistant sublines of Yoshida sarcoma. Neoplasma, 21, 665-669. 14 Voitovitskii, V.K., Edygenova, A.K. and Kabiev, O.K. (1970) Cytogenetic analysis of rat ovarian tumor variants resistant to 6-mercaptopurine and thio-tepa. Vopr. Gnkol., 16, 87-92. 15 Voitovitskii, V.K., Edygenova, A.K. and Kabiev, O.K. (1971) Comparative cytogenetic study of the resistance of tumor cells to sarcolysine. Genetika, 7, 89-93. 16 White, F.R. (1960) Sarcolysin and related compounds. Cancer Chemother. Rep., 6,61-93. 17 Yoshida, T. (1957) Studien uber das ascites-hepatom. Virchows Arch. Pathol. Anat., 330, 85-105. 18 Young, R.C., Canellos, G.P., Chabner, B.A., Schein, P.S., Hubbard, S.P. and DeVita, V.T. (1974) Chemotherapy of advanced ovarian carcinoma: A prospective randomized comparison of phenylalanine mustard and high dose cyclophosphamide. Gynecol. Gncol., 2,489--497.