(CANCER RESEARCH 50. 62-66. January 1. 1990] Changes in c-myc and c-fos Expression in a Human Tumor Cell Line following Exposure to Bifunctional Alkylating Agents1 Bernard W. Futscher and Leonard C. Erickson Section ofHematology•/'Oncology, Departments of Medicine [L. C. E./ and Pharmacology [B. H'. F., L. C. E.], Loyola University of Chicago, Stritch School of Medicine, Mayn-ood, Illinois 60153 ABSTRACT This study was initiated to determine if DNA-damaging chemothera- peutic agents can suppress the expression of oncogenes. The effects of three structurally related bifunctional alkylating agents on the steady state mRNA levels of c-myc, c-fos, N-ras, and 0-actin in the human colon carcinoma cell line Colo320HSR were examined. Colo320HSR has an amplified c-myc oncogene, which is highly overexpressed, and is assumed to be one of the transforming genes of this cell line. Two concentrations of mechlorethamine, i.-phenylalanine mustard, and 4-hydroperoxycyclo- phosphamide, which produced 1 or 3 log cell kills were used to examine the effects of drug exposure on the expression of specific genes. Steady state mRNA levels were measured by Northern blot analysis. Following a I-li drug exposure, RNA was isolated from cells at 0, 6, 12, and 24 h following drug removal. The agents used produced changes in the expres sion of specific genes, and all three did so in a similar fashion. Immedi ately following drug removal, the steady state expression of c-m.tr in treated cells was increased 2- to 3-fold compared to control. At 6 and 12 h following drug removal, c-myc levels were depressed 2.5- to 5-fold. By 24 li. i--m.tr expression approached, but remained below, control levels. Immediately following drug removal, c-fos levels were increased 3- to 4- fold, and from 6 to 24 h following drug removal, c-fos levels gradually returned to, or fell below low basal levels. During the 24-h time course, drug treatment had little or no effect on the steady state levels of N-ras or 0-actin. These data support the hypothesis that alkylating agents may suppress the expression of specific transforming genes. INTRODUCTION In recent years, a number of investigations have demonstrated that perturbations in normal cellular protooncogenes can trans form them into activated oncogenes. These changes may occur by a variety of mechanisms, (e.g., amplification, translocation, point mutation) and typically result in aberrant gene expression or an altered protein product (1-3). Once activated, it appears that oncogenes play a critical role in the tumorigenic phenotype (4-6). Although much is known of the role oncogenes play in carcinogenesis, a paucity of data exists regarding the effects of clinically efficacious antitumor agents on oncogene expression. It is conceivable, however, that different antitumor agents may selectively inhibit the expression of specific oncogenes. For example, a preliminary report indicated that exposure to HN2: suppressed c-myc expression, but did not affect ras expression, in the Burkitt lymphoma cell line Daudi (7). Another report examined the effect of a variety of clinical antileukemic chem- otherapeutic regimens on c-myc expression in patients with leukemia (8). Although no alkylating agents were included in the chemotherapeutic regimens used, this study demonstrated that depression of c-myc expression 24 h after treatment cor- Receivcd 2/15/89; revised 9/7/89; accepted 9/28/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported in part by a fellowship awarded to B. \V. F. by the Arthur J. Schmitt Foundation, and NIH Grants ROI CA 45628 and ROÕ CA47929 awarded to L. C. E. 2 The abbreviations used are: HN2, mechlorethamine; L-PAM. melphalan; 4- HC, 4-hydroperoxycyclophosphamide; SDS. sodium dodecyl sulfate; Ix SSPE. 0.15 M NaCl. 0.015 M NaH2PO4. 0.002 M EDTA. pH 7.4. related with tumor response in patients with acute myelogenous leukemia or acute lymphocytic leukemia (8). These data suggest that drug effects on oncogene expression may serve as indicators of prognosis in specific types of cancer (8). In this report, we have examined the effects of three struc turally related bifunctional alkylating agents on oncogene expression in the colon carcinoma cell line Colo320HSR. Colo320HSR is a human colon tumor cell line of neuroecto- dermal origin isolated from a patient prior to the onset of chemotherapy (9). Contained within the homogeneously stain ing region (HSR) are approximately 30 to 40 copies of the c- myc oncogene, which in turn is highly overexpressed, and is assumed to be one of the transforming genes of this cell line ( 10). Low levels of expression of c-fos and N-ras can also be detected (this study). The compounds used in this study are the clinically useful agents, HN2, L-PAM, and a form of cyclophosphamide active in vitro, 4-HC (11). These electrophilic agents react with a number of cellular macromolecules, including protein and RNA, but it appears that they exert their cytotoxic effects by producing lesions in the DNA, particularly DNA interstrand and intrastrand cross-links (12-16). We have examined the effects of the aforementioned alkylating agents on the expres sion of selected genes in Colo320HSR. These genes are the amplified c-myc oncogene, the N-ras and c-fos oncogenes, as well as the gene encoding £i-actin. The three alkylating agents produced changes in the expression of specific genes, and each did so in a similar fashion. Immediately following drug treat ment, steady state levels of c-myc and c-fos transcripts are increased 2- to 3-fold and 3- to 4-fold, respectively. From 6 to 24 h following drug treatment, c-myc expression decreased to below control levels, and c-fos gradually declined to its low basal levels. Drug treatment did not have any discernible effects on the steady state levels of either N-ras or /S-actin. MATERIALS AND METHODS Cell Culture, Drug Treatment and Survival Assays. The cell line Colo320HSR was purchased from American Type Culture Collection, and cultured in RPMI 1630 (Hazelton-Dutchland) supplemented with 15% heat inactivated bovine calf serum (HyClone), glutamine, penicil lin, and streptomycin at 37°C,95% air/5% CO2. The cells have a doubling time of approximately 24 h and were passaged twice a week. For survival assays and gene expression experiments, exponentially growing cells at 5x 10s cells/ml were exposed to the respective alkylating agents for I h at 37°C,and then washed free of drug. For survival assays, cells were seeded into soft agar culture tubes as de scribed by Chu and Fisher (17). Colony forming efficiency of untreated controls was typically 75%. For measurements of drug effects on gene expression, following drug exposure cells were washed, resuspended in fresh media, and grown at 37°Cbefore cell lysisat designated intervals. Alkylating Agents. HN2 and L-PAM were obtained from the Drug Development Branch, National Cancer Institute. Both compounds were dissolved in sterile filtered 0.1 N HC1 and were maintained as a frozen stock solution. 4-HC was the generous gift of Dr. Michael Colvin of The Johns Hopkins School of Medicine. 4-HC was dissolved in sterile RPMI 1630 immediately prior to use. 62 on May 27, 2015. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from