Changes in C-Myc and C-Fos Expression in a Human Tumor Cell Line Following Exposure to Bifunctional Alkylating Agents1

Home , Myc

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

RNA Isolation. Total cellular RNA was isolated essentially by the procedure of Chirgwin et al. (18). Briefly, cells were washed with ice- cold phosphate buffered saline, and then lysed with a solution composed of 4 M guanidium isothiocyanate (Bethesda Research Laboratories), 0.02 M sodium acetate, 0.01 M dithiothreitol, and 0.5% Sarkosyl (Sigma). Chromosomal DNA was sheared by repeated passage through a 20-gauge needle. RNA was recovered by sedimentation through a 5.7 M CsCl, 0.1 HIM EDTA cushion in an SW 40Ti rotor centrifuged at 35,000 rpm for 16 h. RNA was resuspended in diethyl pyrocarbonate treated deionized water and quantitated by absorbance measurements at 260 nm. Northern Blot Analysis. RNA from control and drug treated cells was denatured by treatment with 50% dimethyl sulfoxide, 1 M glyoxal (Sigma), 0.01 M NaPO4, pH 6.8, at 50Â°Cfor 1 h. Identical amounts of denatured RNA were size fractionated through 1% agarose gels in a recirculated running buffer containing 0.01 M NaPO4, pH 6.8. RNA was transferred to GeneScreenPlus membranes (New England Nuclear) by capillary blotting with lOx standard saline citrate ( 1.5 M NaCl, 0.15 M trisodium citrate) for 18-24 h. The glyoxal reaction was reversed and the membrane was neutralized by sequentially treating the mem brane with 50 m\i NaOH (15s) and 0.2 M Tris (pH 7.5), 2x standard saline citrate (35 s). Prehybridization of the membrane was carried out in 50% formam- ide, 10% dextran sulfate, 5x SSPE 1% SDS, Ix Denhardt's solution (0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% bovine serum Fig. 1. Colony formation ability of Colo320 HSR cells exposed to varying albumin (Pentax Fraction V), and 250 jig/ml denatured salmon sperm concentrations of HN2, L-PAM. and 4-HC for 1 h at 37'C. Cells were washed DNA at 42Â°Cfor 6-24 h in a shaking water bath. Hybridization was free of drug, seeded into agar tubes, and colonies were scored 14 days later. Points, carried out in the same prehybridization solution, at 42Â°Cfor 18-24 h, means of three independent experiments; bars, SD. to which IO6 cpm/ml of denatured "P-labeled DNA probe had been added. Following hybridization, the membrane was first washed in a large volume of 2x SSPE, 0.5% SDS at room temperature. The membrane $ t M was then washed in a large volume of 0.1 x SSPE, 0.5% SDS at 60Â°C (except for c-fos blots, in which case the final wash was 0.2x SSPE, 0.5% SDS at 60Â°C). L-PAM DNA Probes. DNA probes were made by uniformly labeling specific 12.5 pM 25 pM gene fragments with |Â«-"P]dCTP (NEN; specific activity 3000 Ci/ â€”¿2.7kb mmol), utilizing the random primer protocol of Feinberg and Vogel- stein (19). Unincorporated nucleotide was removed by Sephadex G-50 filtration and specific activity was determined by trichloroacetic acid r precipitation. The specific activity of labeled fragments was typically 0.7-1.0 x IO1*cpm//jg. Gene fragments used in hybridization studies were as follows: the 420-base pair Psll exon 2 fragment of human c- 4-HC myc, the 1-kilobase Pst\ fragment of \-fos, a 350-base pair HindlU 40 pM 80 pM fragment containing exon 1 of human N-ras, and the 770-base pair fragment of a chicken cDNA encoding ÃŸ-actin(20-23).

RESULTS In this study we have examined the effects of three clinically HN2 useful antitumor alkylating agents on steady state levels of 2.1 pM 6.3 pM oncogene mRNA. 4-HC was used instead of cyclophosphamide because of the need of cyclophosphamide for microsomal acti * _ 2.7 kb vation. 4-HC spontaneously decomposes in vitro to 4-hydrox- ycyclophosphamide, the same intermediate resulting from the microsomal activation of cyclophosphamide (11). To determine drug exposures to be used for the measurement of effects on Fig. 2. Northern blot analysis of the effect of alkyhiting agents on the expres gene expression, colony formation assays were used (Fig. 1). sion of the 2.7-kilobase (kb) c-myc mRNA. Total cellular RNA was isolated at specified times after drug treatment. RNA samples (20 >

c-fos are shown in Fig. 3. Like c-myc, c-fos expression was 0-actin expression are shown. N-ras, like c-fos, is expressed at elevated immediately following drug removal. In this case, levels very low levels in untreated Colo320HSR. The three alkylating of c-fos transcripts were seen to increase 3- and 4-fold, for 1 agents used in this study appeared to have no discernible effect and 3 log cell kill exposures, respectively. At 6 to 24 h after on N-roi expression over the 24-h time course. Similarly, these drug exposure, c-fos levels returned to, or fell below, the very low basal levels observed in control cultures. Fig. 4 shows Colo320HSR c-myc mRNA levels over a 24-h DC CC time course, after a mock drug treatment. Essentially no alter DC II ations in steady state c-myc mRNA levels are detected, and the I small differences (<20%) between the time points is probably due to experimental variability. Similarly, no differences in c- L-PAM fos levels could be detected (data not shown). 12.5 pM In Figs. 5 and 6, the effects of drug treatment on N-ra5 and

Ã¯ Ã¯ Ã¯ Ã• S 4-HC L-PAM 40 uM 12.5JJM 25 pM â€”¿2.2 Mb

HN2 2.1 pM 6.3 pM 4-HC 40 pM 80 pM ~-4.6 kb

2.2 kb

Fig. 5. Northern blot analysis of the effects of alkylating agents on the expression of the 4.6-kilobase (kb) N-ras mRNA. Conditions were the same as in Fig. 2. except that 40-^g RNA samples were used, and exposures of autoradiographs were 4 to S days with intensifying screens.

HN2 2.1 pM 6.

2.2 kb

L-PAM 12.5 pM

Fig. 3. Northern blot analysis of the effects of alkylating agents on the expression of the 2.2-kilobase (kb) c-fos mRNA. Conditions were the same as in Fig. 2, except that 40-ng RNA samples were used and exposures of the autoradiographs were 3 to 4 days with intensifying screens.

4-HC 40 pM cc I E Ã¯ Ã¯

8 <0 T- OJ

HN2 6.3 pM 2.1 pM 28s â€”¿2.0kb

â€”¿18s

Fig. 6. Northern blot analysis of the effects of alkylating agents on the Fig. 4. Northern blot analysis of c-myc mRNA levels over a 24-h time course, expression of the 2.0-kilobase (kb) ti-actin mRNA. Conditions were exactly the following a mock drug treatment of Colo320HSR. Conditions are exactly as in same as in Fig. 2. Exposures of the autoradiographs were less than 12 h with Fig. 2. intensifying screens. 64

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. ONCOGENE EXPRESSION AFTER ALKVLATING AGENT EXPOSURE compounds had little or no effect on the expression of the closely linked to suppression of c-myc transcripts (42, 43). constitutively expressed "housekeeping gene" ÃŸ-actin. These data imply that reduction of c-myc expression may reflect the loss of cellular proliferation, or in the case of nitrogen mustards, this may be a precursor to cell killing. The cause of DISCUSSION the alkylating agent induced suppression of c-myc transcripts A number of studies have examined the effects of various is yet unanswered; it may be due to a specific cellular response biological and chemical agents on gene expression, but very few to the drug challenge, as appears to be the case for the com have focused on the effects of cytotoxic, DNA damaging anti- pounds mentioned above. Alternatively, suppression of tran tumor agents. In this report we demonstrate that three struc script levels may be due to the accumulation of DNA damage turally related alkylating agents produce strikingly similar re within the c-myc oncogene. Interestingly, the reduced levels of sponses in each of the genes examined. c-myc transcripts observed at 6 and 12 h following drug removal The expression of the c-fos gene can be induced rapidly and are coincident with the time of peak DNA interstrand cross transiently in a variety of normal cell types by a diverse group links produced by these agents. of compounds. These compounds include mitogens, such as Over the 24-h time course, little or no effect of alkylating serum, interleukins, and growth factors (e.g., platelet-derived agent treatment could be seen on the expression of N-ras and growth factor, epidermal growth factor), tumor promoters, such 0-actin. This may not be surprising since very few agents as phorbol esters, and neuroactive compounds (24-28). In the examined to date appear to alter expression of these genes (7, present study we demonstrate that c-fos steady state message is 44,45). It is also possible that the nitrogen mustards may exert increased immediately following cytotoxic exposures to bifunc- their effects, either directly or indirectly, on specific genes. tional alkylating agents. Data presented in this report suggest cytotoxic DNA dam Recently a number of reports have been published which aging agents differentially affect a subset of active cellular genes. begin to define the functional role that the c-fos protein plays Furthermore, these alterations may reflect efficient drug in in the cell. It appears the c-fos protein interacts with the c-jun/ duced cell kills. Future studies will be aimed at determining the AP-1 protein(s), which in turn acts to stimulate or repress direct cause of the initial elevation in c-myc transcripts, as well transcription of genes under the influence of the AP-1 inducible as the subsequent depression of c-myc. In addition it may be enhancer (29-33). Furthermore, it has also been reported that worthwhile to determine if other early growth response genes genes inducible by the DNA damaging agents mitomycin C and (besides c-myc and c-fos), such as EGR2 (46, 47), or other AP- UV light are also AP-1 inducible genes (34-36). From these 1 responsive genes, such as collagenase or metallothionein (34- observations it is possible to speculate that the bifunctional 36), are also induced by the bifunctional alkylating agents. alkylating agents may be initiating a cellular response to DNA Finally, it will be of interest to determine if different types of damage mediated through the induction of c-fos. clinically useful, DNA cross-linking agents (e.g., nitrosoureas Along with the immediate increase in c-fos expression, c-myc and platinum compounds) elicit responses similar to that of the expression is also elevated. The rapid and transient increases nitrogen mustards. in c-fos and c-myc expression are reminiscent of the mitogenic response seen in cells stimulated by a variety of mitogens (37, REFERENCES 38). In fact, it appears that both c-fos and c-myc levels are increased within 15 min following the addition of the bifunc 1. Battey. J., Moulding. C, Taub, R., Murphy, W., Stewart. T., Potter, H., tional alkylating agent (data not shown). Although it is pres Lenoir. G., and Leder, P. The human c-myc oncogene: structural conse quences of translocation into the IgH locus in Burkitt lymphoma. Cell. 34: ently unknown if the increases in c-myc and c-fos steady state 779-787, 1983. messages are due to increased transcription; if they are, then 2. Capon, D., Chen, E., Levinson, A., Seeburg, P., and Goeddel, D. Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its this increase may be due to drug effects on the intracellular normal homologue. Nature (Lond.), 302: 33-37. 1983. mediators of the proliferative response. Furthermore, if the 3. Dalla Pavera, R., Wong-Staal, F., and Gallo. R. Oncogene amplification in immediate increases in c-myc and c-fos do reflect an "inappro promyelocitic leukemia cell line HL-60 and primary leukemic cells of the priate" proliferative response, then it would be interesting to same patient. Nature (Lond.), 299: 61-63, 1982. 4. Schwab. M., Ellison, J., Busch, M., Rosenau, W., Varmus, H., and Bishop, speculate that this response may act to "open up" critical areas J. Enhanced expression of the human gene N-myc consequent to amplifica of the genome necessary for growth, and in turn make these tion of DNA may contribute to malignant progression of neuroblastoma. Proc. Nati. Acad. Sci. USA, 81: 4940-4944. 1984. regions more accessible to drug induced DNA damage. Finally, 5. Brodeur. G., Seeger. R., Schwab, M., Varmus, H., and Bishop, J. Amplifi it is also possible that these increased levels are due to a drug cation of N-m>'c in untreated human neuroblastomas correlates with ad induced stabilization of both of these messages, but this type of vanced disease stage. Science (Wash. DC), 224: 1121-1124, 1984. 6. Almoguera. C., Shibata, D., Forrester, K., Martin, J.. Arnheim, N., and effect may have the same biological consequences as increased Perucho, M. Most human carcinomas of the exocrine pancreas contain transcription. mutant c-K-raj genes. Cell, 53: 549-554. 1988. 7. Watt. R.. Wang, A., and Schein, P. Altered c-myc gene expression in Burkitt Each of the alkylating agents tested was able to profoundly lymphoma cells following nitrogen mustard treatment. Proc. Am. Assoc. suppress the level of c-myc transcripts at 6 and 12 h following Cancer Res., 27: 232, 1986. 8. Venturelli, D., Lange, B., Narni. F.. Selleri, L.. Torelli, U.. and Calabreta, drug removal. It is of interest to note that these are the time B. Prognostic significance of "short term" effects of chemotherapy on MYC points for maximum DNA damage produced by these agents. and histone H3 mRNA levels in acute leukemia patients. Proc. Nati. Acad. Suppression of c-myc transcripts by a number of compounds Sci. USA, Â«5:3590-3594, 1988. has been previously been reported. Specifically, differentiating 9. Quinn, L.. Moore, G.. Morgan. R., and Woods, L.: Cell lines from a human colon carcinoma with unusual products, double minutes, and homogeneously agents such as dibutyryl cyclic 3':5'-AMP, dimethyl sulfoxide, staining regions. Cancer Res., 39: 4914-4924. 1979. and 1,25-dihydroxyvitamin D, reduce c-myc expression in he- 10. Alitalo, K., Schwab, M., Lin, C-, Varmus, K., and Bishop. J. Homogeneously staining chromosomal regions contain amplified copies of an abundantly matological tumor cell lines prior to terminal differentiation expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from (39-41). Also, rt-interferon can cause growth arrest in the a human colon carcinoma. Proc. Nati. Acad. Sci. USA, 80: 1707-1711, 1983. Burkitt lymphoma cell line Daudi, and -y-interferon alone, and 11. Jason, M., Andrews. B., Colvin. M., and Friou, G. In vitro effects of 4- hydroperoxycyclophosphamide on the morphology and function of human in cooperation with tumor necrosis factor, can cause growth peripheral blood mononuclear phagocytic cells (macrophages). Cancer Res., arrest in HeLa cells. In both cases, the growth arrest has been 44:3936-3941. 1984. 65

12. Johnson. J.. and Ruddon. R. Interaction of nitrogen mustard with polyribo- binding affinities. Cell. 55: 917-924. 1988. nucleotides, ribosomes, and enzymes involved in protein synthesis in a cell- 32. Rauscher, F., Cohen, D., Curran, T., Bos, T.. Vogt, P., Bohmann, D., Tjian, free system. Mol. Pharmacol. 3: 195-203. 1967. R., and Pranza. R./os-associated protein p39 is the product of thcy'un proto 13. Menta. J., Przybylski, M., and Ludlum. D. Alkylation of guanosine and oncogene. Science (Wash. DC). 240: 1010-1016. 1988. deoxyguanosine by phosphoramide mustard. Cancer Res., 40: 4183-4186. 33. Sassone-Corsi. P.. Lamph. W.. Kamps. M., and Verma, I. /as-associated 1980. cellular p39 is related to nuclear transcription factor AP-1. Cell. 54: 553- 14. Lawley, P.. and Brookes. P. Further studies of nucleic acids and their 560. 1988. constituent nucleotides. Biochem. J.. 89: 127-144. 1963. 34. Angel. P.. Poting. A.. Mallick. U.. Rahmsdorf. H., Schorpp. M.. and Herrl 15. Erickson. L., and Zlotogorski. C. Induction and repair of macromolecular ich, P. Induction of metallothionen and other mRNA species by carcinogens damage by alkylating agents. Cancer Treat. Rev.. // (Supp. A): 25-35, 1984. and tumor promoters in primary human skin fibroblasts. Mol. Cell. Biol. 6: 16. Zwelling. L., Michaels, S.. Schwartz. H.. Dobson. P.. and Kohn. K. DNA 1760-1766. 1986. crosslinking as an indicator of sensitivity and resistance of mouse L1210 35. Angel. P., Baumann. I.. Stein. B.. Dclius. H.. Rahmsdorf, H., and Herrlich. leukemia to r/.s-diamminedichloroplatinum(ll) and t.-phenyl-alanine mus P. 12-O-Tetra-phorbol-13-acetate induction of the human collagenasc gene tard. Cancer Res.. 40: 640-649, 1980. is mediated by an inducible enhancer element located in the 5'-flanking 17. Chu, M., and Fisher, G. Incorporation of cytosine-'H arabinoside and its region. Mol. Cell. Biol. 7:2256-2266. 1987. effects on the murine leukemia cells (L5178Y). Biochem. Pharmacol., 17: 36. Angel, P.. Imagawa. M., ChiÃ¹. R.. Stein. B.. Imbra. R., Rahmsdorf. H.. 753-768. 1968. Jonat. C., and Karin. M. Phorbol ester-inducible genes contain a common c/'.velement recognized by a TPA-modulated fran.c-acting factor. Cell, 49: 18. Chirgwin. J.. Przybyla. A., MacDonald. R.. and Rutter. W. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. 729-739, 1987. Biochemistry, 18: 5294-5300, 1979. 37. Armelin. H.. Armelin. M.. Kelly, K.. Stewart. T.. Leder, P., Cochran. B., and 19. Feinbcrg. A., and Vogelstein. B. A technique for radiolabelling DNA restric Stiles. C. Functional role for c-myc in mitogenic response to platelet-derived tion endonuclease fragments to high specific activity. Anal. Biochem.. 137: growth factor. Nature (Lond.). 310: 655-660. 1984. 266-267, 1984. 38. Muller. R., Bravo. R.. Burkhardt. J.. and Curran, T. Induction of c-fos gene 20. Colby. W., Chen, E., Smith. D.. and Levinson. A. Identification and nucleo- 312:and protein 716-720". by 1984.growth factors precedes activation of c-myc. Nature (Lond.). tidc sequence of a human locus homologous to the \-myc oncogene of avian myelocytomatosis virus MC29. Nature (Lond.), 301: 722-725, 1983. 39. Siebenblast, U.. Bressler. P., and Kelly. K. Two distinct mechanisms of 21. Curran.T.. Peters, G.. Van Beveren.C..Teich, N..and Vcrma, I. FBJ murine transcriptional control operate on c-mvc during differentiation of HL60 cells. osteosarcoma virus: identification and molecular cloning of biologically active Mol. Cell. Biol.. 8: 867-874. 1988. proviral DNA. J. Virol., 44: 674-682, 1982. 40. Trepel. J.. Colamonici. O.. Kelly, K.. Schwab, G., Watt, R.. Sausville. E.. 22. Taparowsky. E.. Shimizu. K.. Goldfarb. M.. and Wigler. M. Structure and Jaffe. E.. and Neckers L. Transcriptional inactivation of c-ntyc and the activation of the human N-ras gene. Cell, 34: 581-586. 1983. transferrin receptor in dibutyril cyclic-AMP-trcated HL60 cells. Mol. Cell. 23. Cleveland. D.. Lopata. M., MacDonald. R., Cowan. N.. Rutter. W., and Biol., 7:2644-2648. 1987. Kirschner. M. Number and evolutionary conservation of alpha and beta 41. Reitsma, P.. Rothberg, P.. Astrin. S., Trial. J.. Bar-Shavit. Z., Hall, A., tubulin and cytoplasmic beta and gamma uctin genes using specific cloned Teitelbaum, S.. and Kahn. A. Regulation of myc gene expression in HL-60 cDNA probes. Cell, 20: 95-105, 1980. leukemic cells by a vitamin D metabolite. Nature (Lond.), 322: 848-850, 24. Greenberg, M., and Ziff, E. Stimulation of 3T3 cells induces transcriptions 1983. of the c-fos proto-oncogene. Nature (Lond.). 311: 433-438. 1984. 42. Einat, M., Resnitzky. D., and Kimichi, A. Close link between reduction of c- 25. Kovacs, E., Oppenheim, J.. and Young, H. Induction of c-fos and c-myc myc expression by Interferon and G0/Gi arrest. Nature (Lond.). 313: 597- expression in T-lymphocytes after treatment with recombinant interleukin-1 600. 1985. alpha. J. Immunol.. 137: 3649-3651. 1986. 43. Y'arden. A., and Kimichi. A. Tumor necrosis factor reduces c-myc expression 26. Kruiger, W., Cooper, J., Hunter. T., and Verma, I. Platelet-derived growth and cooperates with interferon-gamma in HeLa cells. Science (Wash. DC). factor induces rapid but transient expression of the c-fos oncogene and 234: 1419-1421. 1986. protein. Nature (Lond.). 312: 711-716. 1984. 44. Watanabe. T.. Sariban. E.. Mitchell, T.. and Kufe. D. Human c-myc and N- 27. Greenberg, M.. Ziff, E., and Greene, L. Stimulation of neuronal acetylcholine ras expression during induction of HL-60 cellular differentiation. Biochem. receptors induces rapid gene transcription. Science (Wash. DC). 234: 80-83. Biophys. Res. Commun.. 126: 999-1005. 1985. 1986. 45. Mitchell, T.. Sariban. E., and Kufe. D. Effects of 1-beta-D-arabinofuranosyl- 28. Morgan, J., Cohen, D., Hempstead. J.. and Curran. T. Mapping patterns of cytosine on proto-oncogene expression in human U-937 cells. Mol. Phar c-fos expression in the central nervous system after seizure. Science (Wash. macol., JO: 398-402. 1986. DC), 237: 192-197, 1987. 46. Joseph, L., LeBeau, M., Jamieson. G., Acharya, S.. Shows. T.. Rowley, J.. 29. ChiÃ¹,R., Boyle. W., Meek. J., Smeal. T., Hunter. T., and Karin, M. The c- and Sukhatme, V. Molecular cloning, sequencing, and mapping of EGR2, a fox protein interacts with c-y'un/AP-l to stimulate transcription of AP-I human early growth response gene encoding a protein with "zinc-binding responsive genes. Cell. 54: 541-552. 1988. finger" structure. Proc. Nati. Acad. Sci. USA, 85: 7164-7168, 1988. 30. Distel, R., Ro, H.. Rosen. B., Groves. D.. and Spiegelman, B. Nucleoprotein 47. Sukhatme, V., Cao, X., Chang, L., Tsai-Morris, C., Stamenkovich, D., complexes that regulate gene expression in adipocyte differentiation: direct Ferreira, P., Cohen, D.. Edwards, S., Shows, T.. Curran. T.. LeBeau. M., participation of c-fos. Cell, 49: 835-844, 1987. and Adamson. E. A zinc finger-encoding gene coregulated with c-fos during 31. Halazonetis. T., Geogopoulos. K., Greenberg, M., and Leder. P. c-jun di- growth and differentiation, and after cellular depolarization. Cell. 53: 37-43, merizes with itself and with c-fos, forming complexes of different DNA 1988.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. Changes in c-myc and c-fos Expression in a Human Tumor Cell Line following Exposure to Bifunctional Alkylating Agents

Bernard W. Futscher and Leonard C. Erickson

Cancer Res 1990;50:62-66.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/50/1/62

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/50/1/62. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.