MOLECULAR CARCINOGENESIS 4:203-209 (1991)

DNA Methylation and Oncogene Expression in Methapyrilene-Induced Rat Liver Tumors and in Treated Hepatocytes in Culture Lidia Hernandez, Christos J. Petropoulos, Stephen H. Hughes, and William Lijinsky' Laboratory of Chemical and Physical Carcinogenesis (LH, WL) and Molecular Mechanisms of Carcinogenesis Laboratory (CJe SHH), ABL-Basic Research Program, National Cancer lnstitute-Frederick Cancer Research and Development Center, Frederick, Maryland

Continued exposure of rats t o carcinogenic doses of methapyrilene (MP) leads t o elevated levels of S-methyl- deoxycytidine (SMC) in liver DNA. Since gene expression often correlates with DNA methylation, we investigated these parameters in the MPinduced hepatocellular carcinomas of Fischer 344 rats. DNA was hypermethylated in liver tissue surrounding the tumors relative t o liver tissue of untreated controls of the same age, while tumor DNA was not; DNA methylation declined to normal levels when MP treatment ceased. Gene expression analysis showed measurable levels of mRNA for c-Ki-ras, erbB, erb-B2, hck, src, Iyn, vav, trk, raf-1, I-myc, c-jun, c-yes, c-myc, c-abl, and p53. No significant differences in expression for these and other oncogenes were seen between tumors and surrounding livers, although erbB2 and wav showed visible decreases compared with normal liver. Hypermethylation of DNA and expression of these oncogenes in MP-treated tissues were not correlated. Levels of mRNA for the same genes in MP-treated hepatocytes in culture were similar t o in vivo levels; analysis o f DNA synthesis levels showed that this gene expression pattern occurred in the absence of proliferation bursts or toxicity in these cells, thus suggesting that treatment in vivo may produce the same results.

Key words: Gene expression, carcinogenesis, alkylation

INTRODUCTION The antihistaminic methapyrilene (MP) hydrochloride was

until 1980 a component of many over-the-counter sleep-aids and allergy medications. Its potent hepatocarcinogenicity in rats, however, prompted the removal of this compound from the market [I] and marked the beginning of studies of its mechanism of tumor induction. MP was examined in a variety of genotoxicity assays [2] and appeared to be nongenotoxic. Further studies showed that MP does not bind significantly to liver DNA [3-51, which provoked inter- est in understanding mechanisms of liver carcinogenesis by this compound.

Our laboratory has reported some biological effects of MP treatment that correlate with its carcinogenicity, such as mitochondria1 proliferation [6,7] and lamellar body appearance 181 in hepatocytes. More recently [9], we have reported an increase in liver DNA methylation levels after MP treatment in vivo, which results from high ratios of S-adenosylmethionine (SAM) to S-adenosylhomocysteine in liver cells.

There is sizeable evidence from studies in many systems that changes in methylation of DNA occur during carci- nogenesis [reviewed in 101. Many human tumors and tumor-derived cell lines show decreased 5-methyldeoxy- cytidine(5MC)levelsin DNA[11-141, whileinsomecases, increased DNA methylation has been correlated with tumor development [ I 5,161 or oncogene expression in target tissues.

It is possible that chemical carcinogens may change the methylation status of certain genes, altering their expres- sion and giving rise to tumors. This could represent an indi- rect way by which MP alters DNA expression and has been suggested as a possible mechanism of liver tumor. induc- tion [ I 7-1 91, especially by nongenotoxic carcinogens.

The purpose of our study was to investigate the corre- lation between MP-induced methylation changes in rat liver and the expression levels of several oncogenes, some of which have been implicated in the development of tumors [201. We measured total 5MC levels in genomic DNA and the amount of mRNA for a number of oncogenes in MP-induced hepatocellular carcinomas, in tissue surround- ing the tumors, and in healthy livers of age-appropriate control rats. In parallel with the analyses of rat liver mRNA, we studied the levels of oncogene expression in MP-treated hepatocytes in culture, since such cells represent a spe- cific cell type present in the tumors. Further, since it is

'Corresponding author: National Cancer Institute-Frederick Can- cer Research and Development Center, Frederick, MD 21 702.

Abbreviations: ME methapyrilene; SMC, 5-methyldeoxycytidine; HPLC, high-pressure liquid chromatography; EDTA, ethylenedia- minetetraacetic acid; SSC, standard saline citrate; SAM, S-adeno- sylrnethionine.

By acceptance of this article, the publisher or recipient acknowl- edges the right of the US. Government and its agents and contrac- tors to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

PUBLISHED 1991 WILEY-LISS, INC.

204 HERNANDEZ ETAL.

known that expression of some cellular oncogenes can change during cell proliferation or during liver regenera- tion [21], we measured rates of DNA synthesis in cultured hepatocytes during MP treatment to assess any gene- expression changes that derived from changes in the pro- liferative state of the cells.

MATERIALS AND METHODS An i ma I Treatments

Fischer 344 male rats (7 wk old), obtained from the Fred- erick Cancer Research and Development Center (FCRDC) animal facility, received MP hydrochloride in drinking water (1 g per liter) for 65 wk at a regulated dose of 20 mg/d, 5 d per week, as previously described 1221. Tumors appeared several weeks later. To distinguish beween tumor-associated effects and effects of treatment that might be unrelated to tumor formation, a second group of rats received con- tinuous treatment until death. Control animals weregiven deionized water.

All treated animals developed hepatocellular carcinomas, which were confirmed by histopathological examination by Dr. R.M. Kovatch at our institution.

Each animal wasscheduled for sacrifice when it showed palpable liver tumors or became moribund. Treated ani- mals were killed between 99 and 130 wk of age; untreated animals were killed a t 84 wk. All animals were killed by COz asphyxiation and their livers were removed; liver tumors and surrounding tissue of normal appearance were removed independently. Tissue portions were set aside for histopathology, and tissues were immediately frozen in liq- uid nitrogen and stored at - 80°C.

Purification of DNA and Determination of 5MC Nuclear DNA from hepatocellular carcinomas, surround-

ing tissue, and control livers was extracted and purified as described. Similarly, 5MC was determined in DNA sam- ples by reverse-phase high-pressure liquid chromatogra- phy (HPLC) as described 1231.

Purification of RNA and Determination of mRNA

Tissue portions (3 g frozen liver) were homogenized in RNAzol (CinnaBiotecx, Friendswood, TX) according to the manufacturer's specifications. When cultured cells were used, medium was removed and RNAzol was added directly to the culture plates. The contents of five plates were pooled and extracted together.

Homogenates or cell lysates were shaken vigorously with one-tenth volume of chloroform (J.T. Baker, Phillipsburg, NJ) and subjected to centrifugation for 15 min at 12 000 x g; total RNA was then precipitated with an equal volume of isopropanol for 1 h at - 20°C. The precipitate was pel- leted at 12 000 x gfor 25 min and washed with 70% eth- anol. RNA from tissues was resuspended in one half the homqenate volume of 0.5% SDS, 1 mM ethylenediamine- tetraaceticacid (EDTA), 0.2 M sodium acetate, pH 5.2, and reextracted with an equal volume of buffered phenol/ chloroform followed by reprecipitation with isopropanol. Total RNA was collected by centrifugation at 12 000 x g for 30 min and washed with 70% ethanol.

Table 1. Plasmid DNAs Used in Hybridization Experiments

Gene Plasmid Reference c-Ki-ras-2 c-erbA erb-B

(epidermal growth factor receptor)

(neu-related oncogene)

c-erb- B 2

hck- 1 src fms v-sis v-fos c- fpdfes MJ ret

trk c-raf-1

va v

I-rnyc c-jun c-yes v-mos c-myc

c-Ha-rds-1 c-abl P53 pBR322

Album in a-Fetoprotein Actin

psw11-1 PHE-A1

pHER-A64-1

pMAC117

pHK24 pN1.8

341 -P804 pv-SIS pfos- 1 P80

p2YP2.2 ret2.2 psk 8

pKSmc-raf pDM-17

- pHJ19 pXEyes

MlOC2 PMP

pT24-C3 hab

27.la -

pNKB-26 pRAF87 p-2000

[381

I391 [401

I421 [431 I441 I451 (461 (471 I481

[411

G. Heidecker et al., manuscript in preparation

I491 [Sol (511 I521

C.J. Petropoulos, manuscript in preparation

I531 1541 [551

Bethesda Research Laboratories, Gaithersburg, MD

[561 I571 I581

c-ski KS29 (591

To analyze mRNA levels in the tumors and cell samples for a large number of oncogenes, we used a "reverse-blot" approach. Plasmids (described in Table 1) were denatured at 95°C for 5 min and mixed with 2 vol of 1 M NaOH, 1 mM EDTA. Cloned gene sequences of interest were a gen- erous gift from Drs. Nancy Jenkins, Narayan Bhat, and Pramod Sutrave (FCRDC). Two micrograms of each plas- mid were then slot blotted (Schleicher & Schuell, Inc., Keene, NH) onto Biotrace RP nylon membranes (Gelman Sciences Inc., Ann Arbor, MI). Membranes were then air dried and stored at room temperature until used.

To generate probes for the blots, the total RNA prepara- tions described above were enriched for poly(A)+ RNA species using oligo-dT spin columns (Pharmacia LKB Biotechnology, Piscataway, NJ). Enriched poly(A) + RNA was shown by northern transfer to be intact [23). Single- stranded 32P-labeled cDNAs were generated from the RNA using a commercially available cDNA synthesis kit (Boeh- ringer Mannheim Corp., Indianapolis, IN). Using random

ONCOG EN E EXPR ESSlON 8 Y METHAPYRIL EN E 205

primers and high concentrations (3 mM) of dATP, dGTP, d l l t and high specific-activity [32PldCTP (3000 Ci/mmol, Amersham Corp., Arlington Heights, IL), cDNAs with spe- cificactivitiesof approximately 2 x 1 07cpm/pg were synthe- sized from 1 pg poly(A) + RNA samples. Each cDNA probe containing approximately 1 x 10' cpm was added to a prehybridized membrane and hybridized for 24 h (42"C, 50% formamide). Membranes were then washed for 1 h at room temperature in 2 x standard saline citrate (SSC), O.l%SDS,folIowedby2hinO.5~ SSC,O.l%SDSat55"C. The washed filters were exposed to Kodak XAR-5 film.

Experiments Using Cultured Hepatocytes Experiments were performed with CL9 cells-adult male

Fischer 344 rat hepatocytes (American Tumor Type Collec- tion, Rockville, MDk-between passages 21 and 24. Cells were grown in Kaighn's Ham's F-1 2K medium (Irvine Sci- entific, Santa Ana, CAI containing 10% fetal bovine serum (GIBCO, Grand Island, NY) in a 5% COz atmosphere at 37°C. Generally, 3 x lo6 cells were seeded onto each 1 00-mm dish or six-well plate (Costar, Cambridge, MA) and allowed to grow for 24 h. Cells were treated with fresh medium containing 25 pg/mL MP; control cells received fresh medium only. Cells were reincubated and RNA extracted after 3, 12, or 24 h of treatment.

For toxicity assays, cells from duplicate wells in six-well plates were exposed to various concentrations of MP (see Results) in fresh medium for the same periods of time as the cultures described above. During the last 3 h of each exposure period, i3H]thymidine (5 Ci/mmol; Amersham) was added to each well to a final concentration of 5 pCi/ mL. Radioactivity incorporated into DNA was determined by comparing trichloroacetic acid-precipitated radioactiv- ity with the total amount of protein in the culture, as pre- viously described (241.

RESULTS Deoxycytidine Methylation

Continued MP treatment of rats produces a significant increase in the methylation of DNA after 20 wk, long before tumors appear 191. In the present study, we found that ani- mals that continued to receive the compound in drinking water developed tumors after about 100 wk. As shown in Table 2, these hepatocellular carcinomas had 5MC levels in DNA comparable to those in untreated animals, but the surrounding liver DNA was hypermethylated by 5% ( P <

0.01 1. In contrast, when treatment was discontinued at 65 wk, the resulting hepatocellular carcinomas also had 5MC levels statistically comparable to those in controls, but the surrounding liver DNA was hypomethylated by 25% (P< 0.01).

Oncogene Expression In Vivo Figure 1 shows the typical pattern of oncogene expres-

sion in the livers of untreated rats (Figure 1A) and in tumors (Figure 1 B) and surrounding livers (Figure 1 C) of rats that received MP in drinking water until death. There were measurable levels of c-Ki-ras, erb-B, erbB2, hck, src, /yn, vav(onc-f), trk, raf-1, I-myc, c-jun, c-yes, c-myc, c-abl, and p53 RNAs in all animals tested. The only changes that accompanied MP treatment were a large decrease in the level of erb-82 mRNA and a small decrease in the levels of VJV mRNA. Liver surrounding the tumors showed the same changes. Levels of albumin, a-fetoprotein, actin, and c-ski mRNAS varied only slightly between animals and provided the basis for interpreting changes in onco- gene mRNA levels.

Typical mRNA levels for rats that had MP treatment dis- continued after 65 wk are shown in Figures 1 D and 1 E; similar changes in the expression of erb-B2 and VJV were observed with treatment, and again, there were no dif- ferences between the tumors and surrounding tissues.

The decreases in erb-B2 and vavmRNA levels were con- sistent in all animals and correlated well with MP treat- ment, but were not associated with any of the genomic DNA methylation changes that accompanied treatment.

Oncogene Expression In Vitro As Figure 2 shows, MP treatment of a homogeneous

population of hepatocytes had effects similar to those seen in vivo. After 3 h, the levels of mRNA for oncogenes were not affected. The same pattern was seen in cultures treated for longer periods of time (1 2 h and 24 h; data not shown). These results represent cultures that were not undergo- ing proliferative bursts nor suffering toxic effects of treat- ment, as judged by the levels of DNA synthesis (Figure 3). During treatment with 25 pg/mL of MP, DNA synthesis in CL9 cells remained comparable to that in controls. This dose has been shown 181 to be nontoxic to other normal rat hepatocyte lines in culture, but is capable of inducing most early morphological changes that have been associ- ated with carcinogenesis by this compound.

Table 2. Effect of Methapyrilene on 5MC Levels in Rat Liver DNA*

Continued treatment Discontinued treatment Surrounding Surrounding

Control Tumor liver Tumor liver

3.11 f 0.06 3.14 f 0.01 3.26 k 0.01 t 3.04 2 0.14 2.34 ? 0.26t 'Numbers are average percent of 3-4 rats, calculated at 5MChotal dC ? SE. Control, untreated; surrounding liver, liver tissue of normal appear- ance in vicinity of tumor; tumor, tumor tissue free of normal surrounding liver; continued treatment, MP present in drinking water until death at 99, 100, 104, and 108 wk; discontinued treatment, MP withdrawn from drinking water after 65 wk of treatment and animals killed at 112, 121, and 130 wk. t P < 0.01.

206 HERNANDEZ ETAL.

Figure 1.

ONCOGENE EXPRESSION BY METHAPYRILENE 207

A B

albumin

a-feioproiein

aciin

c-ski

Figure 2. Effect of MP on gene ex ression in vitro. Typical mRNA levels in CL9 cells (A) untreateiand (6) after 3 h treat-

DISCUSSION

Our previous work has shown that preneoplastic livers of rats treated chronically with a carcinogenic dose of MP contain hypermethylated DNA (91. The present study was designed to investigate whether in late stages of carcino- genesis this effect was still present and whether it corre- lated with changes in oncogene expression that might be important in tumor formation.

We not only found that continued MP treatment induced hepatic tumors with normal DNA levels of 5MC but also that nontumorous liver tissue was hypermethylated. This increase was comparable in magnitude to the increase found previously in preneoplastic liver (about 4%) and to the reported decreases in DNA methylation produced by methyl-deficient diets and other carcinogenic treatments that result in liver tumors in rats [18,25].

Discontinuing MP treatment at 65 wk produced a sharp hypomethylation of DNA in liver surrounding the tumors but not of tumor DNA. This suggests that DNA hypermeth- ylation is a direct consequence of the presence of the compound. The data also suggests that the specific cell population giving rise to the tumors is resistant to the cellular effects of MP that result in 5MC level changes. The idea that some carcinogens induce tumors that are resistant to the carcinogens's cytotoxic effects has been of great interest [26], and oncogene-induced multidrug resistance has been demonstrated in rat liver in vivo I271 and in vitro [28].

Earlier work has demonstrated that MP induces signifi-

albumin a-feioproiein

aclin

c-ski

ment with 25 pglmL MR Experimental conditions described in Materials and Methods.

cant elevations of SAM in the liver very early in treatment; also, exogenous SAM administration has been shown to regress liver nodules induced by hepatocarcinogenic regi- mens based on initiation/promotion protocols 1291 and to inhibit the accompanying overexpression of some onco- genes. Overexpression of some oncogenes during hepato- carcinogenesis has been widely reported [30,31 I, as have decreases in methylation of these DNA sequences (18.321. However, we found no significant increase in the expres- sion of 23 oncogenes in MP-induced tumors nor in the surrounding liver containing either hyper- or hypomethy- lated DNA. It is important to note that we obtained similar

3 h

0 12 h

0 24 h

0 48 h

0 10 25 50 75 100 150 f 0

Figure 1. Effect of MP on gene expression in vivo. vpical mRNA levels in (A) liver, (B) tumor, and (C) surrounding liver of a rat that received continued MP treatment, and (D) tumor and (E) surrounding liver of a rat that received discontinued MP treat- ment. Experimental conditions described in Materials and Meth- ods. Onc-f is vav.

Figure3. (3H]Thymidine incor oration in DNAof MPtreated cells. Treatment times corresponfto len ths of exposure of cul- tures to MR Points represent averages o!values from duplicate cultures. Experimental conditions described in Materials and Methods.

208 HERNANDEZ ETAL.

results from the analysis of hypermethylated preneoplastic livers after 34 wk of continued MP treatment (data not shown). The levels of erb-B2 RNA were consistently lower in tumors and surrounding liver, an effect that we cannot explain but that has also been observed in hepatocellular carcinomas induced by other agents [331.

The apparent heterogeneity of oncogene transcript lev- els in liver tumors [34] and the transient changes in the above-listed mRNAs observed during liver regeneration [2 11 prompted our analysis of expression levels during MP treat- ment of hepatocytes in culture. We found that transcript levels in these cells were comparable to those following in vivo treatment. Furthermore, while the concentration of MP chosen for the analysis induced neither toxic nor pro- liferative effects in the hepatocytes, it did produce mito- chondrial and lamellar body accumulation in the cytoplasm (data not shown), parameters previously shown to be asso- ciated with the response of rats to this carcinogen [8]. The present experiments suggest that the levels of oncogene mRNA we measured in the tumors and surrounding livers were representative of a homogeneous cell population and that the patterns of expression for those genes were not likely to be the result of toxic treatment nor of liver regeneration.

Therefore, the hypermethylation of liver DNA induced by MP appears to be a consequence of the presence of the compound, though not correlated with the development of tumors; animals whose MP treatment was discontinued early still developed tumors, and their methylation of liver DNA decreased. These methylation changes, however, were not accompanied by consistent changes in the expression of any of a number of oncogenes in the liver. Further work is needed to determine whether MP-induced changes in methylation may relate to carcinogenesis by affecting the expression of other genes not included in this study.

AC K N OW L E DG M EN TS

This research was sponsored by the National Cancer Insti- tute, Department of Health and Human Services, under contract No. NO1 -CO-74101 with Advanced BioScience Laboratories, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US. Government.

Received October 1, 1990; revised December 18, 1990; accepted February 19, 1991.

REFERENCES

1. Lijinsky W, Reuber MD, Blackwell BN. Liver tumors induced in rats by oral administration of the antihistaminic methapyrilene hydro- chloride. Science 209:817-819, 1980.

2. Mirsalis JC. Genotoxicity, toxicity and carcinogenicity of the anti- histamine methap rilene Mutat Res 185:309-317, 1987.

3. Lijinsky W, Yamasha K.'Lack of binding of methapyrilene and similar antihistamines to rat liver DNA examined by 32P-postlabeling. Cancer Res 48:6475-6477. 1988.

4. Casciano DA, Shaddock JG, Talaska G. The potent hepatocar- cinogen methapyrilene does not form DNA adducts in livers of Fischer 344 rats. Mutat Res 208: 129- 135, 1988.

5. Lijinsky W, Muschik GM. Distribution of the liver carcinogen

methapyrilene in Fischer rats and its interaction with macromole- cules. J Cancer Res Clin Oncol 103:69-73. 1982.

6. Reznik-Schuller HM, Lijinsky W. Morphology of early changes in liver carcinogenesis induced by methapyrilene. Arch Toxicol

7. Reznik-Schuller HM, Lijinsky W. Ultrastructural changes in the liver of animals treated with methapyrilene and some analogs. Ecotox Environ Safety 6:328-335, 1982.

8. type PT, Bucana CD, Kelley SP Carcinogenesis by non-mutagenic chemicals: Early response of rat liver cells induced by methapyri- lene. Cancer Res45:2184-2191, 1985.

9. Hernandez L, Allen PT, Poirier LA, Lijinsky W. 5-Adenosylmethi- onine, 5-adenosylhomocysteine and DNA methylation levels in the liver of rats fed methapyrilene and analogs. Carcinogenesis

10. Jones PA, Buckley JD. The role of DNA methylation in cancer. Adv Cancer Res 54: 1-24, 1990.

1 1. Feinberg AF! Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature

12. Riggs AD, Jones PA. 5-Methylcytosine, gene regulation and can- cer. Adv Cancer Res 40: 1-30. 1983.

49:79-83, 1981.

10:557-562, 1989.

301 :89-92, 1983.

13. Feinberg AP, Gehrke CW, Kuo KC, Ehrlich M. Reduced genomic 5-methylcytosine content in human colonic neoplasia. Cancer Res

14. Mass MJ, Schorschinsky NS, Laslev JA, Beeman DK. Austin SJ. 48: 1 159-1 161, 1988.

Consistent oncogene methylation changes in epithelial cells chem- ically transformed in vitro. Biochem Biophys Res Commun 164:693-699, 1989.

15. Baylin SB, Hoppener JWM, Bustros A, Steenbergh PH, Lips CJM, Nelkin BD. DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas. Cancer Res 46:2917- 2922,1986.

16. Tanaka K, Appella E, Jay G. Developmental activation of the H-2K gene is correlated with an increase in DNA methylation. Cell 35:457-465, 1983.

17. Wilson MT, Shivapukar N, Poirier LA. Hypomethylation of hepatic nuclear DNA in rats fed with a carcinogenic methyl-deficient diet. J Biochem 218:987-990, 1984.

18. Bhave MR, Wilson MT, Poirier LA. c-H-rasand c-K-rasgene hypo- methylation in the livers and hepatomas of rats fed methyl-defi- cientaminoacid-defineddiets. Carcinogenesis9: 343-348,1988.

19. Farrance IK. lvarie R. Ethylation of poly(dC-dG).poly(dC-dG) by ethylmethanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro. Proc Natl Acad Sci USA 8: 1045- 1049, 1985.

20. Barbacid M. Rasgenes. Annu Rev Biochem 56:779-827, 1987. 21. Goyette M, Petropoulos CJ, Shank PR, Fausto N. Expression of

a cellular oncogene during liver regeneration. Science 21 9:510- 512, 1983.

22. Lijinsky W, Kovatch RM. Feeding studies of some analogs of the carcinogen methapyrilene in F344 rats. J Cancer Res Clin Oncol 112:57-60, 1986.

23. Davis LG, Dibner MD, Battey JF (eds). Basic Methods in Molecu-

24. Freshney RI (ed). Culture of Animal Cells: A Manual of Basic Tech- lar Biology, 1986, pp. 143-146.

niques,-l983. pp. 235-237. 25. Shivapukar N. Wilson MT, Poirier LA. Hypomethylation of DNA in

ethionine-fed rats. Carcinogenesis 5:989-992, 1984. 26 Carr BI. Pleiotropic drug resistance in hepatocytes induced by car-

cinogensadministered in rats. Cancer Res47:5577-5583, 1987. 27. Thorgeirsson 55. Huber BE, Sorrel1 S, FOJOA, Pastan I, Gottesman

MM. Expression of the multidrug-resistant gene in hepatocarcino- genesisand regenerating rat liver. Science 236: 1 120-1 122, 1987.

28. Burt RK, Garfield 5 , Johnson K, Thorgeirsson 55. Transformation of rat liver epithelial cells with v-H-ras or v-raf causes expression of MDR-1, glutathione-5-transferase-P and increased resistance to cytotoxic chemicals. Carcinogenesis 9:2329-2332, 1988.

29. Garcea R, Daino L, Pascale R, et al. Proto-oncogene methylation and expression in regenerating liver and preneoplastic liver nod- ules in the rat by diethylnitrosamine: Effect of variations of 5-adenosy1methionine:Sadenosylhomocysteine ratio. Carcinogen- esis 10:1183-1192, 1989.

30. Corcos D, Defer N, Raymondjean M. et al. Correlated increase of the expression of the c-ras genes in chemically induced hepato- carcinomas. BiochemBiophysResCommun 122:259-264,1984.

31. Porsch-Hallstrom I, Blanck A, Eriksson LC, Gustafsson 1. Expres- sion of the c-myc, c-fos and c-H-ras proto-oncogenes during sex- differentiated rat liver carcinogenesis in the resistant hepatocyte model. Carcinogenesis 10:1793-1800. 1989.

ONCOGENE EXPRESS/(

32. Antony A, Rao PM, Rajalakshmi 5, Sarma DSR. Hypomethylation of DNA during early stages of chemical carcinogenesis. Proc Am Assoc Cancer Res 28: 104, 1987. Abstract.

33. Hsieh LL, Hsiao W-L, Peraino C, Maronpot RR, Weinstein IB. Expres- sion of retroviral sequences and oncogenes in rat liver turnon induced bydiethylnitrosamrne. CancerRes47:3421-3424,1987.

34. Suchy BK, Sarafoff M, Kerler R, Rabes HM. Amplification, rear- rangements and enhanced expression of c-myc in chemically induced rat liver tumors in vivo and in vitro. Cancer Res 49:6781-6787, 1989.

35. McCov MS. Baramann CI. Weinbera RA. Human colon carcinoma Ki-rasj oncogene and its corresponding proto-oncogene Mol Cell

36 Spurr NK, Solomon E, Jansson M, et al Chromosomal localiza- BIOI 4 1577-1 582,1984

tion of the human homologues to the oncogenes erbA and B. EMBOJ3:159-163,1984.

37. Ullrich A, Coussens L, Hayflick JS, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 3091418-425, 1984.

38. King CR, Kraus MH, Aaronson SA. Amplification of a novel v-erbs- related gene in a human mammary carcinoma. Science 229:974- 976,1985.

39. Ziegler SF, Marth JD, Lewis DB, Perlmutter RM. Novel protein- tyrosine kinase gene (hck) preferentially expressed in cells of hema- topoietic origin. Mol Cell Biol7:2276-2285, 1987.

40. Martinez R, Mathey-Prevot B, Bernards A, Baltimore D. Neuronal pp60c-src contains a six-amino acid insertion relative to its non- neuronal counterpart. Science 237:411-415, 1987.

41. Sola 8, Fichelson 5, Bordereaux D, Tambourin PE, Gisselbrecht 5. Fim-1 and fim-2: Two new integration regions of Friend murine leukemiavirusin myeloblasticleukemias. J Virol60:718-725, 1986.

42. Robbins KC, Devare SG, Aaronson 5. Molecular cloning of inte- grated simian sarcoma virus: Genome organization of infectious DNA clones. Proc Natl Acad Sci USA 78:2918-2922. 1981.

43. Curran T, Peters G, Van Beveren C, Teich NM, Verma IM. FBJ murine osteosarcoma virus: Identification and molecular cloning of bio- logically active proviral DNA. J Virol44:674-682, 1982.

44. Trus MD, Sodroski JG, Haseltine WA. Isolation and characteriza- tion of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus. J Biol Chem 257:2730-2733, 1982.

45. Yamanashi Y, Fukushige S, Semba K, et al. the yes-related cellu- lar gene Iyn encodes a possible tyrosine kinase similar to p56 Lck. Mol Cell Biol7:237-243, 1987.

3N BY METHAPYRILENE 209 46. Takahashi M, Cooper GM. Rettransforming gene encodes a fusion

protein homologous to tyrosine kinases. Mol Cell Biol 7: 1378- 1385,1987.

47. Katzav S, Martin-Zanca D, Barbacid M. Vav, a novel human oncogene derived from a locus ubiquitously expressed in hema- topoietic cells. EMBO J 8:2283-2290. 1989.

48. Martin-Zanca D, Hughes SH, Barbacid M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyro- sine kinase sequences. Nature 31 9:743-748, 1986.

49. Legou E, DePinho R, Zimmerman K, et al. Structure and expres- sion orthe murine L-mycgene. EMBO J 6:3359-3366,1987.

50. Bohmann D, Bos 1, Admon A, Nishimura T, Vogt PK, Tjian R. Human protooncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science 238: 1386-1 392, 1987.

51. Sukegawa J. Semba K, Yamanashi Y, et al. Characterization of cDNA clones for the human c-yes gene. Mol Cell Biol 7:41- 47, 1987.

52. Wood TG. McGeady ML, Blair DG, Vande Woude GF. Long termi- nal repeat enhancement of v-mos transforming activity: Identi- fication of essential regions. 1 Virol46:726-736, 1983.

53. Pulciani S, Santos E, Lauver AV, Long LK. Barbacid M. Trans- forming genes in human tumors. J Cell Biochem 20:51-61, 1982.

54. Wan JYJ. Ledley F, Goff S, Lee R, Grone Y, Baltimore D. The mouse c-abzocus: Molecular cloning and characterization. Cell 36:349- 356, 1984.

55. Jenkins JT, Rudge K, Redmond S, Wade-Evans A. Cloning and expression analysis of full-length mouse cDNA sequences encod- ing the transformation-associated protein p53. Nucleic Acids Res 12:5609-5626, 1984.

56. Sargent TD, Yang M, Bonner J. Nucleotide sequence of cloned rat serum albumin messenger RNA. Biochemistry 78:243-246, 1981.

57. Jagodzinski LL, Sargent TD, Yan M, Glakin C, Bonner J. Sequence homology between RNAsencdng rat a-fetoprotein and rat serum albumin. Proc Natl Acad Sci USA 783521 -3525, 1981,

58. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, Kirschner MW. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell 20:95-105, 1980.

59. Sutrave P, Hu hes SH. Isolation and characterization of three dis- tinct cDNAs ?or the chicken c-ski gene. Mol Cell 8iol 9:4046- 4051, 1989.