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7 ?So:~B FlLE COPV ~~ Transforming ]Putential Is Detectable in Arteriosclerotic Plaques of Young Animals Arthur Penn, Frank C. Hubbard Jr., and Joan Lee Parkes Tbe carcinogen-treated cockerel is emodel for studying the early stages of arteriosclerotic plaque development. Carcinogen adhninistration accelerates arteriosorlerotic plaque develap- meni In cockerels, and transforming elements are present In DNA from advanced human plaques. In this study, we asked whether transforming elements could also be detected at early stages of plaque development in cockerels. NItH3T3 cells were trantzfected with DNA from plaques isolated Orom carcinogen-treajed cockerels and from the beaithy arterial wall under- lying the plaques. Approximately 5 x 100' cells from each group were injected Into nude miee. Tumors appeared In five of five mice In the plaque DNA groap; no tumors appeared In mice t8roaa the healthy arterial wall group~, All five plaque DNA-associated tumors hybridized to a cockerel genomic probe. Eight cocker+el-speciflc bands were identified In EcoRl digests of first- rourAd (primary) tumors. DNA from b primary tumor was tested in a second round of transfection. Five of five mice developed tumors after Injection with these secondary transfor- tnants. All second-round tumors corituihred cockerel DNA, abd a prominent cockerel-specijic band (> 29 kb) was seen in EcnRl digests of all second-round tumors. lrt addition, a 5.2-kb bao<d appeared prominently In one of five secoitd-round tumors. No evidence was found for activation of the oncogenes Ha ras, Ki-rrrs, srr, or myr In the plaque-astroctated tumors. Similarly, DNA from plaque-associated tumors did not hybridize to probes for Marek disease virus, herpes simplex virus l, or reverse transcriptase, suggesting that neither herpeiviruses nor retroviruses are involved In the transforming actiyit,y of plaque DNA. These results indicate that transform- ing elements are a general property of arteriosclerotic plaques and are detectable In plaques of young animals. (Arteriasclerosifs and iFhrrim6osis 1991;11:1053-1058) 3 T he proliferation of smooth muscle cells (SMCs), a rare event in the normal adult arterial intima, is thought to be critical to the development of arteriosclerotic plaques. Most theo- ries of plaque development have regarded SMC proliferation as a reactive process, occurring in re- sponse to such stimuli as injury, or inflammation.2 Under these conditions the SMCs involved in,pidque formation are regarded as indistinguishable (except for receipt of stimuli that cause proliferation) from the rest of the arterial wall SMCs. According to this view, it is the availability of mitogens or other rrtuo- ulators of cell division rather than any uniqut prop- erty of the responding arterial SMCs that determines whether intimal SMC proliferation and subsequent plaque development will occur. A fundamentally different view is presented by the monoclonal hypothesis.3 This proposes that the pro- liferating SMCs are derived from a stably trans- formed and thus per*nanently altered cell population that is distinct from the bulk of arterial SMCs. One inference from this hypothesis is that there should be demonstrable similarities, on the molecular level, between events associated with the development of both plaques and tumors. Indeed, the prediction has been made that mutagens and viruses should play a role in plaque formation comparable to the role that they play in tumor formation. Studies including those using cockerels as an animal model have provided indirect support for this view. Weekly injections of polynuclear aromatic hydrocarbon carcinogens (PAHCs) at nontumorigenic doses into cockerels markedly accelerate plaque development a-' The in- ducible enzyme systems responsible for the metabo- lism of PAHCs have been identified in the krterywall of both cockerels and mammals.B-10 The PAHC ?,l2-dimethylbenzialanthracene (DMBA), adminis- tered as an "initiator" followed by weekly injections of the a-adrenergic agonist methoxamine (as a pro- moter) yielded microscopic aortic plaques in cocker- From the Institute of Environmental Medicine, New York University Medical Center. N.Y. Supported by National Institute of Environmental Htalth Sci- ences grant Nos. 02143 and 00260. American Heart Astioeiation grant-in-aid No. 87•993. and the Council tor Tobacco Research. Address for reprints: Arthur Penn. PhD. Institute of Environ- mental Medicine. NYU Medical Center. New York, NY i0D166 iteceived September 20. 3990; revision accepted March 21. 1991. PUBLICRTIONS 028943

1054 Arteriosclerosis.nd 7Lrombosis Vol 11, No 4 July/Augusr 1991 els. I I A series of studies during the past decade have Methods also suggested a role €or herpesviruses in plaque formation.12-m Two recent sets of in vitro studies have provided evidence for possible mechanisms of the interaction betvueen viruses and SMCs. In the first of these studies fibroblast growth factor receptor was identified as the site of herpesvirus entry into mammalian cells, including SMCs.21 In the second study rabbit aortic SMCs were transformed by the early region of SV4Q DNA.u Carcinogen and virus studies have thus far provided only indirect support for the predictions of the mono- clonal hypothesis. Direct support requires demonstra- tion of molecular alterations in plaque cells compara- ble to those observed in tumor cells. Earlier, we reported that human coronary artery plaque DNA contained transforming potential and that mouse fi- broblasts (NIH3T3 cells) transfected with human cor- onary artery plaque DNA gave rise to tumors after subcutaneous injection into nude mice?3 The proto- cols followed in these studies were originally devel- oped to demonstrate the presence of dominant trans- forming genes (oncogenes) in tumors and transformed cell lines.r,-26 One limitation associated with these human stud- ies is that the plaques were obtained from older patients in whom atherosclerosis developed over a period of decades. Thus, it could be argued that acquisition of transforming potential is a late-stage event in atherosclerosis and is only indirectly associ- ated with plaque etiology. An advantage of using the carcinogen-treated cock- erel as a model for plaque development is that large plaques can be genetated in the abdominal aortas of these young animals after carcinogen treatment. These plaques arc fibromuscular, with limited lipid involvement and without apparent calcification or necrosis. We have shown that in this system carcin- ogen treatment acts, primarily to accelerate plaque development rather than to initiate it!-7 Morpholog- ical and ultrastructural similarities between fibro- muscular abdominal aortic plaques in cockerels and coronary artery plaques in humans have been docu- mented.27 The objective of the present studies was to determine whether molecular alterations exist in carcinogen-associated cockerel plaques similar to those previously identified in human plaques.23 In this article we report that NIH3T3 cells are transformed by carcinogen-associated cockerel plaque DNA and that injection of these transformed cells into nude mice elicits development of tumors that contain cockerel genomic DNA. Moreover, this plaque transforming potential is transmitted serially. 7hus, there are molecular alterations in the DNA of plaques from diverse sources (human and avian) and from young and old subjects that are similar to alterations that have been identified in tumor DNA. This suggests that somatic cell mutations play a role in plaque etiology. Animals Four-week-old white leghorn cockerels (Kerr Hatcheries, Hightstown, N.l.) were housed in stainless steel cages. Water and standard bnash (Purina Co., St. Louis, Mo.) were available ad libitum. All animal handling and treatment procedures were conducted according to New York University Medical Center guidelines. From 6-22 weeks of age the cockerels received weekly injections of DMBA (20 mg/kg body wt, Sigma Chemical Co., St. Louis, Mo.) into the pectoral muscle. The DMBA was dissolved in dimeth- yl sulfoxidc (Fisher Chemieal Co., Valley Forge, Pa.). Af'ter the eockerels were humanely killed at 23 weeks of age, plaques and healthy arterial wall underlying the plaques were removed under sterile conditions and frozen immediately in liquid Nz. 71kans/'ections AU tissue samples were pulverized in liquid NZ 2' DNA extraction was performed via standard phenol/ yisoamyl alcohol procedures.28 DNA from four CjHClr separate sources was tested in the transfection assay: 1) cockerel aortic plaque, 2) healthy cockerel arterial wall that had underlain the plaques, 3) T24 human bladder carcinoma cell line (positive transfection control), and 4) NIIi3T3 cells (negative transfection control). The DNAs weire cotransfected with pSV2.,,.,29 the hybrid plasmid vector conferring antibiotic resistance, by the standard Ca,(PO,)2 transfection protocol.30 For each sample 40 .u.g genomic DNA was transfected along with 4 µg pSV2,.. into three T 25 flasks, each containing approximately 0.5 x 10° NIa3T3 cells grown in Dulbec- co's modified Eagle medium (DMEM) with 10% fetal bovine serum. Twenty-four hours later cells were split 1:3. Transfe.cted cells were grown in the presence of gentamicirti (G418). Nude Mouse Assay Three weeks after cotransfection the colonies were collected, and 4-5 x 10° cells from each group were injected subcutaneously into the scapular area of five 4-5-week~-old athymic (nu+/nu+) female mice (Har- lan Sprague-Dawley, Indianapolis, Ind.). Animals were checked for the presence of tumors three times per week. The mice were killed when the tumors were at least 15 mm in diameter, and samples of tumors were taken for histological observation, DNA isolation, T nd growth in culture. Southern litlor Hybridizarion DNA was digested with the restriction enzymes EcoRl and BamHI according to the manufacturers' instructions, subjected to electrophoresis overnight in 0.7% agarose (Bio-Rad, Richmond, Calif.) at 20 V, 15 mA per gel, and blotted onto nitrocellulosesl (Schlei- cher and Schuell, Keene, N.H.). pSV2„K, was provided by E. Newcomb, New York University Medical Cen- ter. The probes Ha-ras, Ki-r+as, srr, and myc, consisting PUBLICATIONS • . . • : . • 028944

r TaatE t. 7tiuporlVnic li/Nelenwy of t'taque DNA-71r.o#reetcd Cdls DNA source No. tutnors/No. mice Tumor latency (daye) Arteriosderotbc plaques from DMBA-treated cockerels 515 33-45 Healthy artgrial wall (underiying plaque) from DMBA-treated oockerels /5 .. 774 cells (hytagan bladder carcinoma atll line) 5/5 7-10 NIH3T3 cells 0/5 ... Plaque-asso~.-isied pritnaty tumor CTLJ (foom ftsst source al>oe) 5/5 24-42 T24 cells 5/5 19-23 NIH3T'3 cells 0/5 ... For each grtpup, -5 x 10° cells were injected subcutaneously in the dorsal su'rfsoe of five female nu+inu+ mice. DMBA, dimethyibenz(aJanthrrsoene. of inserts cut out and purified from plasmids, were purchased from Oncor, Gaithersburg, Md.; the re- verse transcriptase (RT) probe, contained in plasmid pRT 432-I. No. 31990, was purchased from the Amer- ican 'lype Culture Collection, Rockville, Md. The herpes sirtiplex virus I (HSV-1) probe was a gift of David Hajjar, Cornell University Medical Center, New York, The Marek disease virus (MDV) probe (BamHl-H insert), which does not hybridize to DNA from uninfected chicken cells, was provided by K. Schat, Cornell University, Ithaca, N.Y. The cockerel genomic probe was prepared from cockerel heart DNA. All the probes were labeled with phosphorus- 32-labeled cytidine triphosphate (New England Nu- clear, Boston, Mass.) by nick translation 32 Hybridization conditions were as follows: for Ha-= myr~ srry and RT, 40% formamide, 40°C 18 hours; for Ki-ras, 45% formamide, 43°C~ 18 hours; for HSV-1 and oockerel 1probe, 50% formamide, 43°C, 18 hours; and for MDV, 50% formamide at 51°C, 30 minutes, and then at 42°C, 24 hours. Washes and exposures were performed by use of standard proc:edures.28 Results Cockerels were injected weekly with DMBA from 6-22 weeks of age and were killed at 23 weeks of age. Four of five DMBA-injected cockerels displayed grossly visible plaques (12x3 mm) in the distal third of the abdominal aorta. These plaques were similar to DMBA-associated plaques that we have described previously4-'t; they were fibromuscular with little lipid involvement. DNA samples obtained from the pooled plaques, healthy arterial wall underlying the plaques, T24 human bladder carcinoma cell line, and NIH3T3 cells were used to cotransfect host NIH3T3 cells. Results of the transfection nude mouse study are presented in Table 1. Tumors developed in all Penn et a/ Transforming Potential in Plaques 1055 23_1. 9A- ss- 4.4. . s.w.,r. FtoURE 1. Hybridfzarion of pldque-associated ttunor Y)NA to a cockerel genomfc probe. EttoRl-digested DNA samples (20 /rg/lane) were Soutiterrr bloned and hybridized to a phosphorus-32-labeled probe prepared fr+vm cockerel heant DNA. Hyb.idisations were perfo'rrned under hfgh strfngency conditions (50% jonnamide, 43°C ) Sources of DNA werr lane 1. 724-associated nude mouse tumor, lane z NIH3T3 cells; lane 3, fast-round cockerkl plaque DNA-associated nude mouse tumor (CTI-3); lanc A CTI-3 pdnwry explant; lane 5, second-round nude mouse tumor (C72-3); lone 4 second-round nude mouse ttunoi (C72-4). Note that fcint bands present in the T24-de.ived tumor and in the N1H3T.1 c•elLs (lanes I and 2) are most 1fkeAv due to cross h3bnidization ojthe cockerel probe to manrmalian nepeutive sequences with which cockend repetitive sequences share homology.s3-35 Nutnbers at leJft are in kb five mice injected with plaque DNA-transfected cells. The tumor latency period was 33-45 days. In contrast, no tumors appeared in any mice injected with cells transfected with DNA from normal arterial wall or from untreated NIH3T3 cells. All five mice in the T24 carcinoma cell line group developed tumors, with a latency period of 7-10 days. Tumors were excised when they reached 15-20 mm in diameter. All tumors had the appearance of poorly differenti- ated fibrosarcomas. DNA from all first-round plaque- associated tumors hybridized to a cockerel genomic probe. At least eight distinct cockerel-specific bands were visible in first-round plaque-associated tumors (see Figure 1). DNA extracted from one of the plaque DNA- associated tumors (CTl-3) was tested in a second round of transfection. All five mice injected with the second-round transfectants developed tumors. La- tency periods ranged from 24 to 42 days (Table l). The positive control group in this study displayed tumors (five of five mice) after 19-23 days. Again, no tumors appeared in the negative controls. DNAs isolated from the setrondary tumors were digested with EcoRl and hybridized to a cockerel genomic probe. All seoondary tumors contained cockerel genomie DNA. A prominent band (>28 kb) was identified in all secondary tumors. EroRI digests PUBLICATIONS 0 28945

1056 Arterioselermsis and Thrombosis ss• •. Vol 11, No 4 JulylAugust 1991 1 2 3 4 5 FTstlrte 2. Absence of cockenl myc sequences in NIH3T3 tmnsjbrinants is shown by Souihem-blotted BamHl-digesxd DNA hybridized to a phosphorus-32-labeled v-myc probe. HybridEtations were perfomied in the presence of 40 % jonn- amide at 40°C. Sourres ef DNA were lane l, cockerel hearr lanes 2 and 3, fast-round plaqae-asaociated nude mouse tnmors; lane 4, nude mouse tumor pimary dplaru; lane 5, N1H3T3 cells. Cockerel myc is mpesented by an intense band (of > A4 kb) in lane l. A less intense mu.fne myc sfgnal appears at -6.6 kb in lanes 2-5. 71ie v-myc probe denved from the avian MC29 vlras has gnrater homology to cockerel myc than it does to mouse myc and therefore hybridres more stnongly to the cockenef proro-oncogene. of two,of these tumors are shown in Figure 1(lanes 5 and 6). In addition, a 5.2-kb band appeared prom- inently in one of five second-round tumors (Figure 1, lane 6). As expected, neither DNA from the T24- associated tumors nor that from N1H3T3 cells hy- bridized to the cockerel genomic probe (Figure 1, lanes 1, and 2). DNA from the first round of plaque-associated tumors was tested for the presence of activated oncogenes. Ki nas, Ha,ras, myc, and src probes were used to screen BamH1-digested DNA from plaque- associated tumors. None of these was responsible for the transforming potential of cockerel arterioscle- rotic plaque DNA (Figure 2 and data not shown). In Figure 2 the intense band (>9.4 kb) in lane I (cockerel heart DNA) is cockerel myc; the less intense bands of approximately 6.6 kb in lanes 2-5 are murine myc The absence of a >9.4-kb cockerel myc signal in the plaque-associated nude mouse tumors indicates that myc is not the cockerel plaque- transforming gene. We also tested the possibility that retroviral infec- tion plays a role in cockerel arteriosclerotic plaque development. DNA from plaque-associated tumors was hybridized to an RT probe. No RT-specific bands were identified (data not presented). Because a single injection of the avian herpes virus MDV to 4-day-old cockerels results in focal plaque formation; = we screened the plaque-associated tumor DNA with MDV. 'lltis probe failed to hybridize to plaque-associated tumor DNA (data not presented). Finally, because herpesviruses have been implicated as etiologic factors in the pathogenesis of human arterio- sclerosis and human HSV-1 has been shovutt to influ- ence lipid accumulation and metabolism in both human and bovine arterial SMCs,t4 we also screened the plaque-associated tumor DNAs with an HSV-l probe. This, too, was negative, suggesting that herpes viral sequences are not associated with the transforming element(s) in cockerel plaque DNA. Discussion Previous;y, we have shown that carcinogen treat- ment accelerates plaque development in cockereis +-'7 We have also demonstrated, via the transfection- nude mouse tumor assay, that human coronary attery plaque DNA has transforming potentiai.23 Recently, we reported that DNA from SMC strains derived from human aortic plaques i.s also positive in the transfection-nude mouse tumor assay.36 The data presented here show that cockerel plaque DNA, like human plaque DNA , contains transform- ing element(s). NIH3T3 cells transfected with oock- erel plaque DNA gave rise to nude mouse tumoi I rs in five of five injected mice. The tumor latency period (33-45 days) was significantly shorter than that for human coronary artery plaque-associated tutators (50-112 days)23 but longer than that for the i'24- as.cociated tumors (7-10 ilays), which contaiti an activated Ha,ros oncogene. As is the case with human coronary artery plaque DNA, cockerel plaque DNA transforming elements are serially transmitted, with DNA from primary plaque-associated tumors gi'ving rise to secondary plaque-associated tumors (Table 1). DNA from both rounds of tumors hybridized io a cockerel genomic probe. A itumber of oockerel- positive bands were present in tcoRl-digested bNA from plaque-associated first-round tumors and their primary explants (Figure 1). When DNA from one of the plaque-associated tumors was tested in a secbnd round of transfection, a prominent cockerel-positive band (>28 kb) was retained in all the resulQing tumors (Figure 1). ln additio>ti, a 5.2-kb band was present in one of the second-round tumors. The retention of eockerel-positive bands in the secdnd- round tumors suggests that the putative transforming gene is associated with these sequences. We are currently investigating this phenomenon. One approach to identifying the transforming garne in cockerel plaque DNA is to screen the plaque- associated tumor DNA with onoogene probes. We tested for the presence of att activated cockerel Ha-r+os gene because mutations in codons 12 and 61 of Ha-ras have been reported in skin cells trans- formed by the PA.HCs benzo[q)pyrene (B[a)P) and DMBA, respectively,'7 and because B[ajP and DMBA markedly accelerate plaque development in cockerels.7 However, we found no evidence of cock- erel Ha-ras in any of the plaque-associated tumors even though the plaques were obtained from DMBA- treated cockerels. Similarly, we found no evidence for the presence of exogenous IC$-ras, sr+c (not shown). or myc (Figure 2) in any of the cockerel plaque- associated nude mouse tumors. 1Ci-,rrts is one of the most commonly activated dominant transforming genes that has been identified in human tumors.`" Activation of src, which is responsible for the trans- forming activity of Rous sarcoma virus, results in malignant transformation of avian celis.39 myc was 11 . PuBLrcRrIoNs 028946

s first identified as the transforming gene of avian myclocytomatosis virus!o In humans, enhanced expression of »iyc has been noted in cases of leuke- mia, lymphoma, and carcinoma of the breast and prostate. A modest elevation of myc expression was also observed in SMCs of spontaneously hypertensive rats compared pvith normotensive rats!t We have recently descriaed a twofold to sixfold enhancement of nryc expression in SMC strains derived from human abdominal nortic plaques compared with healthy human aortic SMCs, although nmyc was not responsible for the transforming potential of the DNA from these cell strains.36 Finally, the absence of RT, MDV, and HSV-1 se- quences in the plaque-associated nude mouse tumors ,suggests that these vpruses are not involved in the etiology of these mocket•el plaques. Although one recent report failed to identify traatsfonning activity in human carotid plaquW2 other investigators have confirmed that serially tran5mitted transforming activity is present in human arteritasclerotic plaques (Reference 43 and R.M.L Zwijsen, personal communication). The results described herein confirm our previ- ous findings with plaque samples of human origin2-' and demonstratee that the presence of transforming elements is a general eharacteristic of plaque DNA. Furthermore, the fact that these activated ele- ments exist in animals only 6 months old demon- strates that transforming potential is detectable in plaques of young animals. Acknowledgments We thank Tom Schat for performing the hybrid- ization with the MDV probe; Alan Bowers and Dule Hubbard for excellent technical assistance; and Sy Garte and C.arroll Snyder for critically reviewing this manuscript. References 1. Ross R: The pathogentsis of atherosclerosis: An update. N Eagl J Med 1986;314:4"- 500 2. Munro JM, Cotrbn RS: The pathogenesis of atherosclerosis: Atherogenesis and in8amroation. Lab Invrst 1988;58:249-2ti0 3. Bendlit EP. Benditt JM: Evidence for a monoclonal origin of human atherosclerotic plaques. Hoc Nail Acad Sci U S A 1973;70:1753-1756 4. Penn A, Batastini G, Al,ben R: Age-dependent changes in prevalence, sire. tind proliferation of arterial lesions in the oockerel: Il. Carcinogen-associated lesions. Arrcry 1981;9: 362-393 S. Penn A, Batastini G, Solomon J, Burns F. Albert R: Dose- dependent size inereases of aortic lesions following chronic exposure to 7,12-dimelhylbenzla)anthracene. Canrer Rea 1981;41:588-592 6. Batastini 0. Penn A: An ultrastructural comparison of carcin- ogen-associated and spontaneous aneriosclerotic lesions in the cockerel. Am J Parhol 1984;114:403-409 7. Penn A, Snyder C Arteriosclerotic plaque development is "prornoted" by polynuclear aromatic hydrocarbons. Carrino- gr.usis 198b:9:2185-2189 8. Bond JA, Kocan RM. Benditt EP. Juchau MR: Metabolism of benzoiajpyrene and 7,12-dimethyibenrlajanthracene in cul- tured human fetal aortic cells. Life Sd 1979;25:425-430 9. Bond JA. Yang H•YL, Majesky MW. Benditt EP. Juchau MR: h.etabolism of benzo[olpyrene and 7,12-dimethylbenzlalan- :Penn el at 7lransfotming Potential in Plagues 1057 thracene in chicken aortas: Monoo>ygenation, bioactivation to mutagens and oovalent binding to DNA in tdr% Toxicol App! Pha.marol 1980:52:323-335 10. Majesky MW. Yang H-YL, Benditt EP, Juchau,MR: Carcino- genesis and atherogenesis: Differences in nionooxygenase inducibility and bioactivation of benzo/aJpyrenC in aorlic and hepatic tissues of atherosclerosis-susceptible versus resistant pigeons. Carrinognuais 1983;4:647-652 11. Majesky MW. Reidy MA. Bendita EP, Juchan MR: Focal smooth muscle proliferation in the aortic intimo produced by an initiation-promotion sequence. b4oc Nar! Acad Sri t/ S A 1985:82:3450-3454 12. Fabricant CG. Fabricant J, Litrenta MM. MiniCk CR: Vituus- indueed atherosclerosis. J Fap Med 1978:148:335-340 13. Hajjar DP. Fabricant C, Minick CR; Fabrieam J: Herpesvirus infection alters aortic cholesterol metabolism acrd aocumula- tion. Arra J Paako! 1988:122:62-70 14. Hajjar DP, Pomerantz KB. Falcone DJ, Wekst~r BB. Grant AJ: Herpas simplex virus infection in human arterial cells. J Clin lnwsr 1987:80:1317-t321 15. Yamashiroya HM, Ghosh L, Yang R, Robertson AL Jr. Herpesviridae in the coronary arteries and aorta of young trauma victims. Am J Parhol 198B;13b:71-79 16. Gyorkey F. Melnick JL, Guinn GA. Gyorkey P. DeBakey ME: "erpesviridae in the endothelial and smooth muscle cells of the proximal aorta in atherosclerosis patients. Erp AfolParlwl 1984;40:328-339 17. Adam E. Melnick Jt., Probstfeld JL, Petrie Bt., Burek 3. Bailey KR. McCollum CH. DeBakey ME: High levels of cytomega- lovirus antibody in patients requiring vascular surgery for etherosderosis. Lancer 1987;8554:291-293 18. Melnick JL, Adam E, DeBakey ME: Possible role of cytomeg- z4lovirus in atherogenesis. JAMA 199tl:263:2204-2267 19. Grattan MT. Moreno-Cabral CE, Starnes VA, Oyer P. Stinson ED. Shumway NE: Cytomegatovirus infection is associated with cardiac altograft rejection and atherosclerosis. JAMA 1989;261:3561-3566 20. McDonald K. Rector RS, Braunlin E1y. Kubo SH, Olivari MT: Association of coronary artery disease in cardiac transplant recipients with cytomegatovirus infection. Anr J Cardio! 1989; 64:359-362 21. Kaner RJ. Baird A. Mansukhani A. Basilico C. Summers BD, Fiorkiewicz RZ. Hajiar DP: Fibroblasi growth factor receptor is a portal of cellular entry for herpes simplex virus type 1. Science 1990;248:1410-1413 22. Nachtigal M. Legrand A, Nagpal ML, Naehtigal SA. Greenspan P: 'IYansformation of rabbit vascular smooth mus- cle cells by transfection with the early region of SV40 DNA. Am J Pathol 1990; t36:297-306 23. Penn A. Garte SJ, Warren 1-, tVesta D. Mindich B: TYansfonn- ing gene in human atherosclerotic plaque DNA. Pyoc Nail Acad Sri U S A 1986;83:7951-7955 24. Pulciani S. Santos E. Lauver A. Long L. Robbins K. Barbacid M: Onoogenes in human tumor cell lines: Molecular ctoningof a transforminggene from human bladdercarcinoma cells.Proc Narl Acad Sri U S A 1982;79:2845-2649 25. Balmain A. Pragnell 1: Mouse skin arcinomas induced in vn+o by chemical carcinogens have a transforming Harvey-ros onco- gene. Nature 19Fi4;303:72-74 26. Guerrero 1, Cafzada P. Mayer A, Pellicer A: A molecular approach to leukemogenesis: Mouse tymphomas contain an activated c-rns oncogene. Proe Narl Aced Sri U S A 1984:81: 202-205 27. Moss NS. t3enditt EP: The ultrastructure of spontaneous and experimentally induced arterial lesions: 11. The spontaneous plaque in the chicken. Lab Int+esr 1970;23231-245 28. Sambrook J, Fritsch E, Maniatis 't': Molecular Clvnin8• A Laboraroay ManuaL Cold Spring Harbor Laboratory Press. Cold Spring Harbor. NY. 1989 29. Southern P. Berg P: Transformation of mammalian cells to anlibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol ApP! Gentt 1982:1:327-341 30. Wigler M. Pellicer A. Sihrerstein S. Axel R. Urlaub G. Chasin t_ DNA-mediated transfer of the adenine phosphoribosyl- PUBL ICRTIONS 028947

.:r.~ 1058 Artaiosdemsis and '!'brombosls Yol ll, No 4 ltdy/Aqgust 1991 transferase locus in mamraalian cells. Ph.c Nad Ac+od Sri U S A 1979t76:1373-1376 31. Shuthern EM: Detection of specific sekiuenoes hmortg DNA tOpne nts separated by gel electrophoresis. l Afol Biol 1975; 9[i:50.i-517 32. Rigby PWJ. Diedctnann M, Rhodes C, Berg P: Labeling dcoxyrt'bonuelele acid to high specific activity in vitro by nick ttanslation with DNA polymerase 1. ! Mol Bioi 1977.113: 2$7-251 33. Weiner AM: An abundant cytoplasmit: 7S RNA is oomple- mentary to the dominant intenpersed middle repetitive DNA scquence family in the human genome. Ce1l 1980:22:209-218 34. Shtmph WE, Kristo P, Tsai M-J, O'MaOey BW: A chicken middle-repetitive DNA sequence which shares homologywith tnammalian ubiquitous repeats. Nucieie Acids Res 19d1;9: 53B3-5397 35. Stumpb WE, Hodgson CP, Tsai M-3. O'Malley BW: Genomic structure and possible retrovirai origin of the chicken CRl repetitive DNA sequence family. Proc Notl Ac:ad Scti U S A 1984;81:6667-6671 36. Pq~rkes JL, Cardell RR, Hubbrrd FC. Hubbard D. Meltrxr A. Pknn A: Glrltured human atherosclerotic plaque smooth mus- ck cells retain transforming potential and display enhanced eupression of the ng+c protooncogene. Am l Parhol 1991:138: 765-775 37. Balmain A. Qrown K: Onohgene activation in chemical car• einogenesis. Adv Cahcttr Res 1988;S1'147-182 38. Dos J: The.as gene family and human eardnogersesis. Murai Res 1988;195:255-271 39. Hanafuss H: Cell transformation by RNA tur»or-vintses. Cpnp V'inml 1977;10:401-48:1 40. Sheiness DK• Hughes SH, Vatstus H@, Stubblefleld E. Bishop JM: The vertcbrate homolog of the putative transforming gene of avian myeEocytootatosis witas: Characteristics of the DNA locus and Its RNA transcript. Vaolop 1980:105:415-424 41. Negoro N, Inanbs H, Intae 7; Kanayama Y, Takeda T: l:wpres- sion of cMr, protoon~etk in beans and cultured smooth muscle cells of spontaneaskly hypertensive rats. ! t4prncns 198g:6(suppl 4):5128-S130 42. Yew PR. Rajavashisth TB, Forrester J. Baralh P. Lush Al: NIH3T3 trankfotming gene is not a general feature of athero- sclerotic plaque DNA. Btodu+n BJophp Res Corrs+ero+ 1989; 165:1067-1071 43. Ahmed AJ. O'MaUey BW. Yatsu FM: Presenee of a puutiim transforming gene in hum.n atherosclerotic plaquss (abstract). Arudosdaadc 1990:10:755a KEY Wottos • arteriosclerotic plaque • transfontdag genes • cockerel • tarcinogens • tumors • transfection . i 0 PUBLICATIONS 028948