Human Papillomavirus Type-Related DNA and c - m y c Oncogene Alterations in Colon Cancer Cell Lines Jhy-Young Cheng, M.D., D.M.Sc.,* Ching-Liang Meng, M.D., D.M.Sc.,-~ Chung-Faye Chao, M.D., Ph.D.,~- Shun-Der Gau, M.D., M.S.,'~ Jih-Chang Lin, M.D.* From the Division of Colon a n d Rectal Surgery, *Department of Surgery, afDepartment of Medical Research, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, R. O. C
ChengJ-Y, Meng C-L, Chao C-F, Gau S-D, LinJ-C. Human papillomavirus type-related DNA and c-myc oncogene alterations in colon cancer cell lines. Dis Colon Rectum 1991;34:469-474. Although squamous-cell epithelium is the most frequent target site of human papillomavirus (HPV) infection, a similar infection is demonstrated in columnar epithelial cells in this paper. The papiHomavirus expression in three cell lines was detected in colorectal adenocarcinoma of Chinese patients. The HPV- 16 and HPV- 18 DNA sequences were found in colorectal cancer cell lines, which might suggest the correlation of HPV to the etiology of colorectal cancers. In addition, c-myc oncogene was identified by amplification in all three colorectal cancer cell lines, but only normal germ-line fragments were found in control tissue. The correlation between HPV and c-myc, and the implications of these findings in colorectal cancers are also discussed. [Key words: HPV; c-myc; Colon cancer] u m a n p a p i l l o m a v i r u s (HPV) DNA has b e e n d e t e c t e d in high p r o p o r t i o n s in b e n i g n tumor, precancer, and c a n c e r of the genital 1 12 and extragenital organs. 13-17 T h e extensive studies on HPV infection including c a r c i n o m a in situ, invasive cancers, and several cell lines f r o m cervical carcin o m a s suggest that this virus m a y act as a causal a g e n t rather than a casual p a s s e n g e r in carcinogenesis. Various cancers w e r e also s c r e e n e d b y m o l e c ular-hybridization analysis for the p r e s e n c e of HPV DNA, including oral cavity, 14 tongue, 16 larynx, ~5 lung, ~3 a n d skin, is and all c o n t a i n e d various a m o u n t s of HPV DNA. Although HPV m o s t l y pres e n t e d in s q u a m o u s - c e l l epithelia, r e c e n t reports
H
This study was presented at the 44th General Scientific Meeting in March 1990, Surgical Association, Taipei, Taiwan, R.O.C. This research was supported by Grant NSC 77-0609-B016-01 from the National Science Council, R.O.C. Address reprint requests to Dr. Cheng: Division of Colon and Rectal Surgery, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan, R.O.C. 469
have d e m o n s t r a t e d their related DNA in cancers of have d e m o n s t r a t e d their related DNA in cancers of c o l u m n a r e p i t h e l i u m , such as the e n d o c e r v i x 12 or c e c u m . 17 Because the c o l o n is part of gastrointestinal tract a n d the m o r p h o l o g y of colonic a d e n o m a m i m i c s that of p a p i l l o m a , the role of HPV in the colorectal c a r c i n o g e n e s i s invites further investigation. HPV-16 and HPV-18 are m o s t f r e q u e n t l y c l a i m e d to b e associated with the majority of cervical carcinoma. HPV-16 was f o u n d in 60 to 90 p e r c e n t of cervical c a n c e r tissues 4'9 w h e r e a s HPV-18 was in o n l y 17 p e r c e n t *~ of invasive cancers. T h e y s e e m to p r e f e r e n t i a l l y i n d u c e lesion with m a r k e d nuclear atypia a n d have high o n c o g e n i c association. ~9 A m o n g the k n o w n cellular o n c o g e n e s , c-myc is o n e of the m o s t f r e q u e n t l y f o u n d in various cell lines d e r i v e d f r o m h u m a n tumors. c-myc has b e e n r e c o g n i z e d as a factor involved in h u m a n m u l t i s t e p t u m o r i g e n e s i s , in that it can either b e a m p l i f i e d or rearranged. 2~ 21 Recently, Yang et aL 22 s u g g e s t e d that the b o v i n e papillomavirus c o n t a i n e d m u l t i p l e t r a n s f o r m i n g genes. We t h e r e f o r e s u s p e c t HPV m a y b e also involved in at least certain steps of colorectal carcinogenesis. T h e p r e s e n c e of HPV DNA s e q u e n c e a n d c-myc oncog e n e in colorectal t u m o r s was r e a s o n a b l y t h o u g h t to b e d e t e c t a b l e Y P-labeled HPV-16, 18, and cmyc p r o b e s w e r e e m p l o y e d to detect the expression of HPV-DNA s e q u e n c e s in the three c o l o n a d e n o c a r c i n o m a cell lines d e v e l o p e d in our laboratory. 23 T h e results of this Southern blot study indicated that h u m a n colorectal tumors, in addition to s q u a m o u s - c e l l tumors, c o n t a i n e d HPV DNA and c-myc o n c o g e n e . T h e r e f o r e , w e b e l i e v e that HPV a n d c - m y c o n c o g e n e m a y play a role in the carcin o g e n e s i s of colorectal cancers.
470
CHENG ET AL MATERIALS AND M E T H O D S Cell Lines
Three human adenocarcinoma cell lines of colon cancer (CC-M1, CC-M2, CC-M3) were used in this study. These cell lines were established from Chinese patients in our laboratoryY They were kept in RPMI-1640 RPMI medium with 10 percent fetal calf serum (GIBCO-Europe Ltd., Paisley, UK), 100 #g/ml of penicillin, and 100 #g/ml of streptomycin (Bristol Laboratories, Langley, Slough), 4 mm ]-glutamine, and 2.5 mm sodium pyruvate (all from GIBCO, Grand Island, NY), and ready to be used.
Dis Colon Rectum, June 1991
hybridized with HPV-16, HPV-18, and c-myc-oncogene probes, with specific activity ranging from 2 x 108 to 2 x 1 0 9 cpm/#g in high-stringency conditions, then washed several times at 55 ~C, and finally exposed to an x-ray film, using an autoradiographic method at -70~ RNA E x t r a c t i o n a n d N o r t h e r n B l o t
Hybridization Total cellular RNA of cell lines was extracted according to Glisin. 26 The hybridized procedures for cellular RNA were similar to that of DNA hybridization. RESULTS
Probes HPV type 16 and 18, and c - m y c p r o b e s were used in this analysis.
DNA Extraction Total cellular DNA was extracted from these cell lines, and the normal epithelium of the colon was used as a normal control by the method previously described.8, 24 Briefly, DNA was extracted and incubated with sodium dodecyl sulfate (SDS) and RNAse at 37 ~ for 3 hours. Subsequently, proteinase K was added and incubated again overnight. Cellular DNA was then extracted with p h e n o l / chloroform/iso-amylalcohol (25:24:1), ether, and dialysed with Tris-EDTA (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) buffer solution. After centrifugation with 1,000 x g at 4 ~ for 20 minutes, the DNA in the supernatant was precipitated by the addition of ethanol. Precipitated nucleic acids were then washed with 70 percent ethanol and dried in the air. The absorbance of DNA at 260 nm was determined by a DU-50 series spectrophotometer (Beckman Instruments, Fullerton, CA) and compared with the absorbance at 280 nm. The DNA absorbance ratio ranged from 1.8 to 2.0 and was stored at 4 ~C until ready for use.
DNA extracted from three colorectal adenocarcinoma cell lines was analyzed by Southern blot hybridization with EcoRI-endonuclease treatment. Under highly stringent conditions, a major HPV16-related DNA band of 4.8 kb was present in all three cancer cell lines, and was especially distinct in CC-M2 cells (Fig. 1). On EcoRI cleavage of the DNA of cancer cells showed a single band of 5 kb by HPV-18 DNA probe (Fig. 2).
Southern Blot H y b r i d i z a t i o n Filter hybridization according to a previous report was carried OUt. 4 Briefly, each DNA was cleaved with the restriction enzyme EcoRI and then electrophoresed in 1 percent agarose gel. After staining with ethidium bromide, the DNA was denatured in situ and transferred to nitrocellulose filter paper by Southern blot. 25 The filters were
Figure 1. High-stringency hybridization of DNA extracts with the HPV-16 DNA probe is shown: 10 #g of DNA were treated with EcoRI, then electrophoresed, transferred to a nitrocellulose paper, and hybridized under high-stringency conditions with a (a2P)-radiolabeled HPV-16 DNA probe. Hybridized probes around 4.8 kb are indicated in Lanes 1, 2, and 3. Lane I:CC-M1, Lane 2: CC-M2, Lane 3:CC-M3, Lane 4: normal colonic epithelium.
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HPV AND c-myc IN COLON CANCER
Figure 2. Autoradiographs of cellular DNA extracts run on 1 percent agarose gels and transferred to a nitrocellulose paper by Southern blot are shown. These DNA were digested with the restrictive endoneuclease EcoRI and hybridized a2P-radiolabeled HPV-18 DNA probe. Lane 1: CC-M1, Lane 2: CC-M2, Lane 3: CC-M3, Lane 4: normal colonic epithelium.
471
Figure 4. Northern blot hybridization for cellular RNA from cell lines of colon carcinoma. RNA (25 #g) were electrophoresed in 1 percent agarose gel, transferred to nitrocellulose paper, and hybridized with c-myc-specific probe. Lane 1: normal colonic epithelium, Lane 2: CC-M1, Lane 3: CC-M2, Lane 4: CC-M3. metric scanning (Fig. 3), whereas in Northern blot hybridization the amplified band of c - m y c was 2.7 kb for all cell lines. CC-M2 ceils demonstrated a 100-fold increase in genome length than the normal colonic mucosa (Fig. 4). DISCUSSION
Figure 3. Analysis of the c-myc oncogene: 10 #g of cellular DNA were digested with EcoRI, blot transferred, and hybridized with c-myc-specific probe. Lane 1: CC-M1, Lane 2: CC-M2, Lane 3: CC-M3, Lane 4: normal colonic epithelium. The Southern blot hybridization of c - m y c showed a single band of 12.6 kb and a more than fivefold increase in CC-M2 and CC-M3 cells compared with the normal colonic mucosa by densito-
HPV-16 and HPV-18 have been extensively studied in various invasive cancers < 9, 10, 17 but seldom in colorectal tumors. By using Southern blot hybridization, we found that all three colorectal cancer cell lines contained HPV-16 and HPV-18 DNA sequences, though they only presented a major band of 4.8 or 5 kb. However, normal control tissues in this study were found to be negative for HPV DNA. These results imply that an integrated or extrachromosomal HPV DNA might exist in colon cancer cells. E1 Awady e t a / . 27 described that viral DNA sequences might delete the 251 bp during integration. In addition, the integrated upstream regulatory region, E6/E7 open reading frames, and the 5' premutation of the viral polyadenylation signals might be the relative prerequisite for cervical transformation during HPV inte-
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CHENG E T A L
gration. This may suggest an intact HPV DNA sequence is not preserved after integration, though HPV DNA appears to be integrated in the majority of cancers. 7 Furthermore, it is not clear yet whether the integration of HPV genomes in chr om os ome is an essential and causative event in malignant conversion.< 9, 28 This study has demonstrated that a considerable amount of HPV-16 and 18 were associated with the colorectal carcinoma cells. Therefore, HPV DNA may be involved at least in the evolution and progression of colorectal tumors. The c-myc o n co ge ne has be e n implicated in the processes of normal cell proliferation and differentiation, i9 Alteration of c - m y c was also found in colon cell lines, 3~ and its protein product was believed to be important in the evolution of colonic neoplasia. 31 Our findings demonstrated that c-myc was amplified not only in DNA, but also in the cellular RNA of human colorectal cancer cell lines. The extent of c - m y c - g e n e amplification in cancer cells is distinctly higher (more than 100-fold) than in normal colonic mucosa (Fig. 4). This result exhibited that c-myc expression may play a particular role in cell transformation and proliferation. It is possible that more than one genetic change may be necessary to produce a tumor. 2~Thus, HPV DNA as well as cellular oncogenes, e.g., c - m y c , could be associated with colorectal tumors. Matlashewski et aL 3i have successfully transformed normal cells by using both HPV-16 DNA and activated ras gene. Besides, this papillomavirus-sequence coding near cellular oncogenes has b e e n shown to produce some cervical carcinomas. 33 Furthermore, HPV might alter the macromolecular machinery of the host cell (perhaps early in the infection), resulting in the alteration of the c- m y c oncogene, and eventually assist c-myc amplification in playing a later role in cancer development. Alitalo et al.,3~ showed that the overproduction of c - m y c mRNA could clearly result from gene amplification; this pathway is, however, unlikely to be the only mechanism by which c-myc activation can be achieved. The distinct amplification of c - m y c mRNA adds strength to this speculation. Because premalignant lesions contain both HPV16 and HPV-18 DNA 5' ~0 but not all premalignant lesions progress to invasive diseases, it is not certain whether HPV association with cancers is causal or casual. Recent reports on a high degree of association of HPV-16/18 with well-differentiated
Dis Colon Rectum, June 1991
precancerous uterine cervical lesions 7 and a much higher HPV-16 DNA expression rate in adenocarcinoma in situ than in adenocarcinoma of the uterine cervix provide further support of this idea. 34 However, all of these only suggest that HPV-16 may play a critical role in the early phase of cancer development. To understand the role of HPV DNA in colorectal carcinogenesis, HPV DNA expression in clinical specimens of colorectal tumors, both benign and malignant, is an essential issue and presently under investigation in our laboratory. In this report, colorectal cancer is claimed to be similar to the carcinogenesis of other cancers, which requires a series of steps and multiple oncogenes to transform the normal primary cells into malignancy, i~ Above all, the finding of a high degree of association of HPV-16 and HPV-18 DNA sequences with colorectal cancer suggests that HPV DNA may play a critical role in colorectal cancer development. Moreover, the increase of HPV-16 DNA, c - m y c gene copies, and c - m y c mRNA encourages us to speculate that both activated c-myc oncogene and HPV DNA sequences are involved in at least certain steps of colorectal carcinogenesis. Further investigations including mapping, cloning, transfection, and sequence determination of HPV DNA on this postulation are urgently needed. Based on this information, new modalities for diagnosis, therapy, prognostic evaluation, and even prevention of colorectal cancers may thus be developed in the future.
ACKNOWLEDGMENTS We thank Dr. K. R. Yu for his technical assistance and Ms. Jean Chuang for proofreading.
REFERENCES 1. ztir Hausen H, Meinhof W, Scheiber W, Bornkamm GW. Attempts to detect virus-specific DNA in human tumors. I. Nucleic acid hybridizations with complementary RNA of human wart virus. Int J Cancer 1974;13:650-6. 2. Maier RC, Norris HJ. Coexistence of cervical intraepithelial neoplasia with primary adenocarcinoma of the endocervix. Obstet Gynecol 1980;56:361-4. 3. Gissmann L, de Villiers E-M, zfir Hausen H. Analysis of human genital warts (condylomata acuminata) and other genital tumors for human papilloma virus type 6 DNA. IntJ Cancer 1982;29:143-6. 4. Dfirst M, Gissmann L, Ikenberg H, zfir Hausen H. A
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HPV AND c - m y c IN COLON CANCER
papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad Sci USA 1983;80:3812-5. McCance DJ, Clarkson PK, Dyson JL, Walker PG, Singer A. Human papillomavirus types 6 and 16 in multifocal intraepithelial neoplasias of the female lower genital tract. Br J Obstet Gynaecol 1985;92:1093-100. Crum CF, Mitao M, Levine RU, Silverstein S. Cervical papillomaviruses segregate with morphologically distinct precancerous lesions. J Virol 1985;54:67581. Dfirst M, Kleinheinz A, Hotz M, Gissmann L. The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours. J Gen Virol 1985;66:1515-22. Yee C, Krishnan-Hewlett I, Baker CC, Schlegel R, Howley PM. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol 1985;119:361-6. McCance DJ, Campion MJ, Clarkson PK, Chesters PM, Jenkins D, Singer A. Prevalence of human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and invasive carcinoma of the cervix. BrJ Obstet Gynaecol 1986;92:1101-5. Lorincz AT, Temple GF, Kurman RJ, Bennett Jenson A, Lancaster WD. Oncogenic association of specific human papillomavirus types with cervical neoplasia. JNCI 1987;79:671-7. Ostrow RS, Manias DA, Clark BA, Okagaki T, Twiggs LB, Faras AJ. Detection of human papillomavirus DNA in invasive carcinomas of the cervix by in situ hybridization. Cancer Res 1987;47:649-53. Tase T, Okagaki T, Clark BA, et al. Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: a study by in situ DNA hybridization. Cancer Res 1988;48:993-8. Bejui-thivolet F, Liagre N, Chignol MC, Chardonnet Y, Patricot LM. Detection of human papillomavirus DNA in squamous bronchial metaplasia and squamous cell carcinomas of the lung by in situ hybridization using biotynlated probes in paraffin-embedded specimens. Hum Pathol 1990;21:111-6. Loning T, Ikenberg H, Becket J, Gissmann L, Hoepfner I, zfir Hausen H. Analysis of oral papillomas, leukoplakias and invasive carcinomas for human papillomavirus type related DNA. J Invest Dermatol 1985;84:417-20. Brandsma JL, Steinberg BM, Abramson AL, Winkler B. Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx. Cancer Res 1986;46:2185-8.
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16. de Villiers E-M, Weidauer H, Otto H, zfir Hausen H. Papillomavirus DNA in human tongue carcinomas. Int J Cancer 1985;36:575-8. 17. Ostrow RS, Manias DA, Fong WJ, Zachow KR, Faras AJ. A survey of human cancers for human papillomavirus DNA by filter hybridization. Cancer 1987;59:429-34. 18. Lutzner MA, Orth G, Dutronquay V. Detection of human papillomavirus type 5 DNA in skin cancers of an immunosuppressed renal allograft recipient. Lancet 1983;2:422-4. 19. z/.ir Hausen HZ. Papillomaviruses in human cancer. Cancer 1987;59:1692-6. 20. Land H, Parada LF, Weinberg RA. Cellular oncogenes and multistep carcinogenesis. Science 1983; 222:771-8. 21. Alitalo K, Koskinen P, Makela TP, Saksela K, Sistonen L, Winqvist R. m y c oncogenes: activation and amplification. Biochim Biophys Acta 1987;907:1-32. 22. Yang YC, Okayama H, Howley PM. Bovine papillomavirus contains multiple transforming genes. Proc Natl Acad Sci USA 1985;82:1030-4. 23. Cheng JY, Meng CL, Lin JC, Tzeng CC, Chin LT, Shen KL. Characterization of four newly established human colorectal adenocarcinoma cell lines from Chinese patients. J surg Oncol 1990;44:260-7. 24. Gross-Bellard M, Oudet P, Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem 1973;36:32-8. 25. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-17. 26. Frazier ML, Mars W, Florine DL, Montagna RA, Saunders GF. Efficient extraction of RNA mammalian tissue. Mol Cell Biochem 1983;56:113-22. 27. E1 Awady MK, Kaplan JB, O'Brien sJ, Burk RD. Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa. Virology 1987;159:389-98. 28. McCance DJ. Human papillomaviruses and cancer. Biochim Biophys Acta 1986;823:195-205. 29. Rather L, Josephs SF, Wong-Staal F. Oncogenes: their role in neoplastic transformation. Annu Rev Microbiol 1985;39:419-49. 30. Alitalo K, Schwab M, Lin CC, Varmus HE, Bishop JM. Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc Natl Acad Sci USA 1983;80:1707-11. 31. Stewart J, Evan G, Watson J, Sikora K. Detection of the c-myc oncogene product in colonic polyps and carcinomas. Br J Cancer 1986;53:1-6. 32. Matlashewski G, Schneider J, Banks L, Jones N, Mur-
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ray A, Crawford L. Human papillomavirus type 16 DNA cooperates with activated r a s in transforming primary cells. EMBO J 1987;6:1741-6. 33. DUrst M, Croce CM, Gissmann L, Schwarz E, Huebner K. Papillomavirus sequences integrate near cel-
Dis Colon Rectum, June 1991
lular oncogenes in some cervical carcinomas. Proc Natl Acad Sci USA 1987;84:1070-4. 34. Nielsen AL. Human papillomavirus type 16/18 in uterine cervical adenocarcinoma in situ and adenocarcinoma. Cancer 1990;65:2588-93.
ChengJ-Y, Meng C-L, Chao C-F, Gau S-D, LinJ-C. Human papillomavirus type-related DNA and c-myc oncogene alterations in colon cancer cell lines. Dis Colon Rectum 1991;34:469-474. Although squamous-cell epithelium is the most frequent target site of human papillomavirus (HPV) infection, a similar infection is demonstrated in columnar epithelial cells in this paper. The papiHomavirus expression in three cell lines was detected in colorectal adenocarcinoma of Chinese patients. The HPV- 16 and HPV- 18 DNA sequences were found in colorectal cancer cell lines, which might suggest the correlation of HPV to the etiology of colorectal cancers. In addition, c-myc oncogene was identified by amplification in all three colorectal cancer cell lines, but only normal germ-line fragments were found in control tissue. The correlation between HPV and c-myc, and the implications of these findings in colorectal cancers are also discussed. [Key words: HPV; c-myc; Colon cancer] u m a n p a p i l l o m a v i r u s (HPV) DNA has b e e n d e t e c t e d in high p r o p o r t i o n s in b e n i g n tumor, precancer, and c a n c e r of the genital 1 12 and extragenital organs. 13-17 T h e extensive studies on HPV infection including c a r c i n o m a in situ, invasive cancers, and several cell lines f r o m cervical carcin o m a s suggest that this virus m a y act as a causal a g e n t rather than a casual p a s s e n g e r in carcinogenesis. Various cancers w e r e also s c r e e n e d b y m o l e c ular-hybridization analysis for the p r e s e n c e of HPV DNA, including oral cavity, 14 tongue, 16 larynx, ~5 lung, ~3 a n d skin, is and all c o n t a i n e d various a m o u n t s of HPV DNA. Although HPV m o s t l y pres e n t e d in s q u a m o u s - c e l l epithelia, r e c e n t reports
H
This study was presented at the 44th General Scientific Meeting in March 1990, Surgical Association, Taipei, Taiwan, R.O.C. This research was supported by Grant NSC 77-0609-B016-01 from the National Science Council, R.O.C. Address reprint requests to Dr. Cheng: Division of Colon and Rectal Surgery, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan, R.O.C. 469
have d e m o n s t r a t e d their related DNA in cancers of have d e m o n s t r a t e d their related DNA in cancers of c o l u m n a r e p i t h e l i u m , such as the e n d o c e r v i x 12 or c e c u m . 17 Because the c o l o n is part of gastrointestinal tract a n d the m o r p h o l o g y of colonic a d e n o m a m i m i c s that of p a p i l l o m a , the role of HPV in the colorectal c a r c i n o g e n e s i s invites further investigation. HPV-16 and HPV-18 are m o s t f r e q u e n t l y c l a i m e d to b e associated with the majority of cervical carcinoma. HPV-16 was f o u n d in 60 to 90 p e r c e n t of cervical c a n c e r tissues 4'9 w h e r e a s HPV-18 was in o n l y 17 p e r c e n t *~ of invasive cancers. T h e y s e e m to p r e f e r e n t i a l l y i n d u c e lesion with m a r k e d nuclear atypia a n d have high o n c o g e n i c association. ~9 A m o n g the k n o w n cellular o n c o g e n e s , c-myc is o n e of the m o s t f r e q u e n t l y f o u n d in various cell lines d e r i v e d f r o m h u m a n tumors. c-myc has b e e n r e c o g n i z e d as a factor involved in h u m a n m u l t i s t e p t u m o r i g e n e s i s , in that it can either b e a m p l i f i e d or rearranged. 2~ 21 Recently, Yang et aL 22 s u g g e s t e d that the b o v i n e papillomavirus c o n t a i n e d m u l t i p l e t r a n s f o r m i n g genes. We t h e r e f o r e s u s p e c t HPV m a y b e also involved in at least certain steps of colorectal carcinogenesis. T h e p r e s e n c e of HPV DNA s e q u e n c e a n d c-myc oncog e n e in colorectal t u m o r s was r e a s o n a b l y t h o u g h t to b e d e t e c t a b l e Y P-labeled HPV-16, 18, and cmyc p r o b e s w e r e e m p l o y e d to detect the expression of HPV-DNA s e q u e n c e s in the three c o l o n a d e n o c a r c i n o m a cell lines d e v e l o p e d in our laboratory. 23 T h e results of this Southern blot study indicated that h u m a n colorectal tumors, in addition to s q u a m o u s - c e l l tumors, c o n t a i n e d HPV DNA and c-myc o n c o g e n e . T h e r e f o r e , w e b e l i e v e that HPV a n d c - m y c o n c o g e n e m a y play a role in the carcin o g e n e s i s of colorectal cancers.
470
CHENG ET AL MATERIALS AND M E T H O D S Cell Lines
Three human adenocarcinoma cell lines of colon cancer (CC-M1, CC-M2, CC-M3) were used in this study. These cell lines were established from Chinese patients in our laboratoryY They were kept in RPMI-1640 RPMI medium with 10 percent fetal calf serum (GIBCO-Europe Ltd., Paisley, UK), 100 #g/ml of penicillin, and 100 #g/ml of streptomycin (Bristol Laboratories, Langley, Slough), 4 mm ]-glutamine, and 2.5 mm sodium pyruvate (all from GIBCO, Grand Island, NY), and ready to be used.
Dis Colon Rectum, June 1991
hybridized with HPV-16, HPV-18, and c-myc-oncogene probes, with specific activity ranging from 2 x 108 to 2 x 1 0 9 cpm/#g in high-stringency conditions, then washed several times at 55 ~C, and finally exposed to an x-ray film, using an autoradiographic method at -70~ RNA E x t r a c t i o n a n d N o r t h e r n B l o t
Hybridization Total cellular RNA of cell lines was extracted according to Glisin. 26 The hybridized procedures for cellular RNA were similar to that of DNA hybridization. RESULTS
Probes HPV type 16 and 18, and c - m y c p r o b e s were used in this analysis.
DNA Extraction Total cellular DNA was extracted from these cell lines, and the normal epithelium of the colon was used as a normal control by the method previously described.8, 24 Briefly, DNA was extracted and incubated with sodium dodecyl sulfate (SDS) and RNAse at 37 ~ for 3 hours. Subsequently, proteinase K was added and incubated again overnight. Cellular DNA was then extracted with p h e n o l / chloroform/iso-amylalcohol (25:24:1), ether, and dialysed with Tris-EDTA (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) buffer solution. After centrifugation with 1,000 x g at 4 ~ for 20 minutes, the DNA in the supernatant was precipitated by the addition of ethanol. Precipitated nucleic acids were then washed with 70 percent ethanol and dried in the air. The absorbance of DNA at 260 nm was determined by a DU-50 series spectrophotometer (Beckman Instruments, Fullerton, CA) and compared with the absorbance at 280 nm. The DNA absorbance ratio ranged from 1.8 to 2.0 and was stored at 4 ~C until ready for use.
DNA extracted from three colorectal adenocarcinoma cell lines was analyzed by Southern blot hybridization with EcoRI-endonuclease treatment. Under highly stringent conditions, a major HPV16-related DNA band of 4.8 kb was present in all three cancer cell lines, and was especially distinct in CC-M2 cells (Fig. 1). On EcoRI cleavage of the DNA of cancer cells showed a single band of 5 kb by HPV-18 DNA probe (Fig. 2).
Southern Blot H y b r i d i z a t i o n Filter hybridization according to a previous report was carried OUt. 4 Briefly, each DNA was cleaved with the restriction enzyme EcoRI and then electrophoresed in 1 percent agarose gel. After staining with ethidium bromide, the DNA was denatured in situ and transferred to nitrocellulose filter paper by Southern blot. 25 The filters were
Figure 1. High-stringency hybridization of DNA extracts with the HPV-16 DNA probe is shown: 10 #g of DNA were treated with EcoRI, then electrophoresed, transferred to a nitrocellulose paper, and hybridized under high-stringency conditions with a (a2P)-radiolabeled HPV-16 DNA probe. Hybridized probes around 4.8 kb are indicated in Lanes 1, 2, and 3. Lane I:CC-M1, Lane 2: CC-M2, Lane 3:CC-M3, Lane 4: normal colonic epithelium.
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HPV AND c-myc IN COLON CANCER
Figure 2. Autoradiographs of cellular DNA extracts run on 1 percent agarose gels and transferred to a nitrocellulose paper by Southern blot are shown. These DNA were digested with the restrictive endoneuclease EcoRI and hybridized a2P-radiolabeled HPV-18 DNA probe. Lane 1: CC-M1, Lane 2: CC-M2, Lane 3: CC-M3, Lane 4: normal colonic epithelium.
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Figure 4. Northern blot hybridization for cellular RNA from cell lines of colon carcinoma. RNA (25 #g) were electrophoresed in 1 percent agarose gel, transferred to nitrocellulose paper, and hybridized with c-myc-specific probe. Lane 1: normal colonic epithelium, Lane 2: CC-M1, Lane 3: CC-M2, Lane 4: CC-M3. metric scanning (Fig. 3), whereas in Northern blot hybridization the amplified band of c - m y c was 2.7 kb for all cell lines. CC-M2 ceils demonstrated a 100-fold increase in genome length than the normal colonic mucosa (Fig. 4). DISCUSSION
Figure 3. Analysis of the c-myc oncogene: 10 #g of cellular DNA were digested with EcoRI, blot transferred, and hybridized with c-myc-specific probe. Lane 1: CC-M1, Lane 2: CC-M2, Lane 3: CC-M3, Lane 4: normal colonic epithelium. The Southern blot hybridization of c - m y c showed a single band of 12.6 kb and a more than fivefold increase in CC-M2 and CC-M3 cells compared with the normal colonic mucosa by densito-
HPV-16 and HPV-18 have been extensively studied in various invasive cancers < 9, 10, 17 but seldom in colorectal tumors. By using Southern blot hybridization, we found that all three colorectal cancer cell lines contained HPV-16 and HPV-18 DNA sequences, though they only presented a major band of 4.8 or 5 kb. However, normal control tissues in this study were found to be negative for HPV DNA. These results imply that an integrated or extrachromosomal HPV DNA might exist in colon cancer cells. E1 Awady e t a / . 27 described that viral DNA sequences might delete the 251 bp during integration. In addition, the integrated upstream regulatory region, E6/E7 open reading frames, and the 5' premutation of the viral polyadenylation signals might be the relative prerequisite for cervical transformation during HPV inte-
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gration. This may suggest an intact HPV DNA sequence is not preserved after integration, though HPV DNA appears to be integrated in the majority of cancers. 7 Furthermore, it is not clear yet whether the integration of HPV genomes in chr om os ome is an essential and causative event in malignant conversion.< 9, 28 This study has demonstrated that a considerable amount of HPV-16 and 18 were associated with the colorectal carcinoma cells. Therefore, HPV DNA may be involved at least in the evolution and progression of colorectal tumors. The c-myc o n co ge ne has be e n implicated in the processes of normal cell proliferation and differentiation, i9 Alteration of c - m y c was also found in colon cell lines, 3~ and its protein product was believed to be important in the evolution of colonic neoplasia. 31 Our findings demonstrated that c-myc was amplified not only in DNA, but also in the cellular RNA of human colorectal cancer cell lines. The extent of c - m y c - g e n e amplification in cancer cells is distinctly higher (more than 100-fold) than in normal colonic mucosa (Fig. 4). This result exhibited that c-myc expression may play a particular role in cell transformation and proliferation. It is possible that more than one genetic change may be necessary to produce a tumor. 2~Thus, HPV DNA as well as cellular oncogenes, e.g., c - m y c , could be associated with colorectal tumors. Matlashewski et aL 3i have successfully transformed normal cells by using both HPV-16 DNA and activated ras gene. Besides, this papillomavirus-sequence coding near cellular oncogenes has b e e n shown to produce some cervical carcinomas. 33 Furthermore, HPV might alter the macromolecular machinery of the host cell (perhaps early in the infection), resulting in the alteration of the c- m y c oncogene, and eventually assist c-myc amplification in playing a later role in cancer development. Alitalo et al.,3~ showed that the overproduction of c - m y c mRNA could clearly result from gene amplification; this pathway is, however, unlikely to be the only mechanism by which c-myc activation can be achieved. The distinct amplification of c - m y c mRNA adds strength to this speculation. Because premalignant lesions contain both HPV16 and HPV-18 DNA 5' ~0 but not all premalignant lesions progress to invasive diseases, it is not certain whether HPV association with cancers is causal or casual. Recent reports on a high degree of association of HPV-16/18 with well-differentiated
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precancerous uterine cervical lesions 7 and a much higher HPV-16 DNA expression rate in adenocarcinoma in situ than in adenocarcinoma of the uterine cervix provide further support of this idea. 34 However, all of these only suggest that HPV-16 may play a critical role in the early phase of cancer development. To understand the role of HPV DNA in colorectal carcinogenesis, HPV DNA expression in clinical specimens of colorectal tumors, both benign and malignant, is an essential issue and presently under investigation in our laboratory. In this report, colorectal cancer is claimed to be similar to the carcinogenesis of other cancers, which requires a series of steps and multiple oncogenes to transform the normal primary cells into malignancy, i~ Above all, the finding of a high degree of association of HPV-16 and HPV-18 DNA sequences with colorectal cancer suggests that HPV DNA may play a critical role in colorectal cancer development. Moreover, the increase of HPV-16 DNA, c - m y c gene copies, and c - m y c mRNA encourages us to speculate that both activated c-myc oncogene and HPV DNA sequences are involved in at least certain steps of colorectal carcinogenesis. Further investigations including mapping, cloning, transfection, and sequence determination of HPV DNA on this postulation are urgently needed. Based on this information, new modalities for diagnosis, therapy, prognostic evaluation, and even prevention of colorectal cancers may thus be developed in the future.
ACKNOWLEDGMENTS We thank Dr. K. R. Yu for his technical assistance and Ms. Jean Chuang for proofreading.
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