Jian-Min Yang, Rong-Quan Wang, Bao-Guo Bu, Zi-Cheng Zhou, Dian-Chun Fang and Yuan-Hui Luo, male, born on 1956-06-21 in Linhai, Zhejiang Province, graduated from Third Military Medical University in 1983, earned doctor degree in 1993, now associate professor and tutor of postgraduates, mainly engaged in the basic and clinical research on hepatocellular carcinoma, as first and second or third author having 30 and 20 papers published, co-edited 3 monographs, which were awarded both the 2nd and 3rd Class prizes for scientifi c and technological progress of the PLA.
*Supported by the PLA Eighth 5-year Scientific Research Fund for Youths.
Correspondence to: Prof Jian-Min Yang. Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Telephone: +86-23-68754124(O), 68754270(H)
Received: 1998-09-17

Subject headings: carcinoma; hepatocellular/etiology; hepatit is C-like viruses/pathogenicity; oncogenes/genetics; genes, suppressor; tumor/genetics; immunohistochemistry/methods

Yang JM, Wang RQ, Bu BG, Zhou ZC, Fang DC and Luo YH. Effect of HCV infection on expression of several cancer-associated gene products in HCC. World J Gastroentero,1999;5(1):25-27

Abstract
AIM: To study hepatocarcinogenesis of hepatitis C virus (HCV).

METHODS: Expression of HCV antigens (CP10, NS3 and NS5) and sev eral cancer-associated gene products (ras p21, c-myc,c-erbB-2, muta_ted p53 and p16 protein) in the tissues of hepatocellular carcinoma (HCC, n=46) and its surrounding liver tissue were studied by the ABC (avidin-biotin complex) immu nohistochemical method. The effect of HCV infection on expression of those gene products in HCC was analyzed by comparing HCV antigen-positive group with HCV antigen negative group.

RESULTS: Positive immunostaining with one, two or three HCV antigens was found in 20 (43.5%) cases, with either of two or three HCV antigens in 16 (34.8%) cases, and with three HCV antigens in 9 (19.6%) cases. Deletion rate of p16 protein expression in HCC with positive HCV antigen (80%, 16/20) was significantly higher than that in HCC with negative HCV antigen. Wh ere as no significant difference of the other gene product expression was observed between the two groups.

CONCLUSION: HCV appears related to about one-third of cases of HCC in Chongqing, the southwest of China, and it may be involved in hepatocar cinogen  esis by inhibiting the function of p16 gene, which acts as a negative regulator of cell cycle.

INTRODUCTION
Our previous studies by seroepidemiological, mole_cular epidemiological and immunopathological methods have revealed that hepatitis C virus (HCV) infection i s closely linked to development of hepatocellular carcinoma (HCC) and HCV may be the se_cond important factor in association with HCC-etio_logy in Chongqing , the southwest of China^［1-5］. But the molecular mechanisms involved in hepatocar cinogenesis of HCV remain poorly understood. Up to now, many authors believe that HCV can not directly change the structure of the host genes like hepatitis B virus by integration because HCV is a RNA virus. Therefore, the effect of HCV on factors of controlling cell growth and development is an important field in the hepatocarcinogenesis stu_dies. In this study, expression of several oncogene and tumor suppressor gene products in HCV-associated and non-HCV-associa ted HCC was investiga_ted, so as to identify if HCV infection can affect expression of these gene products.

MATERIALS AND METHODS
Specimens
HCC specimens of 46 cases were randomly selected from partial hepatectomy in 199 4 in this hospital. Of them, 38 cases contained pericancerous liver tissues. All specimens were fixed in 100mL/L-formalin, embedded in paraffin and sequentially sectioned with a thickness of 5μm.

Reagents
Mouse monoclonal antibodies (mAb) to HCV NS3 and NS5 were kindly provided by Pr ofessor TAO Qi-Min (The Institute of Hepatology, Beijing Medical University). M ouse mAb against HCV CP10 was kindly presented by Professor LI Meng-Dong (Depa rtment of Infectious Diseases, the Southwest Hospital). Mouse mAbs to human ras p21, C-myc, C-erbB-2 and mutated p53 protein were purchased from Fuzhou Maxi m Biotechnical Company. Mouse McAb to human p16 protein was purchased from Beiji ng Zhongshan Biotechinical Company. Avidin-biotin complex (ABC) kits were purch ased from Fuzhou Maxim Biotechnical Company and Vector company.

Immunostaining
Immunostaining of HCV antigens CP10, NS3, NS5 and cancer-associated gene produc ts ras p21, c-myc, c-erbB-2, mutated p53 and p16 proteins was performed by the ABC method in each case. The procedures of ABC staining were taken according to the manufacturer′s recommendations as previously described^［5］. The color was developed with diamino-benzidine and hematoxylin. Positive and negative con trols were simultaneously used to ensure specifity and reliability of the staini ng.

RESULTS
Expression of HCV antigens
In the 46 cases of HCC, positive HCV antigen was found in 20 (43.5%) cases, of which 4 cases with one positive HCV antigen, 7 cases with two positive HCV antig ens, and 9 cases with three positive HCV antigens. The positive stainin g of HCV antigen CP10, NS3 and NS5 in the cancer tissues was observed in 10 (21.7%), 10 (21.7%) and 7 (15.2%) cases, respectively, while in its pericancerous liver tissues in 14 (36.8%), 13 (34.2%) and 12 (31.6%) cases. Although the expression rates were higher in the pericancerous tissues than in the cancer tissues, no statistical significance was obtained (P＞0.05) (Table 1). The immunostaining of each HCV antigen was mainly seen in HCC and hepatocyte cytoplasms, seldom in the cell membranes, none in the nuclei. The positive-staining cells were distributed mostly in scattered or focalized patterns, seldom in diffused patter n.

Table 1 Expression of HCV antigens in HCC tissue and its surrounding liver tissue (%, positive rate)

The effect of HCV infection on expression of the gene products
On the one hand, positive rates of ras p21 and mutated p53 in HCC (58.7%, 27/46; 28.3%, 13/46) were significantly higher than in the pericancero us tissues (34.2%, 13/38; 7.9%, 3/38, P＜0.05), whereas the positive rate of p16 in HCC (41.3%, 19/46) was significantly lower than in the peric ancerous tissues (63.2%, 24/38, P＜0.05). But the expression rates of c -myc and c-erbB-2 did not show significant difference between the cancer and pericancerous groups (P＞0.05). On the other hand, it attracted our attention that the positive rate of P16 protein in HCV antigen-positive HCC(20%,4/20)significantly lower than in HCV antigen- negati ve HCC (57.7%, 15/26,P＜0.025), even though the expression rates of ras p21, C-myc, C-erbB-2 and mutated p53 showed no significant difference between HCV-associated and non HCV-associated HCC (Table 2).

Table 2 Relationship of HCV antigens with expression of cancer-asso ciated gene products(CAGP)(n, positive cases)

DISCUSSION
In the previous studies, we found that HCV RNA could be detected in 36.6% (34/93) serum samples of patients with primary hepatic carcinoma and 37.5% (21/56) cases of HCC tissues^［1,3］. In this study, using three McAbs to dif ferent HCV antigens and immunohistochemical ABC method, we found that the positive immunostaining with either one, two or three HCV antigens was found in 20 (43.5%) cases, with either two or three HCV antigens in 16 (34.8%) cases and with three HCV antigens in 9 (19.6%) cases among the 46 cases of HCC. The present data are consistent with our previous studies and further indicate that about one-third of HCC seems to be related to HCV infection in Chongqing, the southwest of China. Up to now, a lot of affirmative evidences in seroepidemi ology, molecular epidemiology and immunopathology have been obtained concerning the association of HCV infection with HCC development in this area.
      Recent studies have shown that the molecular mechanisms of hepatocarcinogenesis are involved in oncogene activation and anti-oncogene inactivation like many other tumors. The role of ras, c-myc, c-erbB-2, p53 and p16 gene in the development and progression of HCC have been noted by many workers. To understand the potential hepatocarcinogenesis of HCV, we studied the expression of these gene products in HCV-associated and non-HCV associated HCC tissues. The results showed that the expression of ras p21, c-myc, c-erbB-2 and mutat ed p53 was not significantly different between HCV antigen-positive and HCV-ant igen- negative groups, but the deletion rate of p16 protein expression in HCV antigen-positive HCC (80%, 16/20) was significantly higher than in HCV antige n-negative HCC (42.3%, 11/26, P＜0.025). It implicates that the molecu lar mechanisms involved in HCV hepatocarcinogenesis seems to be connected with the repression of p16 gene function.
      The p16 gene is a new negative regulator of cell cycle and tumor supressor gene found recently, which is located in chromosome 9p21 with 8.5kb long and encoding for a nucleus phosphoprotein with 16kD-P16 protein. P16 protein can bind to cycle dependent kinase 4 (CDK4), preventing their interaction with cyclin D and thereby preventing cell cycle progression from G1 to S phase. Many authors proposed that when p16 gene function is repressed, the activity of cyclin D/CDK4 complex will increase because of the CDK4 being free from the inhibition of P16 protein, thereafter cell proliferation will be out of control and tumor may develop at last^［6,7］. Recently, Ray et al reported that HCV core protein can act as an effector in the promotion of cell growth by repression trancription of the another negative regulator of cell cycle and inhibitor of cyclin D/CDK4 complex p21 (WAF1/Cip1/Sid1) gene thr ough unknown cellular factors^［8］. Therefore, the role of p16 gene in molecular mechanisms of HCV hepatocarcinogenesis deserves further studies.

ACKNOWLEDGEMENT We thank Professor TAO Qi Min for kindly providing the HCV, NS3, NS5, mAbs and Professor LI Meng-Dong for kindly providing the HCV CP10 mAb.

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