Abstract
Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver‐enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBPα using real‐time reverse‐transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B‐17/Icrj‐scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF‐3β, HNF‐4α, HNF‐1α, and C/EBPα were specific to HCCs with well‐differentiated function and morphology. Furthermore, among these four transcription factors, HNF‐4α and HNF‐1α expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF‐4α and HNF‐1α are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBPα in HCCs.
References
- 1. Maxwell Parkin D. Global cancer statistics in the year 2000. Lancet Oncol 2001; 2: 533–43. [DOI] [PubMed] [Google Scholar]
- 2. Lerose R, Molinari R, Rocchi E, Manenti F, Villa E. Prognostic features and survival of hepatocellular carcinoma in Italy: impact of stage of disease. Eur J Cancer 2001; 37: 239–45. [DOI] [PubMed] [Google Scholar]
- 3. Pietrangele A, Shafritz DA. Gene expression during pathophysiologic states. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, editors The liver: biology and pathobiology. Third ed. New York : Raven Press Ltd; 1994. p. 85–95. [Google Scholar]
- 4. Courtois G, Morgan JG, Campbell LA., Fourel G, Crabtree GR. Interaction of a liver‐specific nuclear factor with the fibrinogen and α1‐antitrypsin promoters. Science 1987; 238: 688–92. [DOI] [PubMed] [Google Scholar]
- 5. Cereghini S, Blumenfeld M, Yaniv M. A liver‐speciflc factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes & Dev 1988; 2: 957–74. [DOI] [PubMed] [Google Scholar]
- 6. Hardon E, Frain M, Paonessa G, Cortese R. Two distinct factors interact with the promoter regions of several liver specific genes. EMBO J 1988; 7: 1711–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Costa RH, Grayson DR, Darnell JE Jr. Multiple transthyretin and α1‐anti trypsin genes. Mol Cell Biol 1989; 9: 1415–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Liang X, Lijian H, Shaohui W, Juakeu G, Ying J, Yanping W, Yuan J, Kin W, Zheguang H, Gengxi H. Expression profiling suggested a regulatory role of liver‐enriched transcription factors in human hepatocellular carcinoma. Cancer Res 2001; 61: 3176–81. [PubMed] [Google Scholar]
- 9. Kalkuhl A, Kaestner K, Buchmann A, Schwarz M. Expression of hepatocyte‐enriched nuclear transcription factors in mouse liver tumours. Carcinogenesis 1996; 17: 609–12. [DOI] [PubMed] [Google Scholar]
- 10. Flodby P, Liao DZ, Blanck A, Xanthopoulos KG, Hallstrom IP. Expression of the liver‐enriched transcription factors C/EBP alpha, C/EBP beta, HNF‐1 and HNF‐4 in preneoplastic nodules and hepatocellular carcinoma in rat liver. Mol Carcinog 1995; 12: 103–9. [DOI] [PubMed] [Google Scholar]
- 11. Wang W, Hayashi Y, Ninomiya T, Ohta K, Nakabayashi H, Tamaoki T, Itoh H. Expression of HNF‐1 alpha and HNF‐1 beta in various histological differentiations of hepatocellular carcinoma. J Pathol 1998; 184: 272–8. [DOI] [PubMed] [Google Scholar]
- 12. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 1982; 42: 3858–63. [PubMed] [Google Scholar]
- 13. Nakabayashi H, Taketa K, Yamane T, Miyazaki M, Miyano K, Sato J. Phenotypical stability of a human hepatoma cell line, HuH‐7, in long‐term culture with chemically defined medium. Gann 1984; 75: 151–8. [PubMed] [Google Scholar]
- 14. Alexander JJ, Bey EM, Geddes EW, Lecatsas G. Establishment of a continuously growing cell line from primary carcinoma of the liver. S Afr Med J 1976; 50: 2124–8. [PubMed] [Google Scholar]
- 15. Yano H, Kojiro M, Nakashima T. A new human hepatocellular carcinoma cell line (KYN‐1) with a transformation to adenocarcinoma. In Vitro Cell Dev Biol 1986; 22: 637–46. [DOI] [PubMed] [Google Scholar]
- 16. Haramaki M, Yano H, Fukuda K, Momosaki S, Ogasawara S, Kojiro M. Expression of CD44 in human hepatocellular carcinoma cell lines. Hepatology 1995; 21: 1276–84. [PubMed] [Google Scholar]
- 17. Yano H, Maruiwa M, Murakami T, Fukuda K, Ito Y, Sugihara S, Kojiro M. A new human pleomorphic hepatocellular carcinoma cell line, KYN‐2. Acta Pathol Jpn 1988; 38: 953–66. [DOI] [PubMed] [Google Scholar]
- 18. Murakami T, Maruiwa M, Fukuda K, Kojiro M, Tanaka M, Tanaka M, Tanikawa K. Characterization of a new human hepatoma patient [Abstract]. Proceedings of the 47th annual meeting. Jpn J Cancer Res( Gann ) 1988; 79: 292. [Google Scholar]
- 19. Osada T, Sakamoto M, Ino Y, Iwamatsu A, Matsuno Y, Muto T, Hirohashi S. E‐Cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma. Hepatology 1996; 24: 1460–7. [DOI] [PubMed] [Google Scholar]
- 20. Hirohashi S, Shimosato Y, Kameya T, Koide T, Mukojima T, Taguchi Y, Kageyama K. Production of alpha‐fetoprotein and normal serum proteins by xenotransplanted human hepatomas in relation to their growth and morphology. Cancer Res 1979; 39: 1819–28. [PubMed] [Google Scholar]
- 21. Shimoyama Y, Gotoh M, Ino Y, Sakamoto M, Kato K, Hirohashi S. Characterization of high‐molecular‐mass forms of basic fibroblast growth factor produced by hepatocellular carcinoma cells: possible involvement of basic fibroblast growth factor in hepatocarcinogenesis. Jpn J Cancer Res 1991; 82: 1263–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Noguchi M, Hirohashi S. Cell lines from non‐neoplastic liver and hepatocellular carcinoma tissue from a single patient. In Vitro Cell Dev Biol-Animal 1996; 32: 135–7. [DOI] [PubMed] [Google Scholar]
- 23. Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB. Controlled synthesis of HBsAg in a differentiated human liver carcinoma‐derived cell line. Nature 1979; 282: 615–6. [DOI] [PubMed] [Google Scholar]
- 24. Beurton F, Bandyopadhyay U, Dieumegard B, Barouki R, Aggerbeck M. Delineation of the insulin‐responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor‐3 and nuclear factor I. Biochem J 1999; 343: 687–95. [PMC free article] [PubMed] [Google Scholar]
- 25. Zvibel I, Fiorino AS, Brill S, Reid LM. Phenotypic characterization of rat hepatoma cell lines and lineage‐specific regulation of gene expression by differentiation agents. Differentiation 1998; 63: 215–23. [DOI] [PubMed] [Google Scholar]
- 26. Tomizawa M, Watanabe K, Saisho H, Nakagawara A, Tagawa M. Down‐regulated expression of the CCAAT/enhancer binding protein alpha and beta genes in human hepatocellular carcinoma: a possible prognostic marker. AnticancerRes 2003; 23: 351–4. [PubMed] [Google Scholar]