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Comparison of protein expression patterns between hepatocellular carcinoma cell lines and a hepatoblastoma cell line

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Abstract

Hepatocellular carcinoma (HCC) and hepatoblastoma (HB) are malignancies of the liver with different etiologies, but the HB cell line HepG2 has been frequently used in various studies of HCC. In this study, we compare the protein expression patterns between HepG2 cells and three HCC cell lines, HKCI-2, HKCI-3, and HKCI-4, respectively. The cell lysates of individual cell lines were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots in the gel images were quantified and compared by image analysis software. The differentially expressing proteins were then identified by tryptic peptide, mass fingerprinting. Compared with the HepG2 cells, the normalized quantities of 49 and 58 protein spots were found to be at least twofold higher and twofold lower, respectively, in all three HCC cell lines. The differentially expressed proteins can be grouped into structural proteins (annexins, transgelin, laminin receptor), stress-induced proteins (HSP27, 60, and 70), enzymes (aldehyde dehydrogenase, pyruvate kinase, α-enolase, ect.), and transcription factors (far upstream element binding protein 2, GTP-binding nuclear protein RAN). Some of these proteins play important roles in regulating homeostasis, drug resistance, apoptosis, cell differentiation, cell growth, and metastasis. In conclusion, our proteomic data indicate that there are considerable differences in the protein expression patterns between HepG2 cells and the HCC cells, suggesting differences in cellular properties. Hence, HepG2 may not be a good cell line model for studying HCC.

References

  1. 1.

    Ferlay, J., Bray, F., Pisani, P., and Parkin, D.M. (2001). GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalance Worldwide, version 1.0, ed. 1.0. IARC Press, Lyon, France.

  2. 2.

    Stuver, S.O. (1998). Towards global control of liver cancer. Semin. Cancer Biol. 8:299–306.

  3. 3.

    Schnater, J.M., Kohler, S.E., Lamers, W.H., von Schweinitz, D., and Aronson, D.C. (2003). Where do we stand with hepatoblastoma? A review. Cancer 98:668–678.

  4. 4.

    DeBaun, M.R. (1998). Risk of cancer during the first four years of life in children from The Beckwith-Wiedemann Syndrome Registry. J. Pediatr. 132:398–400.

  5. 5.

    Giardiello, F.M., Offerhaus, G.J., Krush, A.J., Booker, S.V., Tersmette, A.C., Mulder, J.W., et al. (1991). Risk of hepatoblasma in familial adenomatous polyposis. J. Pediatr. 119:766–768.

  6. 6.

    Ovid MEDLINE(R) Database 1966 to October Week 1 2004. Available at: http://gateway.ut.ovid.com/gw1. Accessed October 12, 2004.

  7. 7.

    Pang, E., Wong, N., Lai, P.B.S., To, K.F., Lau, W.Y., and Johnson, P.J. (2002). Consistent chromosome 10 arrangements in four newly established human hepatocellular carcinoma cell lines. Genes Chromosomes Cancer 33:150–159.

  8. 8.

    Zhang, W. and Chait B. T. (2000). ProFound: an expert system for protein identification using mass spectrometric peptide mapping information. Anal. Chem. 72:2482–2489.

  9. 9.

    Seow, T.K., Ong, S.E., Liang, R.C., Ren, E.C., Chan, L., Ou, K., et al. (2000). Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated protein by mass spectrometry. Electrophoresis 21:1787–1813.

  10. 10.

    Han, J., Yoo, H.Y., Choi, B.H., and Rho, H.M. (2000) Selective transcriptional regulations in the human liver cell by hepatitis B viral X protein. Biochem. Biophys. Res. Commun. 272:525–530.

  11. 11.

    Liang, R.C., Neo, J.C., Lo, S.L., Tan, G.S., Seow, T.K., and Chung, M.C. (2002). Proteome database of hepatocellular carcinoma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 774:303–328.

  12. 12.

    Ou, K., Seow, T.K., Liang, R.C., Ong, S.E., and Chung, M.C. (2001). Proteome analysis of a human hepatocellular carcinoma cell line, HCC-M: an update. Electrophoresis 22:2804–2811.

  13. 13.

    Kang, D.G., Ha, N.Y., and Ahn, J.S. (2002). Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins. Electrophoresis 23:4142–4156.

  14. 14.

    Yu, L.R., Zeng, R., Shao, X.X., Wang, N., Xu, Y.H., and Xia, Q.C. (2000). Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry. Electrophoresis. 21:3058–3068.

  15. 15.

    Park, S.G., Lee, S.M., and Jung, G. (2003). Antisense oligodeoxynucleotides targeted against molecular chaperonin Hsp60 block human hepatitis B virus replication. J. Biol. Chem. 10:39851–39857.

  16. 16.

    Schneider, J., Jimenez, E., Marenbach, K., Romero, H., Marx, D., and Meden, H. (1999). Immunohistochemical detection of HSP60-expression in human ovarian cancer. Correlation with survival in a series of 247 patients. Anticancer Res. 19:2141–2146.

  17. 17.

    Yin, Y., Qin, Q., Zhang, W., Zhao, J., Zhang, C., and Yu, J. (2001). Overexpression of heat shock protein 70 and spontaneous cancer cell apoptosis in hepatocellular carcinoma. Zhonghua Gan Zang Bing Za Zhi 9:84–85.

  18. 18.

    Chuma, M., Sakamoto, M., Yamazaki, K., Ohta, T., Ohki, M., Asaki, M., et al. (2003). Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology 37:198–207.

  19. 19.

    Zheng, D.L., Peng, B.W., Huang, Q.L., and Lin, J.Y. (2003). Expression of 67kD laminin receptor in human hepatocellular carcinoma cells [Article in Chinese]. Ai Zheng 22:248–252.

  20. 20.

    Seow, T.K., Ong, S.E., Liang, R.C., Ren, E.C., Chan, L., Ou, K., et al. (2000) Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry. Electrophoresis 21:1787–1813.

  21. 21.

    Liu, J.B. (1990). M2-type pyruvate kinase in the diagnosis of hepatocarcinoma—a pilot study [Article in Chinese]. Zhonghua Zhong Liu Za Zhi 12:166–169.

  22. 22.

    Heinz, S., Krause, S.W., Gabrielli, F., Wagner, H.M., Andressen, R., and Rehli, M. (2002). Genomic organization of the human gene HEP27: alternative promoter usage in HepG2 cells and monocyte-derived dendritic cells. Genomics 79:608–615.

  23. 23.

    Suto, K., Kajihara-Kano, H., and Yokoyama, Y. (1999). Decreased expression of the peroxisomal bifunctional enzyme and carbonyl reductase in human hepatocellular carcinomas. J. Cancer Res. Clin. Oncol. 125:83–88.

  24. 24.

    Buendia, M.A. (2002) Genetic alterations in hepatoblastoma and hepatocellular carcinoma: common and distinctive aspects. Med. Pediatr. Oncol. 39:530–535.

  25. 25.

    Wong, N., Lai, P., Pang, E., Leung, T.W., Lau, J.W., and Johnson P.J. (2000). A comprehensive karyotypic study on human hepatocellular carcinoma by spectral karyotyping. Hepatology 32:1060–1068.

  26. 26.

    Nishihara, J.C. and Champion, K.M. (2002). Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis 14:2203–2215.

  27. 27.

    Giometti, C.S., Gemmell, M.A., Tollaksen, S.L., and Taylor, J. (1991). Quantitation of human leukocyte proteins after silver staining: a study with two-dimensional electrophoresis. Electrophoresis 12:536–543.

  28. 28.

    White, I.R., Pickford, R., Wood, J., Skehel, J.M., Gangadharan, B., and Cutler, P. (2004). A statistical comparison of silver and SYPRO Rudy staining for proteome analysis. Electrophoresis 25:3048–3054.

  29. 29.

    Ludwig, A., Dietel, M., and Lage, H. (2002). Identification of differentially expressed genes in classical and atypical multidrug-resistant gastric carcinoma cells. Anticancer Res. 22: 3213–3221.

  30. 30.

    Chung, Y.M., Park, S., Park, J.K., Kim, Y., Kang, Y., and Yoo, Y.D. (2000). Establishment and characterization of 5-fluorouracil-resistant gastric cancer cells. Cancer Lett. 16:95–101.

  31. 31.

    Schneider, J., Jimenez, E., Marenbach, K., Marx, D., and Meden, H. (1998). Co-expression of the MDR1 gene and HSP27 in human ovarian cancer. Anticancer Res. 18:2967–2974.

  32. 32.

    Concannon, C.G., Gorman, A.M., and Samali, A. (2003). On the role of Hsp27 in regulating apoptosis. Apoptosis 8:61–70.

  33. 33.

    Takayama, S., Reed, J.C., and Homma, S. (2003). Review: Heat-shock proteins as regulators of apoptosis. Oncogene 8:9041–9047.

  34. 34.

    Kapranos, N., Kominea, A., Konstantinopoulos, P.A., Sayva, S., Artelaris, S., Vandoros, G., et al. (2002). Expression of the 27-kDa heat shock protein (HSP27) in gastric carcinomas and adjacent normal, metaplastic, and dysplastic gastric mucosa, and its prognostic significance. J. Cancer Res. Oncol. 128:426–432.

  35. 35.

    Lemieux, P., Oesterreich, S., and Lawrence, J.A. (1997). The small heat shock protein hspa27 increases invasiveness but decreases motility of breast cancer cells. Invasion Metastasis 17:113–123.

  36. 36.

    Um, J.H., Kang, C.D., Hwang, B.W., Ha, M.Y., Hur, J.G., Kim, D.W., et al. (2003). Involvement of DNA-dependent protein kinase in regulation of the mitochondrial heat shock proteins. Leuk. Res. 27:509–516.

  37. 37.

    Samadi, A., Cai, J., Zhivotovsky, B., Jones, D.P., and Orrenius, S. (1999). Presence of a preapoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells. EMBO J. 15:2040–2048.

  38. 38.

    Johnsson, A., Zeelenberg, I., Min, Y., Hilinski, J., Berry, C., Howell, S.B., et al. (2000_. Identification of genes differentially expressed in association with acquired cisplatin resistance. Br. J. Cancer 83:1047–1054.

  39. 39.

    Inoue, Y., Sato, Y., Nishimura, M., Seguchi, M., Zaitsu, Y., Yamada, K., et al. (1999). Heat-induced drug resistance is associated with increased expression of Bcl-2 in HL60. Anticancer Res. 19:3989–3992.

  40. 40.

    Ravagnan, L., Gurbuxani, S., Susin, S.A., Maisse, C., Daugas, E., Zamzami, N., et al. (2001). Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat. Cell Biol. 3: 839–843.

  41. 41.

    Deng, X.L., Chen, W., Cai, M.Y., and Wei, D.P. (2003). Expression of class I MHC molecule, HSP70 and TAP in human hepatocellular carcinoma. World J Gastroenterol 9:1853–1855.

  42. 42.

    Hendrick, J.P. and Hart, F.U. (1993). Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62:349–384.

  43. 43.

    Schlesinger, M.J. (1990). Heat shock proteins. J. Biol. Chem. 265:12111–12114.

  44. 44.

    Shi, Y., Zhai, H., Wang, X., Wu, H., Ning, X., Han, Y., et al. (2002). Multidrug-resistance-associated protein MGr1-Ag is identical to the human 37-kDa laminin receptor precursor. Cell. Mol. Life Sci. 59:1577–1583.

  45. 45.

    Dell Gaspera, B., Braut-Boucher, F., Bomsel, M., Chatelet, F., Guguen-Guillouzo, C., Font, J., et al. (2001). Annexin expressions are temporally and spatially regulated during rat hepatocyte differentiation. Dev. Dyn. 222:206–217.

  46. 46.

    Maxwell, P.J., Longley, D.B., Latif, T., Boyer, J., Allen, W., Lynch, M., et al. (2003). Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling. Cancer Res. 63:4602–4606.

  47. 47.

    Cole, S.P., Pinkoski, M.J., Bhardwaj, G., and Deeley, R.G. (1992). Elevated expression of annexin II (lipocortin II, p36) in a multidrug resistant small cell lung cancer cell line. Br. J. Cancer 65:498–502.

  48. 48.

    Chiang, Y., Rizzino, A., Sibenaller, Z.A., Wold, M.S., and Vishwanatha, J.K. (1999). Specific down-regulation of annexin II expression in human cells interferes with cell proliferation. Mol. Cell. Biochem. 199:139–147.

  49. 49.

    Wang, W., Xu, J., and Kirsch, T. (2003). Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis. Biol. Chem. 278:3762–3769.

  50. 50.

    Liu, J.W., Shen, J.J., Tanzillo-Swarts, A., Bhatia, B., Maldonado, C.M., Person, M.D., et al. (2003). Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration. Oncogene 22:1475–1485.

  51. 51.

    Shields, J.M., Rogers-Graham, K., and Der, C.J. (2002). Loss of transgelin in breast and colon tumors and in RIE-1 cells by Ras de-regulation of gene expression through Raf-independent pathways. Biol. Chem. 277: 9790–9799.

  52. 52.

    Tsujimoto, Y. and Shimizu, S. (2002). The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 84: 187–193.

  53. 53.

    Feo, S., Arcuri, D., Piddini, E., Passantino, R., and Giallongo, A. (2000). ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1). FEBS Lett. 473:47–52.

  54. 54.

    Yang, X.W., Wang, W., Fu, J.X., Cen, J.N., Guo, F., Xia, X.M., et al. (2002). Aldehyde-dehydrogenase gene-transduced hematopoietic cell line K562 overcomes the cytoxicity of cyclophoshamide in vitro. Zhongguo Shi Yan Xue Ye Xue Za Zhi 10:205–208.

  55. 55.

    Kato, S., Tajiri, T., Matsukura, N., Matsuda, N., Taniai, N., Mamada, H., et al. (2003). Genetic polymorphisms of aldehyde dehydrogenase 2, cytochrome p450 2E1 for liver cancer risk in HCV antibody-positive japanese patients and the variations of CYP2E1 mRNA expression levels in the liver due to its polymorphism. Scand. J. Gatroenterol. 38:886–893.

  56. 56.

    Takeshita, T., Yang, X., Inoue, Y., Sato, S., and Morimoto, K. (2000). Relationship between alchohol drinking, ADH2 and ALDH2 genotypes, and risk for hepatocellular carcinoma in Japanese. Cancer Lett. 149:69–76.

  57. 57.

    Durany, N., Joseph, J., Jimenez, O.M., Climent, F., Fernandez, P.L., Rivera, F., et al. (2000). Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase, creatine kinase and enolase activity and isoenzymes in breast carcinoma. Br. J. Cancer 82:20–27.

  58. 58.

    Durany, N., Joseph, J., Campo, E., Molina, R., and Carreras, J. (1997). Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymes in lung, colon and liver carcinomas. Br. J. Cancer 75: 969–977.

  59. 59.

    Durany, N., Joseph, J., Cruz-Sanchez, F.F., and Carreras, J. (1997). Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and creatine kinase activity and isoenzymes in human brain tumours. Br. J. Cancer 76: 1139–1149.

  60. 60.

    Yeung, K., Seitz, T., Li, S., Jonosch, P., McFerran, B., Kaiser, C., et al. (1999). Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 401:173–177.

  61. 61.

    Yoo, B.C., Ku, J.L., Hong, S.H., Shin, Y.K., Park, S.Y., Kim, H.K., et al. (2004). Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines. Int. J. Cancer 108:532–539.

  62. 62.

    Boopathi, E., Anandatheerthavarada, H.K., Bhagwat, S.V., Biswas, G., Fang, J.K., and Avadhani, N.G. (2000). Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs. Biol. Chem. 275:34415–34423.

  63. 63.

    Donadel, G., Garzelli, C., Frank, R., and Gabrielli, F. (1991). Identification of a novel nuclear protein synthesized in growth-arrested human hepatoblastoma HepG2 cells. Eur. J. Biochem. 195:72372–72379.

  64. 64.

    Gabrielli, F., Donadel, G., Bensi, G., Heguy, A., and Melli, M. (1995). A nuclear protein, synthesized in growth-arrested human hepatoblastoma cells, is a novel member of the short-chain alcohol dehydrogenase family. Eur. J. Biochem. 232:473–477.

  65. 65.

    Yamagata, M., Mori, M., Begum, N.A., Shibuta, K., Shimoda, K., and Barnard, G.F. (1998). Glyceraldehyde-3-phosphate dehydrogenase mRNA expression in hepatocellular carcinoma. Int. J. Oncol. 12:677–683.

  66. 66.

    Gong, Y., Cui, L., and Minuk, G.Y. (1996). Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28s-ribosomal RNA gene expression in human hepatocellular carcinoma. Hepatology 23:734–737.

  67. 67.

    Castaldo, G., Calcagno, G., Sibillo, R., Cuomo, R., Nardone, G., Castellano, L., et al. (2000). Quantitative analysis of aldolase A mRNA in liver discriminates between hepatocellular carcinoma and cirrhosis. Clin. Chem. 46:901–906.

  68. 68.

    Bischoff, F.R. and Ponstingl, H. (1991). Mitotic regulator protein RCC1 is complexed with a nuclear ras-related polypeptide. Proc. Natl. Acad. Sci. USA 88:10830–10834.

  69. 69.

    Milano, J. Jr. and Strayer, D.S. (1998). Effects of overexpression of Ran/TC4 mammalian cells in vitro. Exp. Cell Res. 239:31–39.

  70. 70.

    Daniel, R., Chung, S.W., Eisenstein, T.K., Sultzer, B.M., and Wong, P.M. (2001). Specific association of Type I c-Abl with Ran GTPase in lipopolysaccharide-mediated differentiation. Oncogene 20:2618–2625.

  71. 71.

    Hendrick, J.P. and Hart, F.U. (1993). Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62:349–384.

  72. 72.

    Menard, S., Tagliabue, E., and Colnaghi, M.I. (1998). The 67-kDa laminin receptor as a prognostic factor in human cancer. Breast Cancer Res. Treat. 52:137–145.

  73. 73.

    Frohlich, M., Motte, P., Galvin, K., Takahashi, H., Wands, J., and Ozturk, M. (1990). Enhanced expression of the protein kinase substrate p36 in human hepatocellular carcinoma. Mol. Cell. Biol. 10:3216–3223.

  74. 74.

    Zheng, S., Ruan, Y., Wu, Z., and Tang, J. (1997). The relationship between 67KD laminin receptor expression and metastasis of hepatocellular carcinoma. J. Tongji Med. Univ. 17:200–202, 224.

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Correspondence to Terence C. W. Poon.

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Pang, R.T.K., Poon, T.C.W., Wong, N. et al. Comparison of protein expression patterns between hepatocellular carcinoma cell lines and a hepatoblastoma cell line. Clin Proteom 1, 313–331 (2004) doi:10.1385/CP:1:3-4:313

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Key Words

  • Hepatocellular carcinoma
  • hepatoblastoma
  • proteome
  • two-dimensional polyacrylamide gel electrophoresis
  • cell line models