Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray

Abstract Purpose. To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide a suitable model for the study of lung-metastasis-related molecular mechanisms. Methods. HCC clone cells MHCC97-H were inoculated into BALB/c nude mice, and the pulmonary metastatic lesions were harvested and re-implanted into nude mice for the second round of in vivo selection. The same procedure was repeated twice. A new cell line from the third round of lung metastases was established. Results. A human HCC cell line with unique metastatic characteristics was established by in vivo selection. This cell line, designated as HCCLM3, was polygonal epithelial cell with hypotriploid karyotype and population doubling time of 34.9 h. The cells were positive for alpha fetoprotein (AFP), albumin, cytokeratin 8 (CK8), and negative for hepatitis B surface antigen (HBsAg) by immunocytochemistry. Fluorescence polymerase chain reaction (PCR) showed HBV DNA integration in the cellular genome. When 5×106 cells were injected subcutaneously into nude mice, tumorigenicity was 100%, with a latency period of 11±1 days. Five weeks after s.c. injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 days, widespread loco-regional and distant metastases occurred, with 100% abdominal wall metastases, 80% intra-abdominal cavity metastases, 100% intrahepatic metastases, 70% diaphragm metastases, and 100% pulmonary metastases. The median number of lung metastatic lesions was 268 per mouse. Gene expression profile of HCCLM3 was compared by cDNA microarray with MHCC97-L, a clonal cell strain from the same parental cell line but with low metastatic potential; 25 differentially expressed genes were identified, 18 of which showed decreased expression and seven increased expression in HCCLM3, including the decreased expression of cell cycle control gene Rb2, mismatch repair gene hMSH2, and signal transduction gene protein kinase C β2, and increased expression of signal transduction gene MAP kinase, kinase 6. Conclusions. A new HCC cell line characterized by high pulmonary metastases via s.c. and orthotopic inoculation was established, which provides a new model for the study of liver cancer metastasis. Its gene expression profile could help in the understanding of the mechanism of metastasis and provide potential targets for anti-metastasis intervention.

[1]  Zeng-chen Ma,et al.  Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues , 1996, International journal of cancer.

[2]  R. Davis,et al.  MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway , 1996, Molecular and cellular biology.

[3]  A. Zijno,et al.  Mismatch repair and differential sensitivity of mouse and human cells to methylating agents. , 1999, Carcinogenesis.

[4]  A. Giordano,et al.  Who is the boss in the retinoblastoma family? The point of view of Rb2/p130, the little brother. , 2001, Cancer research.

[5]  J. Lord,et al.  Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids , 1995, International journal of cancer.

[6]  S. Ye,et al.  New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis , 1999, British Journal of Cancer.

[7]  P. Meltzer,et al.  Metastasis-associated differences in gene expression in a murine model of osteosarcoma. , 2001, Cancer research.

[8]  S. Fukushima,et al.  Induction of hepatocellular carcinoma with high metastatic potential in WS/Shi rats: discovery of an inbred strain highly susceptible to the liver carcinogen N-nitrosomorpholine. , 2001, Oncology Research.

[9]  A. Giordano,et al.  Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer. , 1997, Clinical cancer research : an official journal of the American Association for Cancer Research.

[10]  A. Giordano,et al.  Differential expression of the retinoblastoma gene family members in choroidal melanoma: prognostic significance. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[11]  H.-B. Tang,et al.  Invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor , 2000, Journal of Cancer Research and Clinical Oncology.

[12]  H. Yoshiji,et al.  Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver , 1999, International journal of cancer.

[13]  J. Neoptolemos,et al.  Protein kinase C isoenzyme patterns characteristically modulated in early prostate cancer. , 1999, The American journal of pathology.

[14]  K. Klinga-Levan,et al.  The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19. , 1993, Oncogene.

[15]  M. Duffy,et al.  Urokinase plasminogen activator as a prognostic marker in different subgroups of patients with breast cancer , 1994, Cancer.

[16]  Eric S. Lander,et al.  Genomic analysis of metastasis reveals an essential role for RhoC , 2000, Nature.

[17]  J. Griffith,et al.  Binding of mismatched microsatellite DNA sequences by the human MSH2 protein. , 1994, Science.

[18]  Dihua Yu,et al.  Pathway Enhances Endothelial Cell Migration 1-activated p 38 Signaling β Cancer Cells by the Heregulin-Up-Regulation of Vascular Endothelial Growth Factor in Breast , 2001 .

[19]  J. Ferlay,et al.  Estimates of the worldwide mortality from 25 cancers in 1990 , 1999, International journal of cancer.

[20]  S. Hirohashi,et al.  Loss of Cell-Cell Contact Is Induced by Integrin-Mediated Cell-Substratum Adhesion in Highly-Motile and Highly-Metastatic Hepatocellular Carcinoma Cells , 2000, Laboratory Investigation.

[21]  S. Ye,et al.  Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. , 2001, World journal of gastroenterology.

[22]  N. Tanaka,et al.  Immunohistochemical investigation of new suppressor oncogene p130 in oral squamous cell carcinoma , 1999 .

[23]  Y. Nagamine,et al.  Regulation by p38 mitogen-activated protein kinase of adenylate- and uridylate-rich element-mediated urokinase-type plasminogen activator (uPA) messenger RNA stability and uPA-dependent in vitro cell invasion. , 1999, Cancer research.

[24]  S. Elledge,et al.  Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease , 2003, Nature Genetics.

[25]  D. Tarin,et al.  Identification of metastasis-associated genes by transcriptional profiling of a pair of metastatic versus non-metastatic human mammary carcinoma cell lines. , 2001, Anticancer research.

[26]  T. Shuin,et al.  Human mismatch repair gene (hMSH2) product expression in relation to recurrence of transitional cell carcinoma of the urinary bladder , 1999, Cancer.

[27]  G. Hannon,et al.  Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. , 1993, Genes & development.

[28]  K. Milde-Langosch,et al.  Expression of Rb2/p130 in breast and endometrial cancer: correlations with hormone receptor status , 2001, British Journal of Cancer.

[29]  S. Sampson,et al.  Patterns of protein kinase C isoenzyme expression in transitional cell carcinoma of bladder. Relation to degree of malignancy. , 2001, American journal of clinical pathology.

[30]  S. Ye,et al.  Modulation of apoptosis, tumorigenesity and metastatic potential with antisense H-ras oligodeoxynucleotides in a high metastatic tumor model of hepatoma: LCI-D20. , 2000, Hepato-gastroenterology.

[31]  M. Tatematsu,et al.  Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice , 2001 .

[32]  A. Duncan,et al.  The adenovirus E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family and physically interacts with cyclins A and E. , 1993, Genes & development.