Functional Localization Of Metastasis Suppressor Gene For Liver Cancer On Human Chromosome 8 And Preliminary Research On TEY1 Gene In Liver Cancer

BACKGROUND & OBJECTIVE : (1)Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer, whose functional evidence is imperatively demanded. (2)In order to functionally localize metastatic suppressor genes on human chromosomes, we tried to establish a spontaneous metastatic model of rat liver cancer in nude mice. In the mean time, the experimental metastatic behavior of C5F was studied. (3)TEY1 was a newly discovered metastasis suppressor gene for prostate cancer located on human chromosome 8p. In order to explore whether TEY 1 gene could be taken as a metastasis suppressor gene for liver cancer, the expression of TEY 1 was quantitated in liver cancer cell strains with different metastatic potentials and surgical specimens of 5 hepatocellular carcinoma (HCC) patients. METHODS: (1)The rat liver cancer cell line C5F was subcutaneously implanted in 4-week-old male and 7-week-old female BALB/cA nude mice respectively. Subcutaneous tumor formation was observed by measuring the MTD weekly. 6-7 weeks thereafter, the moribund mice were sacrificed and autopsy was performed to check for macroscopic metastases. Serial sections of the fixed lung tissues were done to grade and count lung metastases. Histological examination was done for suspicious organs. For experimental metastasis assay, C5F was injected into 6-7 week old female nude mice through the tail vein, 6-7 weeks later these mice were sacrificed and checked for macroscopic and microscopic metastases.(2)Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selection of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. STS-PCR and WCP-FISH were used to confirm the introduction. The microcell hybrid clone Neo8/C5F-1～10 and the C5F cell line were subcutaneously implanted in 6-7 week-old female BALB/cA nude mice toperform the spontaneous metastasis assay. Subcutaneous tumor formation and macroscopic lung metastases were observed. Serial dissections of the lung tissue were done to count the microscopic metastatic foci. The number of the metastatic foci were compared with oneway ANOVA assay. (3) RNA was extracted from the liver cancer cell strains, MHCC97-H,MHCC97-L and HCCLM3, the normal liver cell line L02 and the surgical HCC specimens. The first strand cDNA was synthesized by reverse transcription, which was then used as template to perform fluorescent quantitative PCR. The quantity of the expression of TEY 1 gene was normalized by dividing with that of the housekeeping gene, GAPDH, for each sample. One-way analysis of variance (ANOVA) was performed for the normalized values. RESULTS: (1)The rate for the subcutaneous tumor formation was 100% (10/10). In the female nude mice, 100% (6/6) overt metastases were observed. The median numbers of gross and microscopic metastatic foci were 45 and 755 per nude mouse respectively. No metastasis was found in the abdominal organs such as the liver, spleen and kidney. In contrast, neither macroscopic nor microscopic metastasis was found in male nude mice. In the experimental metastasis assay, no macroscopic or microscopic metastatic foci were found (0/6). (2)Microcell hybrids resistant to HAT and G418 were obtained and 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome. The spontaneous metastasis assay for these microcell hybrid clones showed that 6 clones displayed prominent metastasis suppression(p<0.05). Among 2 of them, metastases had been totally suppressed. (3)The normailized initial cDNA concentration of TEY1 of MHCC97-H,MHCC97-L,HCCLM3 and L02 were(3.57±2.62)...