人肝癌转移抑制基因染色体功能定位与TEY1基因的研究
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摘要
【摘要】背景与目的 （1）我所的前期工作发现8号染色体短臂上的缺失与肝癌的转移相关，提示人染色体8p上存在肝癌转移抑制基因的可能性，但缺乏其存在的功能证据。（2 ）为寻求肝癌转移抑制基因的功能证据，必须建立异种动物自发转移动物模型。大鼠肝癌细胞系C5F细胞系在KSN裸鼠自发转移实验中只形成显微镜下的小转移灶，但未形成肉眼可见的肺转移灶，尚需改进实验条件在BALB/cA裸鼠中建立大体肺转移动物模型。（3） TEY 1基因是最近美国NCI Barrett实验室从人染色体8p21-p12区域克隆出新的前列腺癌转移抑制基因，为研究TEY1基因作为肝癌转移抑制基因的可能性，我们对不同转移潜能肝癌细胞株和5例肝细胞癌手术标本中TEY 1基因的表达水平进行了定量分析。方法 （1） 大鼠肝癌细胞系C5F皮下接种4周龄雄性和7周龄雌性BALB/cA裸鼠，观察皮下成瘤及肺部和腹腔脏器的大体转移灶形成情况，取可能转移的器官做组织学检查，肺组织连续切片并计数镜下转移灶。（2）为寻求人8号染色体上存在肝癌转移抑制基因的功能证据，通过微细胞介导的染色体转移方法（MMCT）将随机标记有耐药基因neo的正常人8号染色体导入到大鼠肝癌高转移细胞系C5F中，用G418和HAT进行药物筛选微细胞杂交克隆，单细胞分离克隆方法进行单细胞克隆，STS-PCR和WCP-FISH方法对导入的8号染色体进行证实。将获得的微细胞杂交克隆和C5F细胞分别接种裸鼠皮下，观察皮下成瘤性、大体和显微肺转移灶形成情况的差异，结果进行单因素方差统计分析。（3）提取肝癌细胞株MHCC97-H，MHCC97-L，HCCLM3、正常细胞系L02和肝细胞癌手术标本的RNA，反转录合成第一链cDNA，反转录产物进行荧光定量PCR。用看家基因GAPDH的cDNA起始浓度作为参照标化TEY1 的cDNA起始浓度，标化结果进行单因素方差分析。结果 （1）裸鼠皮下成瘤率100% (10/10)，雌性裸鼠中肺表面肉眼可见明显转移灶，转移率为100% (6/6)，肺表面大体转移灶中位数45个/裸鼠，镜下转移灶中位数755个/裸鼠，腹腔、肝脏、脾脏、肾脏和心脏均未见转移灶。雄性裸鼠未见大体和镜下转移灶。（2）获得具有G418和HAT双重抗性的微细胞杂交细胞，通过单细胞分离克隆方法获得15个具有双重抗性的微细胞杂交克隆，STS-PCR发现导入到C5F中的人8号染色体发生了染色体片段丢失, WCP-FISH发现导入的人8号染色体与大鼠染色体发生了稳定的重组。
微细胞杂交克隆的自发转移实验发现有6个杂交克隆出现转移表型的显著抑制(p<0.05)，其中2个杂交克隆转移表型发生完全抑制。（3）MHCC97-H，MHCC97-L，HCCLM3和L02标化后的TEY1 cDNA起始浓度分别为(3.57±2.62)×10-4，(5.02±1.95)×10-4，(2.82±0.78)×10-5，(15.20±0.58)×10-4。肝癌细胞株的TEY1基因表达水平和正常细胞系之间存在显著差异(p<0.001)，HCCLM3的TEY1基因表达水平与MHCC97-H和MHCC97-L的相比存在显著差异(p<0.05) ，MHCC97-L的TEY1基因表达水平尽管是MHCC97-H的1.4倍，但是两者无显著的统计学差异(p>0.05)。在两例有门脉癌栓的病例中，癌旁组织中TEY1的表达水平显著高于癌组织以及门静脉癌栓中的表达水平(p<0.05)，癌组织中的TEY1表达水平分别是癌栓中的1.76和3.50倍，但差别无显著性(p>0.05)。在另外3例肝内播散灶病例中，播散灶中TEY1表达水平和癌旁组织中的差别无显著性(p>0.05)。结论 （1）C5F细胞系在雌性BALB/cA裸鼠中能充分表达自发性转移潜能，肺脏是其优势转移器官。成功建立的大鼠肝癌自发肺转移裸鼠模型为肝癌转移抑制基因的染色体功能定位研究提供了适宜的动物模型。（2）人8号染色体对大鼠肝癌高转移细胞系C5F的成功导入改变了C5F的转移表型，提供了人8号染色体上存在肝癌转移抑制基因的直接的功能证据，并为其在人8号染色体上的进一步定位奠定了基础。（3）在体外培养的细胞中，TEY 1基因表达水平与肝癌细胞的转移潜能负相关。门脉癌栓病例手术标本中TEY1的检测结果提示TEY1基因的表达水平与肝癌的转移潜能负相关。播散灶病例中，未发现此种负相关性，但这些播散灶是否为多中心发生的癌灶尚需进一步鉴别。基于此发现，可以推测TEY 1基因作为肝癌转移抑制基因的可能性。
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 to
perform 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)
微细胞杂交克隆的自发转移实验发现有6个杂交克隆出现转移表型的显著抑制(p<0.05)，其中2个杂交克隆转移表型发生完全抑制。（3）MHCC97-H，MHCC97-L，HCCLM3和L02标化后的TEY1 cDNA起始浓度分别为(3.57±2.62)×10-4，(5.02±1.95)×10-4，(2.82±0.78)×10-5，(15.20±0.58)×10-4。肝癌细胞株的TEY1基因表达水平和正常细胞系之间存在显著差异(p<0.001)，HCCLM3的TEY1基因表达水平与MHCC97-H和MHCC97-L的相比存在显著差异(p<0.05) ，MHCC97-L的TEY1基因表达水平尽管是MHCC97-H的1.4倍，但是两者无显著的统计学差异(p>0.05)。在两例有门脉癌栓的病例中，癌旁组织中TEY1的表达水平显著高于癌组织以及门静脉癌栓中的表达水平(p<0.05)，癌组织中的TEY1表达水平分别是癌栓中的1.76和3.50倍，但差别无显著性(p>0.05)。在另外3例肝内播散灶病例中，播散灶中TEY1表达水平和癌旁组织中的差别无显著性(p>0.05)。结论 （1）C5F细胞系在雌性BALB/cA裸鼠中能充分表达自发性转移潜能，肺脏是其优势转移器官。成功建立的大鼠肝癌自发肺转移裸鼠模型为肝癌转移抑制基因的染色体功能定位研究提供了适宜的动物模型。（2）人8号染色体对大鼠肝癌高转移细胞系C5F的成功导入改变了C5F的转移表型，提供了人8号染色体上存在肝癌转移抑制基因的直接的功能证据，并为其在人8号染色体上的进一步定位奠定了基础。（3）在体外培养的细胞中，TEY 1基因表达水平与肝癌细胞的转移潜能负相关。门脉癌栓病例手术标本中TEY1的检测结果提示TEY1基因的表达水平与肝癌的转移潜能负相关。播散灶病例中，未发现此种负相关性，但这些播散灶是否为多中心发生的癌灶尚需进一步鉴别。基于此发现，可以推测TEY 1基因作为肝癌转移抑制基因的可能性。
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 to
perform 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)
引文
1. Yoshida BA, Sokoloff MM, Welch DR, et al. Metastasis-suppressor genes: a review and perspective on an emerging field. J Natl Cancer Inst, 2000; 92: 1717-1730
2. Overhauser J. Somatic Cell Hybrid Mapping Panels: Resources for Mapping Disease Genes. Human Genome Methods Edited by Adolph K.W. CRC Press LLC USA 1998, pp258-260
3. Dong JT, Lamb PW, Rinker-Schaeffer CW, et al. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science, 1995; 268: 884-886
4. Ichikawa T, Ichikawa Y, Dong J, et al. Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome11. Cancer Res, 1992; 52(12): 3486-3490
5. Gao AC, Lou W, Dong JT, et al. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13. Cancer Res, 1997; 57(5): 846-849
6. Gao AC, Lou W, Ichikawa T, et al. Suppression of the tumorigenicity of prostatic cancer cells by gene(s) located on human chromosome 19p13.1-13.2. Prostate,1999; 38(1): 46-54
7. Nihei N, Kouprina N, Larionov V, et al. Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60-kilobase region on human chromosome 8p21-p12. Cancer Res, 2002; 62:367 - 370.
8. Ichikawa T, Hosoki S, Suzuki H, et al. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer. Asian J Androl, 2000; 2(3):167-171
9. Chekmareva MA, Hollowell CM, Smith RC, et al. Localization of prostate cancer metastasis suppressor activity on human chromosome 17. Prostate, 1997; 33(4):271-280
10. Mashimo T, Watabe M, Cuthbert AP, et al. Human chromosome 16 suppresses metastasis but not tumorigenesis in rat prostatic tumor cells. Cancer Res, 1998;58(20):4572-4576
Luu HH, Zagaja GP, Dubauskas Z, et al. Identification of a novel metastasis
11. suppressor region on human chromosome 12. Cancer Res, 1998; 58(16): 3561-3565
12. Miele ME, Robertson G, Lee JH, et al. Metastasis suppressed, but tumorigenicity and local invasiveness unaffected, in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6. Mol Carcinog, 1996; 15(4): 284-299
13. Qin LX, Tang ZY, Sham JS, et al.The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res, 1999; 59:5662-5665
14. Sun FX, Tang ZY, Liu KD, et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer, 1996; 66: 239-240
15. Tian J, Tang ZY, Ye SL, et al. New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer, 1999; 81: 814-821
16. Qin LX ,Tang ZY, Ye SL, et al. Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared. J Cancer Res Clin Oncol, 2001; 127(8) : 482-488
17. Ogawa K, Nakanishi H, Takeshita F, et al. Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int J Cancer, 2001; 91(6): 797-802
2. Overhauser J. Somatic Cell Hybrid Mapping Panels: Resources for Mapping Disease Genes. Human Genome Methods Edited by Adolph K.W. CRC Press LLC USA 1998, pp258-260
3. Dong JT, Lamb PW, Rinker-Schaeffer CW, et al. KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science, 1995; 268: 884-886
4. Ichikawa T, Ichikawa Y, Dong J, et al. Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome11. Cancer Res, 1992; 52(12): 3486-3490
5. Gao AC, Lou W, Dong JT, et al. CD44 is a metastasis suppressor gene for prostatic cancer located on human chromosome 11p13. Cancer Res, 1997; 57(5): 846-849
6. Gao AC, Lou W, Ichikawa T, et al. Suppression of the tumorigenicity of prostatic cancer cells by gene(s) located on human chromosome 19p13.1-13.2. Prostate,1999; 38(1): 46-54
7. Nihei N, Kouprina N, Larionov V, et al. Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60-kilobase region on human chromosome 8p21-p12. Cancer Res, 2002; 62:367 - 370.
8. Ichikawa T, Hosoki S, Suzuki H, et al. Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer. Asian J Androl, 2000; 2(3):167-171
9. Chekmareva MA, Hollowell CM, Smith RC, et al. Localization of prostate cancer metastasis suppressor activity on human chromosome 17. Prostate, 1997; 33(4):271-280
10. Mashimo T, Watabe M, Cuthbert AP, et al. Human chromosome 16 suppresses metastasis but not tumorigenesis in rat prostatic tumor cells. Cancer Res, 1998;58(20):4572-4576
Luu HH, Zagaja GP, Dubauskas Z, et al. Identification of a novel metastasis
11. suppressor region on human chromosome 12. Cancer Res, 1998; 58(16): 3561-3565
12. Miele ME, Robertson G, Lee JH, et al. Metastasis suppressed, but tumorigenicity and local invasiveness unaffected, in the human melanoma cell line MelJuSo after introduction of human chromosomes 1 or 6. Mol Carcinog, 1996; 15(4): 284-299
13. Qin LX, Tang ZY, Sham JS, et al.The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res, 1999; 59:5662-5665
14. Sun FX, Tang ZY, Liu KD, et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer, 1996; 66: 239-240
15. Tian J, Tang ZY, Ye SL, et al. New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer, 1999; 81: 814-821
16. Qin LX ,Tang ZY, Ye SL, et al. Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared. J Cancer Res Clin Oncol, 2001; 127(8) : 482-488
17. Ogawa K, Nakanishi H, Takeshita F, et al. Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int J Cancer, 2001; 91(6): 797-802