ANALYSIS OF GENETIC ALTERATIONS DURING HEPATOCARCINOGENESIS

对可能的p53抑癌基因的突变进行了分析 9个黄曲霉毒素B1(AFLB1)诱导的猴肿瘤系列，6个AFLB1 转化的大鼠肝细胞系以及一组19人 启东、中国和17例肝癌组织中的肝细胞癌 食物诱变剂2-氨基-3-甲基咪唑并[4，5-f]喹啉(IQ) 在食蟹猴身上。在AFLB1诱导的猴肿瘤中，只有一种 检测到175密码子的单点突变(G到T颠换) 通过对四个保守结构域(II)的测序分析，仅在一个肝癌中发现了 到V)在p53基因中。在智商诱发的猴子肿瘤中，有3/17是相同的 在密码子159、175和248处检测到G到T易位。数列 6株AFLB1中P53基因的RT-PCR扩增产物分析 转化的大鼠肝细胞系显示出相同的点突变， 密码子173中从G到A的转变(CGC到CAC)，导致 编码的氨基酸由精氨酸变为组氨酸。没有野性- 在所有AFLB1细胞系中都观察到P53基因的类型等位基因 通过基因组DNA分析。突变型p53蛋白在体内的半衰期 AFLB1转化子的时间大于3小时，而正常情况下为30分钟 P53蛋白。启东地区19种HCs的PCR-SSCP分析 RFLP分析显示，19例肝癌中有10例(53%)存在P53突变。分成两份 肿瘤单个突变发生在第5和第6外显子，而在8例肝癌中发生G~ 外显子7的249位密码子第三位发生T突变。 P53和视网膜母细胞瘤(Rb)基因杂合性缺失(LOH) 分别在7/9(78%)和3/6(50%)的信息性病例中检出。 在10个信息性的腺瘤病结肠息肉病基因座上没有观察到杂合性丢失 案子。16号染色体16Q和16P杂合性缺失分别发生在10/18(56%)和 信息性病例分别为6/13例(46%)。这些数据表明， P53基因的选择性突变和频繁杂合性缺失在肿瘤发生中起重要作用 启东地区肝癌的发生发展及至少三种附加肿瘤 抑癌基因可能参与了人肝癌的发生。

Mutations in the putative p53 tumor suppressor gene were analyzed in a series of 9 aflatoxin B1 (AFLB1) induced monkey tumors, 6 AFLB1 transformed rat liver cell lines, as well as a group of 19 human hepatocellular carcinomas (HCC) obtained from Qidong, China, and 17 HCCs induced by the food mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in cynomolgus monkeys. Among the AFLB1 induced monkey tumors only a single point mutation at codon 175 (G to T transversion) was identified in only one HCC by sequencing analysis of the four conserved domains (II to V) in the p53 gene. In 3/17 of the IQ induced monkey tumors the same G to T transversion was detected at codons 159, 175, and 248. Sequence analysis of amplified cDNA by RT-PCR of p53 mRNA from the six AFLB1 transformed rat liver cell lines showed the same point mutation, a transition from G to A in codon 173 (CGC to CAC) which results in a change of the encoded amino acid from arginine to histidine. No wild- type alleles of the p53 gene were observed in any of the AFLB1 cell lines by genomic DNA analysis. The half life of the mutant p53 protein in the AFLB1 transformants was greater than 3 hours versus 30 minutes for normal p53 protein. Analysis of 19 HCCs from Qidong using PCR-SSCP as well as RFLP analysis revealed that 10/19 HCCs (53%) showed p53 mutation. In two tumors single mutations occurred in exons 5 and 6, while in 8 HCCs a G to T mutation at the third position of codon 249 in exon 7 was observed. Loss of heterozygosity (LOH) of the p53 and retinoblastoma (RB) genes was detected in 7/9 (78%) and 3/6 (50%) of informative cases, respectively. No LOH of adenomatis polyposis coli locus was observed in 10 informative cases. LOH of chromosomes 16q and 16p was detected in 10/18 (56%) and 6/13 (46%) informative cases, respectively. These data suggest that the selective mutation and frequent LOH of the p53 play an important role in the development of HCCs from Qidong and at least three additional tumor suppressor genes might be involved in the human hepatocarcinogenesis.