dispose the hepatocytes to uncontrolled growth and malig-We analyzed the p16 INK4 status of 6 hepatocellular carnant transformation as a result of disruption of the cell cycle's cinoma (HCC) cell lines and 32 primary HCC tumors, regulatory system. Mammalian cell-cycle progression is reguincluding 9 early-stage tumors, to determine whether lated by the sequential activation and inactivation of various p16 INK4 tumor-suppressor gene inactivation participates cyclin-dependent kinases (cdk) at different stages of the cell in hepatocarcinogenesis. p16 INK4 was studied at its procycle. 10 The p16 INK4 tumor-suppressor gene was cloned retein level through Western blotting, at its messenger cently and mapped to the 9p21 region. [11][12][13] The p16 INK4 gene RNA (mRNA) level through reverse-transcriptase polyproduct p16 protein was originally isolated as a protein that merase chain reaction analysis (RT-PCR) and Northern associated with cdk complexes 14 and was thought to exert a blotting, and at its genomic level through Southern blotnegative type of control on cell proliferation through its bindting and PCR-single-strand conformation polymoring to cdk4, thereby preventing cdk4 from forming an active phism analysis. The p16 protein was absent from 3 of 6 complex with cyclin D protein. 11,15 Deletion of the p16 INK4 cell lines (50%) and 11 of 32 primary tumors (34%), but gene in several types of tumor cell lines has been reported. 12,13 present in noncancerous tissues, indicating that p16 INK4 Although subsequent investigations demonstrated a high freis involved in hepatocarcinogenesis. Furthermore, we quency of p16 INK4 mutation in primary esophageal 16 and bilisuggest that the p16 protein loss may contribute to the ary tract 17 cancers, both mutant and homozygous deletions following: (1) early-stage hepatocarcinogenesis, because in pancreatic cancer, 18 and squamous cell carcinoma of the it was observed in 22% of early stage tumors; and (2) bladder, 19 these genomic alterations were detected rarely or tumor progression, because it occurred approximately not at all in other tumor types, such as breast, 20 nasopharyntwice as often in advanced rather than in early stage geal, 21 and gastric 22 cancers. On the other hand, p16 INK4 inactumors (40%). It was striking that neither p16 INK4 homotivation involving transcription loss associated with de novo zygous deletion and mutation nor loss of p16 INK4 mRNA methylation of the 5-CpG island in gliomas and breast, coexpression were observed in HCC cell lines and primary lonic, head and neck cancers, 23,24 and transitional cell carcinotumors, including those specimens from which the p16 mas of the bladder was reported recently. 25 Thus, previous protein was absent except the Li7HM cell line, in which studies showed various mechanisms of p16 INK4 inactivation p16 INK4 mRNA was not detected. These results suggest in several types of human cancer. However, to our knowledge, that p16 INK4 in HCC is inactivated predominantly by no data on HCC have been reported. Therefore, we analyzed posttranscriptional regulation rather than by genomic the p16 gene, as well as its messenger RNA (mRNA) and aberrations and lack of transcription. (HEPATOLOGY protein expression in HCC cell lines, and a series of primary 1996;24:575-579.)
HCC tumors, including early stage tumors, to determine whether p16 INK4 is involved in hepatocarcinogenesis. We and others have shown that genetic alterations occur in multistage hepatocarcinogenesis, including the inactiva-
Methods
tion of tumor-suppressor genes, such as p53 and Rb, [1][2][3][4] and Cell Lines and Tumor Specimens. WI-38 (human diploid cell line the loss of distinctive chromosomal regions, such as 4q, 8p, from normal embryonic lung tissue) and six HCC cell lines, HepG2, 13q, 16q, and 17p. [4][5][6][7][8][9] However, these events are likely to play PLC/PRF/5, Li7NM, Li7HM, Li21, and Li23, were used in our study. roles in the late stages of hepatocarcinogenesis, because they Li7NM, Li7HM, Li21, and Li23 26 were established in our laboratory. have not been observed in early-stage hepatocellular carcino-Thirty-two HCC tumors and noncancerous liver tissues from 28 pa- mas (HCC). The early genetic events in hepatocarcinogenesis tients were obtained from specimens surgically resected at the Na- remain to be determined.