Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inactivation of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor p16INK4A may be caused by gene deletion, mutation or promoter hypermethylation. We have previously reported that p16INK4A in hepatocellular carcinoma (HCC) tissues and cell lines is inactivated predominantly by promoter hypermethylation rather than genomic aberrations. In the present experiments, we have studied the effects of the demethylating agent, 5-aza-2'-deoxycytidine (5-AZA/decitabine), on the expression of aberrant p16INK4A RNA transcripts and the CDK-retinoblastoma gene pathway in HCC cell lines with p16INK4A promoter hypermethylation. The expression of aberrant p16INK4A RNA transcripts was down-regulated and p16INK4A protein was strongly re-expressed in the HCC cell lines, SNU 354, 398, 423 and 475 after 5-AZA/decitabine treatment for 5 days. The re-expressed p16INK4A was functional, because it bound to and inhibited CDK4 kinase activity, and increased the concentrations of the hypophosphorylated form of retinoblastoma protein (pRB) in cells with a wild type RB gene. Moreover, treatment with the demethylating agent led not only to G1 cell cycle arrest, but also to the increased expression of the senescence-associated marker beta-galactosidase. This up-regulation of p16INK4A mRNA and protein correlated with demethylation of the p16INK4A promoter, and with the down-regulation or disappearance of aberrant p16INK4A transcripts. These results suggest that the aberrant p16INK4A RNA transcript can be transcribed from the methylated p16INK4A gene, and endogenous reactivation of functional p16INK4A mRNA by a demethylating agent can restore the pRB pathway in HCC, and foster the terminal differentiation of the malignant cells. Therefore, demethylating agents, such as 5-AZA/decitabine, may have potential in the treatment of HCC.
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PMID:5-Aza-2'-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines. 1109 88

Inactivation of the p16INK4a (p16) tumor suppressor gene by promoter hypermethylation and mutation within exon 3 of beta-catenin represent two of the more common gene alterations in human hepatocellular carcinoma (HCC). One exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liver parenchyma. Treatment of rats with high doses of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes liver toxicity and a high incidence of tumors. The purpose of the current investigation was to define the prevalence of genetic alterations in p16 and beta-catenin in NNK-induced rat liver cancer to determine if the molecular mechanisms seen in human tumors are the same in this animal model. DNA isolated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR. p16 methylation was detected in five of 15 adenomas and eight of 14 carcinomas (45% of all tumors). Methylation of p16 was extensive within the 5'-untranslated region and exon 1alpha, areas shown to correlate with loss of gene transcription. Liver tumors were also screened for mutations within exon 3 of beta-catenin. Single strand conformation polymorphism and DNA sequencing revealed five mutations in four of 29 tumors (14%). Mutations were present in three adenomas and one carcinoma and were located within codons 33, 36 or 37. All mutations resulted in amino acid substitutions; three of these mutations occurred at potential serine phosphorylation sites. Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common genetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic alterations critical to the initiation of HCC.
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PMID:p16INK4a and beta-catenin alterations in rat liver tumors induced by NNK. 1123 87

The genesis of hepatocellular carcinoma is promoted by changes in the regulatory MDM2-P14ARF system. The incidence of such changes has to date not been analysed in non-tumourous livers showing regenerative proliferation. In the present study, 24 cirrhotic livers of alcohol-, autoimmue disorder- or HCV-caused genesis were screened for MDM2-P14ARF alterations at the level of protein, DNA and mRNA. Using confocal laser scanning microscopy, the absence of MDM2 and P14ARF expression was detected in all samples except three HCV-infected livers (four livers) which contained hepatocytes overexpressing MDM2 (P14ARF) protein. In two of the samples lacking P14ARF expression, laser microdissection and PCR demonstrated deletion of the P14ARF gene. The P14ARF gene amplified from other specimens did not carry mutations. MDM2 splicing variants were present in tissues from alcohol- and autoimmune disorder-induced cirrhoses. Sequencing of full-size mRNA revealed a MDM2 mis-sense mutation in an alcohol-induced cirrhosis. One sample contained regenerative nodules with genetic instability occurring at MDM2 locus D12S83 according to the data of automatic PCR fragment analysis. In summary, this study gives first evidence for different types of MDM2 and P14ARF alterations in cirrhotic livers. We suggest that the changes impair the regulatory MDM2-P14ARF system, thus possibly favouring regenerative proliferation and transformation.
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PMID:Cirrhotic livers reveal genetic changes in the MDM2-P14ARF system of cell cycle regulators. 1195 87

Despite the recent introduction of real-time PCR methods and cDNA microarrays, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. In this study, we developed a construct, termed tumor suppressor-internal standard (TS-IS) that produced polycompetitive RNA templates as an internal standard to quantify cellular RNA concentration of tumor suppressor genes. This construct is composed of not only sets of primers for detecting the expression of several tumor suppressor genes (such as pRB, p16(INK4A) 15(INK4B), p14(ARF) p53, and p21(WAF1)), but also HPRT as an endogenous marker. Using an internal standard RNA that was synthesized from the TS-IS construct, we were able to establish optimized conditions for the quantification of tumor suppressor genes with minimal amounts (50 ng) of cellular RNA. In addition, the usefulness of this method was confirmed by analyzing the expression levels of tumor suppressor genes in fourteen hepatoma cell lines as a model. The TS-IS assay that we used was inexpensive and a widely applicable method that permitted the reliable and accurate quantification of tumor suppressor genes.
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PMID:Quantification of tumor suppressor mRNA expression by poly-competitive RT-PCR using a TS-IS that contained multiple internal competitors. 1213 90

One of the main regulatory pathways reported to be altered in hepatocellular carcinoma (HCC) is that of cell cycle control involving RB1 gene-related cell inhibitors. We investigated p14(ARF), p15(INK4B), p16(INK4A), p18(INK4C), and RB1 genes in a series of HCCs and associated cirrhosis with the goal of ascertaining their pattern of inactivation by gene methylation. Thirty-three HCCs, adjacent nonneoplastic cirrhotic tissues, and 6 HCC cell lines were studied. Cirrhoses (25 of 33, 76%), HCCs (31 of 33, 94%), and 3 of 6 (50%) cell lines showed 1 or more methylated genes. Cirrhoses (17 of 33, 51%) had more frequently than HCCs (11 of 33, 33%, P =.01) only 1 methylated gene. With the exception of p18(INK4C) the genes under study showed promoter methylation with frequency ranging from 82% (p16(INK4A) in HCC) to 33% and 39% (p15(INK4B) and p16(INK4A) in cirrhoses). In cases with only 1 methylated gene, p15(INK4B) in cirrhosis (8 of 17, 47%) and p16(INK4A) in HCC (10 of 11, 91%) were the more frequently altered. An optimal correlation was found between p15 and p16 gene methylation and complete protein loss in HCC detected by immunocytochemistry, whereas a partial loss of the same proteins was a feature of methylated cirrhoses. Inactivation by DNA methylation of several genes of the RB1 pathway is common to cirrhosis and HCC. An early pattern of methylatory events (1 methylated gene) is a feature of cirrhosis rather than HCC, whereas an advanced one (> or = 3 methylated genes) is characteristic of malignancy. Early methylation changes seem to involve p15(INK4B) and p16(INK4A) in cirrhosis and p16(INK4A) in HCC. In conclusion, a stepwise progression of methylating events is a feature of the sequence cirrhosis-HCC and contributes to the process of hepatic carcinogenesis with potential clinical implications.
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PMID:Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. 1214 52

The INK4alpha/ARF locus encodes p14(ARF) and p16(INK4alpha) , that function to arrest the cell cycle through the p53 and RB pathways, respectively. Genetic alterations of p14ARF and their relationship with p16(INK4a) or p53 inactivation have not been characterized in hepatocellular carcinoma (HCC). We examined 40 pairs of HCCs/noncancerous liver tissues for homozygous deletions (HD), methylation and mutations of the INK4a/ARF locus and for mutations of p53, and analyzed their clinicopathological correlation. p16(INK4a), p53 and p14(ARF) were inactivated in 62.5% (25 out of 40), 42.5% (17 out of 40) and 20% (8 out of 40) of HCCs, respectively. Inactivation of p14(ARF) was always associated with the concomitant inactivation of p16(INK4a) and occurred more frequently in hepatitis C virus (HCV)-associated HCC (p=0.042). Inactivation of p16INK4a) occurred more frequently in older patients (p=0.0027. The predominant mechanism of inactivation of p14(ARF) was homozygous deletion (7 out of 8), while that of p16(INK4a) was methylation (21 out of 25). Although statistically insignificant, genetic alterations of p14(ARF) tended to occur in tumors with wild-type p53. Genetic alterations of the INK4alpha/ARF locus could occur in small HCCs. In contrast, p53 mutations occurred more frequently in advanced HCCs (p=0.041). Inactivation of either p14(ARF)/p53 or p16(INK4a) occurred in 80% (32 out of 40) and concomitant disruption of both pathways occurred in 40% (16 out of 40) of HCCs, respectively. These results suggest that p14(ARF), p16(INK4a) and p53 are differentially disrupted through distinct molecular mechanisms at different stages in HCC and that p14(ARF) and p53 appear to function in the same tumor suppression pathway in HCC
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PMID:Genetic alterations of INK4alpha/ARF locus and p53 in human hepatocellular carcinoma. 1216 36

Inhibitors of differentiation and DNA binding-1 (Id-1) have been demonstrated to oppose Ets-mediated activation of p16INK4a. As p16INK4a protein is inactivated in hepatocellular carcinoma (HCC), we aimed to investigate the role of Id-1 in regulating p16INK4a expression during the development of HCC in HCC patients and direct ectopic Id-1 introduction into the PLC/PRF/5 HCC cell line. Sixty-two HCC samples were recruited for evaluation of Id-1 and proliferating cell nuclear antigen (PCNA) protein expression. The messenger RNA (mRNA) expression of Id-1 and p16INK4a was detected by quantitative reverse transcription-polymerase chain reaction. For in vitro Id-1 transfection, five Id-1 transfected clones were isolated and the effect of ectopic Id-1 introduction was investigated by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, immunostaining and western blot. Our results showed that Id-1 was over-expressed in HCC specimens both at mRNA and protein levels. Over-expression of Id-1 protein was correlated with PCNA (r = 0.334, P = 0.033). HCC samples showing low Id-1 protein expression had a lower Id-1 mRNA level (340.2 versus 1467%, P = 0.039) and higher p16INK4a expression (195 versus -78.6%, P = 0.039) than samples with high Id-1 protein expression. In the PLC/PRF/5 HCC cell line study, ectopic Id-1 expression resulted in proliferation of HCC cells and an increased percentage of S phase cells and PCNA expression. The results showed that over-expression of Id-1 induces cell proliferation in HCC through inactivation of p16INK4a/retinoblastoma pathway. In conclusion, the results provided an insight for the understanding of the role of Id-1 in functional inactivation of p16INK4a in HCC.
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PMID:Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. 1294 53

The inactivation of the p16INK4A (p16) gene by promoter hypermethylation has been reported in many human cancers. We previously reported that aberrant p16 RNA transcripts are expressed in hepatocellular carcinoma (HCC) cell lines having hypermethylated p16 promoters. In this study, we investigated the functional roles of aberrant p16 RNA transcripts in HCC cells to elucidate molecular events underlying hepatocarcinogenesis. The aberrant p16 RNA transcripts encoded key peptides (amino acids 84-103) involved in binding with cyclin-dependent kinase (CDK) 4. GST-aberrant p16 fusion proteins were found to interact with endogenous CDK4 in vitro. Furthermore, overexpression of these aberrant p16 RNA transcripts resulted in decreased cell proliferation rate, enlargement of cell shape and reduced level of hyperphosphorylated forms of pRb. Overall, our results suggest that the aberrant p16 RNA transcripts have functions similar to those of wild type p16 in controlling cell cycle.
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PMID:Aberrant p16INK4A RNA transcripts expressed in hepatocellular carcinoma cell lines regulate pRb phosphorylation by binding with CDK4, resulting in delayed cell cycle progression. 1295 83

In our study, we examined whether human hepatocellular carcinoma (HCC) expresses peroxisome proliferator-activated receptor gamma (PPARgamma) and the effects of PPAR gamma activation by its selective ligands on cell growth and cell invasion in HCC cells. RT-PCR and Western blot analysis revealed that HCC-derived cell lines, HepG2 and HLF, express PPARgamma mRNA and protein. Luciferase assay in HLF cells showed that troglitazone, a selective ligand for PPAR gamma, transactivated the transcription of a peroxisome proliferator response element-driven promoter in a dose-dependent manner, suggesting that the expressed PPARgamma functions as a transcriptional factor. Not only troglitazone but pioglitazone dose-dependently inhibited cell growth in HepG2 and HLF cells. Invasion assay using a transwell chamber demonstrated that troglitazone also inhibited cell invasion in HCC cells. To examine the mechanism of the troglitazone-induced growth inhibition, we determined p27(Kip1), a cyclin dependent kinase inhibitor, expression by Western blot analysis in troglitazone-treated HLF cells. Troglitazone increased p27(Kip1) in time- and dose-dependent manners, suggesting that p27(Kip1) may be involved in the growth inhibition by troglitazone in HLF cells. To further examine the mechanism of the troglitazone-induced p27(Kip1) protein accumulation, 2 major systems for regulation of p27(Kip1) protein, proteasome activity and Skp2, an F-box protein that targets p27(Kip1) for degradation, were evaluated. Troglitazone potently inhibited proteasome activity and decreased Skp2 protein levels. All these results suggest that human HCC cells express functional PPAR gamma and PPARgamma activation resulted in growth inhibition. The growth inhibition was mediated by p27(Kip1) accumulation, which is induced by both inhibition of ubiquitylation of p27(Kip1) and reduction of degradation activity of p27Kip1 by proteasome.
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PMID:Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells. 1461 13

To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found that while remained unmethylated the ABL, CAV, EPO, GATA3, LKB1, NEP, NFL, NIS and p27KIP1 genes, varying extents of the HCC specific hypermethylation were found associated with the ABO, AR, CSPG2, cyclin a1, DBCCR1, GALR2, IRF7, MGMT, MT1A, MYOD1, OCT6, p57KIP2, p73, WT1 genes, and demethylation with the MAGEA1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more frequently than in their neighboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for the association with, the hypermethylation of the cyclin a1 gene was more prevalent in the non-cirrhosis group (P=0.021) while the hypermethylated p16INK4a gene was more common in the cirrhosis group (P=0.017). The concordant methylation behaviors of nineteen genes, including the four previously studied and their association with cirrhosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us to shape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, as well as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.
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PMID:Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. 1467 55


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