Molecular analysis of PinX1 in human hepatocellular carcinoma.

PinX1 is located at 8p23, a region with frequent loss of heterozygosity in hepatocellular carcinomas (HCCs). Overexpression of PinX1 is known to inhibit telomerase activity, shorten telomeres and induce crisis while its depletion increases tumorigenesis in nude mice. These results suggest that PinX1 might be critical for hepatocarcinogenesis. In this study, we assessed transcript expression of PinX1, the correlation between PinX1 mRNA level and telomere length and telomerase activity, as well as sequence alteration, in 24 HCCs and their adjacent non-HCC tissues from patients with B viral chronic hepatitis/cirrhosis. There was no significant difference between the levels of PinX1 mRNA in HCCs and those in non-HCCs. The PinX1 mRNA tended to increase as the telomere shortened in the HCCs (p=0.067, R(2)=0.166), but no correlation was found in non-HCCs. The PinX1 level revealed no significant relationship with telomerase activity in HCCs and non-HCCs. The missense mutations of PinX1, at the 254 and 265 residues, were found in 17% of the HCCs and their adjacent non-HCCs. The mutations were located in the non-conserved region and revealed no relation with PinX1 expression, telomere length and telomerase activity, suggesting that they are likely polymorphisms. Our findings suggest that PinX1 may not play a major role in hepatocarcinogenesis as a target tumor suppressor gene. PinX1, however, might be involved in the telomere length regulation of HCCs.

Expression of Smad7 in hepatocellular carcinoma and dysplastic nodules: resistance mechanism to transforming growth factor-beta.

BACKGROUND/AIMS: Smad7 is an inhibitory Smad of the transforming growth factor (TGF)-beta signaling pathway. To study resistance mechanisms to antiproliferating effect of TGF-beta in human multistep hepatocarcinogenesis, Smad7, Smad4, TGF-beta1 and TGF-beta receptor II were investigated. METHODOLOGY: Smad7 and Smad4 were evaluated in 15 low-grade dysplastic nodules (DNs), 9 high-grade DNs, 6 early hepatocellular carcinomas (eHCCs) and 41 advanced HCCs by immunohistochemistry and in 37 HCCs and corresponding non-HCCs with fresh tissue, the mRNAs of TGF-beta1 and TGF-beta receptor II were analyzed by RT-PCR. RESULTS: Smad7 immunoreactivity in tumor cells was found in 25 (61%) advanced HCCs, in contrast to none of DNs and eHCCs. Smad7 expression was significantly higher in advanced HCCs with increased TGF-beta1 or no decrease of TGF-beta receptor II compared to those of corresponding non-HCCs (p=0.044, p=0.027). Smad4 expression in stellate cells was present in 28 (68%) advanced HCCs, which was higher in smaller sized and better differentiated HCCs. CONCLUSIONS: Smad7, expressed in tumor cells, is considered to be one of resistance mechanisms to increased TGF-beta1 in late stage hepatocarcinogenesis, especially in advanced HCCs without reduced TGF-beta receptor II. Smad4, in stellate cells of HCCs, might be involved in the host resistance to hepatocarcinogenesis.

p53 and p21WAF1/CIP1 in hepatitis B virus related hepatocarcinogenesis.

BACKGROUND/AIMS: This study was undertaken to determine the expression p21WAF1/CIP1 and p53, the main regulator of G1 restriction point, in hepatitis B virus related hepatocarcinogenesis and their role in regulation of cell dynamics. METHODOLOGY: Immunohistochemistry for p21WAF1/CIP1, p53 and Ki-67 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling were preformed in 8 low grade dysplastic nodules, 7 high grade dysplastic nodules, 58 hepatocellular carcinomas, and 62 chronic hepatitis/cirrhosis. In 33 hepatocellular carcinomas with fresh frozen tissue, p21WAF1/CIP1 mRNA was studied by reverse transcriptase polymerase chain reaction. RESULTS: Immunostaining for p21WAF1/CIP1 and p53 was positive in 28% and 38% of hepatocellular carcinomas and higher in poorly differentiated hepatocellular carcinomas, whereas it was negative in all of chronic hepatitis/cirrhosis and dysplastic nodules. In hepatocellular carcinomas with p21WAF1/CIP1 mRNA amplified, 31% showed the protein expression indicating post-transcriptional regulation. The rate of apoptosis and proliferation showed significant correlation with p53 status, however not with p21WAF1/CIP1 in hepatocellular carcinomas. Induction of p21WAF1/CIP1 was regulated by both p53 dependent and independent pathway. CONCLUSIONS: In hepatitis B virus related hepatocarcinogenesis, p21WAF1/CIP1 and p53 are suggested to be involved in late stage of tumor progression rather than in early stage of dysplastic nodules and p53 is considered to be the main regulator of the cell dynamics.

Induction of telomerase activity during an early burst of proliferation in pancreatic regeneration.

Telomerase activity (TA) and telomerase reverse transcriptase (rTERT) were investigated in Sprague-Dawley rats, killed at 6 and 12 h, and 1, 2, 3, 7, and 14 days after 90% pancreatectomy (px), by TRAPeze enzyme-linked immunosorbent assay telomerase detection and reverse transcriptase-polymerase chain reaction. TA increased at 2 days and reached a maximum at 3 days after px, when the small ductules showed the highest proliferation activity. After 3 days, TA decreased to basal levels as the ductules differentiated into new endocrine and exocrine pancreas. The expression of rTERT showed a correlation with TA. These results suggest telomerase is actively regulated during pancreatic regeneration.