Adjacent non-HCC Tissues Research Articles

Expression of cancer-testis antigen (CTA) in tumor tissues and peripheral blood of Chinese patients with hepatocellular carcinoma

To investigate the expression of cancer-testis antigen (CTA) in Chinese patients with hepatocellular carcinoma (HCC), and the relationship between CTA gene expression and clinical indexes, we used one-step reverse transcription polymerase chain reaction (RT-PCR). The expression of the CTA mRNA was investigated in the tissues of HCC and corresponding peripheral blood of 37 patients with HCC. Fifteen samples of cirrhotic tissues and 15 normal tissues were examined with the same method. Two kinds of CTA (SSX-2 and SSX-5) showed high-specific and high-frequent expression in HCC tissues, but neither of them could be detected in adjacent non-HCC tissues. In corresponding peripheral blood of HCC tissues, the positive expression rate of the SSX-2 and SSX-5 mRNA was not very high. No relationship was found between the expression of CTA and clinical indicators such as age, sex, tumor size, TNM staging, serum AFP level and infection with hepatitis virus. In 15 patients with cirrhosis and 15 other non-tumor patients, none of the SSX-2 and SSX-5 mRNA was detected in liver tissue or peripheral blood. High frequency and specificity of CTAs in HCC indicates that their products may be new potential promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of the two genes in HCC provides a possibility of polyvalent vaccinations for HCC. Specific expression of CTAs was observed in AFP-negative HCC, suggested applying their mRNA as tumor markers to detect circulating HCC cells as adjuvant diagnostic tool and as indicators of recurrence and prognosis.

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.

Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma.

Silencing of the suppressor of cytokine signaling ( SOCS-1) by aberrant methylation at the CpG island in the coding region gene has been reported in hepatocellular carcinoma (HCC). However, principally, it is methylation in the 5'-noncoding region but not that in the coding region which determines the regulation of gene expression. Methylation-specific PCR was performed for the analysis of methylation status both in the 5'-noncoding region and the CpG island of SOCS-1 from 22 HCC tissue samples with adjacent non-HCC tissue samples and from two cell lines. Using primers in the CpG island, 9 of 22 HCC samples exhibited aberrant methylation of SOCS-1, while only 1 of 22 adjacent non-HCC samples did so. The unmethylation pattern was detected in 1 of 22 HCC and in 5 of 22 non-HCC samples. Thus, aberrant methylation of SOCS-1 was significantly associated with HCC ( P = 0.0076 by Fisher's exact test). Using primers in the 5'-noncoding region, aberrant methylation was observed in 12 of 22 HCC and in 2 non-HCC samples. The unmethylated pattern was observed in 5 of 22 HCC and in 10 of 22 non-HCC samples ( P = 0.0042). There was no significant correlation between the methylation status of SOCS-1 and clinicopathological findings, such as the presence or absence of cirrhosis or the histological grade of HCC. Aberrant methylation of the SOCS-1 had a significant correlation with HCC. The rate of aberrant methylation was similar in the 5'-noncoding region and in the CpG island. Aberrant methylation of SOCS-1 may be associated with hepatocarcinogenesis, although further studies are necessary.

Expression of cancer-testis antigens in hepatocellular carcinoma.

To investigate the expression of cancer-testis (CT) antigens MAGE-1, SSX-1,CTp11 and HCA587 genes in hepatocellular carcinoma (HCC) and the possibility of applying these antigens as targets for specific immunotherapy for HCC. Expression levels of MAGE-1, SSX-1, CTp11 and HCA587 mRNA were detected with reverse transcription polymerase chain reaction (RT-PCR) in HCC tissues and corresponding adjacent non-cancerous tissues from 105 HCC patients, 40 samples of cirrhosis and normal liver tissues. Genes of five samples with positive PCR results were sequenced. Of 105 HCC tissues, MAGE1, SSX-1,CTp11 and HCA587 mRNA expressions were detectable in 75.2%(79/105), 72.4%(76/105), 62.9%(66/105) and 56.2%(59/105) of HCC samples, respectively. About 93.3%(98/105), 72.4%(76/105), 48.6%(51/105) and 37.1%(39/105) of HCC tissues positively expressed at least one, two, three, and four members of CT antigens, respectively. Conversely, only SSX-1 could be detectable in 2.9%(3/105) of the corresponding adjacent non-HCC tissues in which no metastatic lesion was found. Of the latter 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated no expression of SSX-1 mRNA in these two samples. In addition, none of 40 samples of cirrhotic and normal liver tissues expressed CT antigen gene mRNA. DNA sequences confirmed that the RT-PCR products were true target cDNA. No relationship was found between expression of CT antigens and clinico pathological indicators such as age, gender, tumor size, degree of tumor differentiation, serum alpha-fetoprotein level and infection of hepatitis B virus or hepatitis C virus (P>0.05). CT antigens genes (MAGE-1, SSX-1, CTp11 and HCA587) are expressed with high percentage and specificity in HCC and their products are promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of multiple members of CT antigens in HCC provides possibility of polyvalent vaccinations for HCC.

Analysis of differentially expressed genes in human hepatocellular carcinoma using suppression subtractive hybridization.

The genetic basis of hepatocellular carcinoma (HCC) has not yet been fully understood. Although various methods have been developed to detect differentially expressed genes in malignant diseases, efficient analysis from clinical specimens is generally difficult to perform due to the requirement of a large amount of samples. In the present study, we analysed differentially expressed genes with a small amount of human HCC samples using suppression subtractive hybridization (SSH). Total RNA were obtained from the hepatitis C virus-associated HCC and adjacent non-HCC liver tissues. cDNA was synthesized using modified RT-PCR, and then tester cDNA was ligated with 2 different kinds of adaptors and hybridized with an excess amount of driver cDNA. Tester specific cDNA was obtained by suppression PCR and the final PCR product was subcloned and sequenced. We identified 7 known genes (focal adhesion kinase, deleted in colon cancer, guanine binding inhibitory protein α, glutamine synthetase, ornithine aminotransferase, M130, and pepsinogen C) and 2 previously unknown genes as being overexpressed in HCC, and 1 gene (decorin) as suppressed in HCC. Quantitative analysis of gene expression using quantitative RT-PCR demonstrated the differential expression of these genes in the original and other HCC samples. These findings demonstrated that it is possible to identify the previously unknown, differential gene expression from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in HCC pathogenesis, developing the new diagnosic markers, or determining novel therapeutic targets. © 2001 Cancer Research Campaign http://www.bjcancer.com

Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal RNA gene expression in human hepatocellular carcinoma

The gene responsible for transcribing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is commonly used as a reporter gene to estimate the amount of RNA present in Northern analyses. However, recent data suggest that GAPDH gene expression may vary with the extent of cell proliferation and differentiation. 28S-ribosomal RNA (28S-rRNA) has also been employed to normalize Northern blots prepared with total RNA. In the present study, we compared the expression of GAPDH messenger RNA (mRNA) with 28S-rRNA by Northern blot analyses in human hepatocellular carcinoma tissues (HCC) and adjacent non-HCC tissues from eight patients with chronic viral hepatitis-induced cirrhosis and normal liver tissue from eight healthy control subjects. The results of the study revealed that GAPDH mRNA levels in HCC were significantly higher (14×-16×) than those in adjacent non-HCC and normal liver tissues. Conversely, 28S-rRNA levels did not vary among HCC, adjacent non-HCC, and normal liver tissues. We also demonstrated that the 28S-RNA signal was proportional to the amount of RNA loaded. These findings indicate that 28S-rRNA, rather than GAPDH mRNA, should be used as RNA loading controls for Northern blot analyses involving HCC and nontumor tissues. The findings also raise the possibility that GAPDH mRNA gene expression might serve as a diagnostic indicator for human HCC. (Hepatology 1996 Apr;23(4):734-7)