Liver Cancer Samples Research Articles

The imprinting status of genetic imprinted gene PEG10 in human hepatocellular carcinomas

To study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines. Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing. It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05). PEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.

Abstract 158: The landscape of DNA hypomethylation in liver cancer, its correlation with other cancers, and its potential role in cancer

Abstract DNA demethylation, although a common hallmark of cancer, has been relatively ignored. We used hepatic cellular carcinoma, which is one of the most common human cancers, as a model to delineate the landscape of hypomethylation in cancer, unravel the driving forces of hypomethylation and determine its potential role in cancer progression, metastasis and invasion. Using liver cancer cell lines as a model system, we tested whether the demethylation observed in liver cancer is driven by MBD2, a protein previously implicated in demethylation, which we found to be overexpressed in liver cancer. We used genome-wide promoter arrays and methylated DNA immunoprecipitation to map the sites in the genome that lost methyl marks in liver cancer patients. Affymetrix arrays were used for gene expression studies. The array data were validated by pyrosequencing and QPCR. For functional analyses, we used siRNA transfection, soft agar and invasion assays. We discovered that about 3700 transcript-encoding genes were demethylated in liver cancer samples compared to adjacent normal tissue. At least 350 of these 3700 genes were significantly induced in tumors. In the group of demethylated and induced genes, we identified a distinct subgroup of over 100 genes with CpG dense promoters that were highly methylated in normal liver, but demethylated in the tumors. Gene ontology analyses revealed that these genes are involved in cell growth, cell adhesion, signal transduction and invasion. Examination of the Ensemble methylome data suggests that these promoters are differentially methylated in a tissue specific manner but are always methylated in liver. All of the demethylated genes in liver cancer were found to be demethylated in other cancers suggesting that the demethylation of these genes is a basic requirement in cancer. The experiments in HepG2 and SkHep1 cells showed that knock-down of MBD2 with siRNA results in suppression of the majority of the hypomethylated genes and in a relevant decrease in cancer cell growth and invasion. Furthermore, the down-regulation of these genes was accompanied by an increase in promoter methylation and a decrease in MBD2 binding. It suggests that MBD2 is required for demethylation and up-regulation of these genes, which appear to be putative targets for MBD2. Our results demonstrated that DNA hypomethylation drives the coordinated expression of several gene circuitries involved in cancer growth, survival and metastasis. We showed that MBD2 is involved in driving this processes by affecting methylation and expression of important players in liver cancer growth and metastasis. Our studies established for the first time the rules governing hypomethylation in liver cancer and defined the potential functional role of hypomethylation. This study was supported by a grant from the MDEEI program of the government of Quebec and the National Cancer Institute of Canada to MS and MOST to ZGH Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 158.

Organ‐specific endoglin (CD105) expression in the angiogenesis of human cancers

Some markers of angiogenic endothelial cells are emerging as targets for cancer therapy. The present study compared the expression of CD105 with that of other endothelial markers in cancers from various organs. Surgically resected cancer tissues from 188 patients comprising brain (n = 17), lung (n = 38), breast (n = 30), stomach (n = 30), colon (n = 31), liver (n = 32), and kidney (n = 10) cancers were immunohistochemically analyzed on tissue microarrays using a panel of eight endothelial markers. CD31 was expressed in vascular endothelial cells in cancer lesions as well as in non-cancerous areas (30-100%) in all core tissue samples. CD105 expression was intense and restricted to capillary endothelial cells in cancer lesions (>73%). In contrast, positive expression of CD105 was seen in <20% of non-cancerous areas in the same organs. However, no significant difference in CD105 expression in vascular endothelial cells between cancer lesions and non-cancerous areas from liver and renal cancer samples was found. Vascular endothelial growth factor (VEGF), Flt1, and Flk1 were also expressed, but only sporadically and in few samples (<30%), and transforming growth factor (TGF)-beta1 and TGF-betaRII were negative in vascular endothelial cells but generally positive in cancer cells. CD44 was strongly expressed in sinusoidal endothelial cells of the liver (90-100%). These results show that CD105 is expressed specifically in the tumor angiogenesis of brain, lung, breast, stomach, and colon cancers.

Identification of two novel human dynein light chain genes, DNLC2A and DNLC2B, and their expression changes in hepatocellular carcinoma tissues from 68 Chinese patients

Two full-length cDNAs, DNLC2A and DNLC2B, were cloned and characterized. Their open reading frames respectively encode 96 amino acids which are most closely homologous to roadblock/LC7, one member of an ancient dynein light chain protein family, conserved in nematode, fruit fly, mouse and rat. The DNLC2A was expressed in 12 of 16 human tissues examined, with especially strong expression in heart, liver and brain, whereas there was weak expression in lung, prostrate, testis, small intestine and colon. The expression of DNLC2B was generally high compared with that of DNLC2A except in liver. Northern blotting and/or semi-quantitative RT–PCR analysis examined the expression changes of DNLC2A and DNLC2B in 68 hepatocellular carcinoma tissue samples. It was revealed that DNLC2A was up-regulated (45 out of the 68 cases) while DNLC2B was down-regulated (44 out of 68 cases), compared with their adjacent tumor-free liver tissues. Interestingly, among the total 68 liver cancer samples tested, DNLC2A was up-regulated while DNLC2B was down-regulated in 28 cases; DNLC2A was up-regulated while no obvious change was observed for DNLC2B in 10 cases; no obvious change was observed for DNLC2A while DNLC2B was down-regulated in 14 cases. Although the underlying mechanism is not clear to date, the apparent up-regulation of DNLC2A and down-regulation of DNLC2B suggest that these genes might be involved in tumor progression. On the other hand, the different expression changes of the two homologous genes indicate that hepatocellular carcinomas are caused by different pathological mechanisms. In addition, DNLC2A was assigned to human chromosome 20q12–q13.11 near the marker D20S106 by radiation hybrid mapping.

The thermal activity of normal and malignant tissues. Microcalorimetric response of liver cancer to hepatic artery ligation, cryosurgery, adriamycin and norcantharidin.

The usefulness of metabolic heat measurements in quantifying the response of a solid tumour to anticancer treatment was evaluated. The heat production characteristic of malignant tissues, as measured from human stomach, breast and liver cancer samples, was observed to be inconsistent, and its value could be higher or lower than that of its normal tissue of origin. The various thermal activity responses of an experimental rat hepatoma to hepatic artery ligation, cryotherapy, intra-arterial (i.a) Adriamycin (2.4 mg/ kg), i.a. Norcantharidin (0.5 mg/kg) were next studied. The tumour/liver (T/L) ratio of untreated tumour-bearing rats was 0.83 but this fell to a minimum at 24 h in both the hepatic artery ligation and the cryosurgery groups. In these two groups marked fluctuations in the heat production of normal liver occurred with poor recovery of the T/ L ratio even at 2--3 weeks. In the Adriamycin group, the T/L ratio dropped to a minimum at 5 days, and in the Norcantharidin group, at 3 days. Minimal disturbances in the thermal activity of liver tissue occured in these two chemotherapy groups and the T/L ratio recovered by 3 weeks. Norcantharidin appeared as efficacious as Adriamycin in the treatment of hepatoma when evaluated in terms of thermal activity.

Overexpression of glutamine synthetase in human primary liver cancer

Background/Aims: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. Methods: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. Results: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3′ untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. Conclusions: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

High prevalence of mutations at codon 249 of the p53 gene in hepatocellular carcinomas from Senegal.

In hepatocellular carcinoma, mutation within the p53 gene occurs mainly at codon 249 and its frequency has been associated with exposure to aflatoxin. As Senegal is a country where liver cancer incidence is one of the highest in the world and where people are highly exposed to aflatoxin, we screened 15 liver cancer samples from this country for mutation at codon 249 of the p53 gene. Non-tumoral DNA from the patients showed a wild type genotype. Mutation at codon 249 of the p53 gene was detected in 10 of the 15 tumour tissues tested (67%). This frequency of mutation in codon 249 of the p53 gene is the highest described. These results confirmed that there is an association between countries of high aflatoxin intake and a high frequency of mutation in codon 249 of p53 gene, and that HBV alone does not contribute to these base changes.