人端粒酶催化亚基基因启动子调控的肿瘤细胞特异表达载体

为获得端粒酶阳性肿瘤细胞特异表达载体用于癌症的基因治疗 ,克隆并构建了人端粒酶催化亚基 (hTERT)基因启动子调控的萤光素酶报告载体 .用脂质体转染法将其分别转染肿瘤细胞和正常细胞 ,检测其在肿瘤细胞和正常细胞中的转录活性 .hTERT启动子在所检测的 4种端粒酶阳性的肿瘤细胞中具有明显的转录活性 ,平均为阳性对照的 4 4 3% ;而在端粒酶阴性的正常人胚肺成纤维细胞中则无明显的转录活性 .提示hTRET启动子的转录活性在端粒酶阳性的肿瘤细胞中明显上调 ,由hTERT启动子构建的载体可能是一种新颖和有前景的肿瘤细胞特异性表达的基因治疗载体     

Increased protein stability causes DNA methyltransferase 1 dysregulation in breast cancer

We report that DNA methyltransferase 1 (DNMT1) expression is dysregulated in breast cancer. The elevated protein levels are not a result of increased mRNA levels, but rather an increase in protein half-life. We found that DNMT1 protein levels were elevated in breast cancer tissues and in MCF-7 breast cancer cells relative to normal human mammary epithelial cells (HMECs) without a concomitant increase in DNMT1 mRNA or proliferative fraction. Although DNMT1 mRNA levels were properly S-phase-regulated in both cell types, DNMT1 protein levels did not follow S-phase fraction in MCF-7 cells. Rather, an increase in DNMT1 protein stability was found for MCF-7 cells relative to HMECs, and a destruction domain was mapped to the N-terminal 120 amino acids of DNMT1, which was required for its proper ubiquitination and degradation in HMECs. Furthermore, overexpression of DNMT1 with this deleted destruction domain in HMECs resulted in significantly increased genomic 5-methylcytosine levels relative to overexpression of the full-length protein. The regulation of DNMT1 destruction via this domain may be dysfunctional in cancer cells leading to subsequent cytosine hypermethylation in the genome.     

Genomic organization of claudin-1 and its assessment in hereditary and sporadic breast cancer

Human claudin-1 is an integral protein component of tight junctions, a structure controlling cell-to-cell adhesion and, consequently, regulating paracellular and transcellular transport of solutes across human epithelia and endothelia. Recently, a claudin-1 (CLDN1) cDNA has been isolated from human mammary epithelial cells (HMECs). CLDN1 expression in HMECs, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to CLDN1 as a possible tumor-suppressor gene. In order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, we have characterized its genomic organization and have screened the four coding exons for somatic mutations in 96 sporadic breast carcinomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, we have compared the 5'-upstream sequences of the human and murine CLDN1 genes to identify putative promoter sequences and have examined both the promoter and coding regions of the human gene in the breast cancer cell lines showing decreased CLDN1 expression. In the sporadic tumors and hereditary breast cancer patients, we have found no evidence to support the involvement of aberrant CLDN1 in breast tumorigenesis. Likewise, in the breast cancer cell lines, no genetic alterations in the promoter or coding sequences have been identified that would explain the loss of CLDN1 expression. Other regulatory or epigenetic factors may be involved in the down-regulation of this gene during breast cancer development.     

Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.