Differentially expressed genes throughout the cellular immortalization processes are quite different between normal human fibroblasts and endothelial cells

It is widely accepted that activation of telomerase and maintenance of telomeres play central roles in cellular immortalization for most cancer cells. However, they seem to be insufficient for normal human cells. To elucidate critically responsible genes for telomerase mediated cellular immortalization in non-cancerous cells, we explored the genes that are differentially expressed throughout the immortalization process of normal human cells using cDNA microarrays with novel normalization procedures. We found that the number of genes, differentially expressed during cellular immortalization after ectopic expression of telomerase, dramatically increased in a later phase, especially in fibroblasts. We identified 18 and 20 genes/ESTs dysregulated throughout the cellular immortalization processes in fibroblasts and endothelial cells, respectively, but none of them overlapped. Only BGN and COL5A2 were commonly downregulated, except for at early phase in fibroblasts, and a few genes showed controversial expression changes, with regard to previous reports in cancer cells. These findings indicate that normal somatic cells would require cell-type specific events in addition to telomerase activation, and a rare population that eventually experience such events would acquire immortality. The key molecules that distinguish the immortalization mechanisms in cancerous and non-cancerous cells may become crucial targets for anticancer therapy and regenerative therapy.     

Differential gene expression of human telomerase-associated protein hTERT and TEP1 in human hematopoietic cells

The maintenance of telomere length is crucial for the survival of cells. Recently, genes for proteins that consist of human telomerase have been cloned and the results have indicated a close relationship between telomerase activity and its gene expression. We studied the mRNA expression of the telomerase-associated genes, hTERT and TEP1, in hematopoietic cells in order to clarify the relation between them and telomerase activity using semiquantitative RT-PCR. In polymorphonuclear cells and monocytes isolated from peripheral blood, which had no detectable telomerase activity, no hTERT mRNA expression was seen. On the other hand, lymphocytes and CD34-positive cells both demonstrated hTERT mRNA expression. TEP1 mRNA was detected in all samples, showing no differential expression. We then assessed hTERT and TEP1 mRNA expression in CD34-positive cells cultured in vitro with growth factors. After 4 weeks of culture, all the cells showed myeloid differentiation and the telomerase activity was downregulated. hTERT mRNA was expressed in CD34-positive cells, but was downregulated in 4-week-cultured cells. TEP1 showed no apparent differential expression. We conclude that hTERT mRNA expression is downregulated in accordance with telomerase downregulation during the course of myeloid differentiation, which suggests that it plays a crucial role in the expression of enzyme activity, while TEP1 has a much smaller role to play, if any.     

hTERT gene dosage correlates with telomerase activity in human lung cancer cell lines.

Maintenance of telomeres, most often by telomerase, is a necessary prerequisite for immortality of eukaryotic cells. To better understand the mechanisms of telomerase up-regulation during tumorigenesis, we analysed the gene dosage of hTERT on chromosome 5p15, a region known to be overrepresented in a variety of malignancies, in 20 lung cancer cell lines by Southern blotting, fluorescence in-situ hybridization, and comparative genomic hybridization. We found a significant correlation between hTERT gene dosage, hTERT mRNA expression and telomerase activity. Imbalances of chromosome 5p may exert functionally relevant hTERT gene dosage effects in human lung cancer.     

Haploinsufficiency of hTERT leads to telomere dysfunction and radiosensitivity in human cancer cells

One of the most consistent differences between cancer cells and normal somatic cells is the continuous expression of telomerase, an enzyme that is important for maintenance of chromosome ends, or telomeres. It is believed that telomerase expression allows cancer cells to maintain their telomeres after many cell divisions and thereby avoid replicative senescence. We have tested this hypothesis by targeting the gene encoding the catalytic subunit of the telomerase holoenzyme, hTERT, in a human cancer cell line. Heterozygous disruption of hTERT led to a reduction in telomerase activity, telomere shortening, activation of DNA damage signaling and the appearance of a subpopulation of cells that displayed features of senescence. Targeted cells were radiosensitive, as compared with parental controls that had two intact hTERT alleles, and expressed a classical marker of senescence after irradiation. These results suggest that telomerase inhibitors might be useful in the sensitization of cancer cells to DNA damaging agents.     

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.     

端粒酶逆转录酶在脐静脉内皮细胞耐受曲古抑菌素A诱导凋亡中的作用

目的观察曲古抑菌素A（TSA）对脐静脉内皮细胞及官颈癌细胞凋亡、端粒酶逆转录酶（hTERT）表达的影响,并探讨hTERT在脐静脉内皮细胞耐受TSA中的作用。方法磺酰罗丹明B （RSB）法检测药物动力学特征;流式细胞仪检测周期改变和凋亡;RT-PCR检测hTERT和p21^Waf1基因表达变化;免疫荧光结合流式细胞术检测hTERT蛋白表达变化;转染hTERT质粒后,PCR-TRAP- ELISA法检测转染细胞端粒酶活性;AnnexinV／PI检测转染细胞在TSA作用下的早期凋亡。结果在大剂量TSA作用脐静脉内皮细胞后,增殖抑制、周期阻滞,但凋亡并不显著;HeLa细胞在相同剂量的TSA作用下凋亡明显。脐静脉内皮细胞经TSA诱导后,hTERT表达上调,p21^Waf1则无明显变化;而HeLa细胞p21^Waf1表达上升,hTERT表达下降。转染显性负突变hTERT的脐静脉内皮细胞,其端粒酶活性显著低于对照组。TSA作用转染不同质粒的脐静脉内皮细胞的凋亡率与对照组差异有统计学意义。结论脐静脉内皮细胞可以耐受大剂量TSA诱导的凋亡,hTERT表达上调可能是脐静脉内皮细胞耐受TSA诱导凋亡的重要机制之一。