Alteration of hTERT full-length variant expression level showed different gene expression profiles and genomic copy number changes in breast cancer

We analyzed hTERT splicing patterns with respect to telomerase activity in breast cancer. Using a cDNA microarray in 22 cell lines, we observed the difference in expression profiling based on the different levels of full-length variant expression with 71 selected genes. Using 33 known genes that act with the telomerase complex, we performed unsupervised clustering with all cell lines, and found a clustering tendency related to the full-length variant expression level. Using array-based CGH, highly altered genomic copy number changes were found more often in MCF-7 (159 genes) than in MDA-MB-231 (109 genes) and MDA-MB-435 (49 genes), suggesting more genomic changes in MCF-7 cells. On comparing MCF-7 with MDA-MB231 and MDA-MB-435 cell lines, we identified 8 genes with different copy numbers, including dystroglycan, which is located in the p12-21.2 area of chromosome 3. In conclusion, alterations in the level of the full-length variant of hTERT showed different gene expression profiles and genomic copy number changes in breast cancer, which require further study into their cause-and-effect relationship.     

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.     

DNA copy number changes in young gastric cancer patients with special reference to chromosome 19

Only a few cytogenetic and genetic studies have been performed in gastric cancer patients in young age groups. In the present study we used the comparative genomic hybridisation (CGH) method to characterise frequent DNA copy number changes in 22 gastric cancer patients of 45 years or younger and three gastric cancer cell lines established from patients younger than 45 years. Analysis of DNA copy number changes revealed frequent DNA copy number increases at chromosomes 17q (52%), 19q (68%) and 20q (64%). To confirm the CGH results and to characterise the amplicon region on the most frequently amplified chromosome, chromosome 19, we carried out fluorescence in situ hybridisation (FISH) analysis and Southern blot analysis. Fluorescence in situ hybridisation with the bacterial artificial chromosome (BAC) clone mapped to 19q12 indicated a copy number increase in all eight tumour specimens studied. Southern blot analysis of six tumour specimens and three tumour cell lines, with five probes mapped to the 19q12-13.2 region, suggested cyclin E to be one of the candidate target genes in the 19q region for gastric cancer tumorigenesis. Cyclin E protein overexpression was verified in tumours with amplification on chromosome 19. Further studies are required to investigate the biological and clinical significance of 19q amplicon and cyclin E upregulation in gastric cancer of young patients.     

Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines

We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(-) 'conditionally immortal' HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57(Kip2). We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(-/-) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(-) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.     

Functional evidence for a telomerase repressor gene on human chromosome 10p15.1

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.     

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.     

Activation of the hTERT expression in squamous cell cervical carcinoma is not associated with gene amplification

The hTERT gene encodes the telomerase catalytic subunit that plays a key role in cancer cell immortalization. Earlier, hTERT amplification was detected in squamous cell cervical carcinomas (SCC), however possible relations between elevated hTERT mRNA level and gene amplification was not studied. Here, we compared the hTERT expression and copy number in the same tumors by quantitative real-time PCR. The hTERT DNA copy number was virtually unchanged in all 33 studied tumors, when compared to normal tissues. This result was confirmed using two reference genes beta-actin and beta-D-glucuronidase. Nevertheless, the activation of hTERT expression was found in 80% of cases (37/46, p<0.001). There was no correlation between the degree of mRNA increase and the tumor size and/or presence of metastases. No hTERT gene expression was observed in 20% of cases (9/46), while the control GADPH expression was unchanged. The detected elevation of the hTERT mRNA level was found using primers specific to functionally active full-length isoform of mRNA. Similar results were obtained with SCC cell lines carrying human papilloma virus (HPV) genomes. We conclude that frequent activation of hTERT expression in SCC is not associated with gene amplification.     

端粒酶在人胚肾上皮细胞转化中的作用

目的:利用原代人胚肾上皮细胞转化模型阐明端粒酶在细胞癌变过程中的作用。方法:用逆转录病毒介导的基因导入法,使人端粒酶催化亚基(humantelomerasecatalyticsubunit,hTERT)、HRas癌基因、猿猴病毒40(simianvirus40,SV40)编码的早期抗原在原代人胚肾上皮细胞中稳定地表达,观察细胞的生长特性、染色体畸变率以及细胞转化特征。结果:端粒酶的激活虽然能延长细胞的寿命,但不能使细胞永生化。如果细胞同时表达端粒酶和SV40编码的大T抗原(largeTantigen,LT),细胞就能获得永生化。在此永生化细胞株中导入HRas癌基因以及SV40编码的小T抗原(smallTantigen,ST),细胞发生恶性转化,表现为在软琼脂上形成克隆并在裸鼠皮下形成肿瘤。与原代细胞相比,永生化细胞株的染色体畸变率无改变,而恶性转化细胞的畸变率明显增加。此外在转化细胞和几种肿瘤细胞中表达阻抑人端粒酶的特异性siRNA,能显著地抑制肿瘤细胞的生长、诱导细胞凋亡并减少软琼脂上形成的克隆数目。结论:细胞永生化是癌变过程的必要阶段,端粒酶的激活不仅是细胞永生化的重要环节,而且在维持肿瘤细胞生长中起重要的作用。