Experimental Studies On Human Telomerase Reverse Transcriptase Expression And Regulation Mechanism In Senescence Modal Of Human Embryo Kidney

Structure and function of telomere as well as mechanism of cell senescence are important research subjects in biology. As the development of molecular technique, the relationship between telomere or telomerase and cell senescence becomes the hot area of biological research. So much research work has confirmed that the telomere serves as mitotic clock and genetic time bomb in cell replication and cell cleavage.Telomerase is a rib nucleoprotein enzymatic complex that adds telomere repeat sequences TTAGGG onto the chromosome ends, compensates for the telomeric loss that occurs with cell division and stabilizes. Telomerase consists of three components: human telomerase reverse transcriptase (hTERT), an RNA template-telomerase RNA component (hTR), another subunit is telomere associate protein (TP1). The hTR and TP1 is ubiquitously exist in both normal and malignant tissues and cells, but the hTERT component is undetectable in most normal human tissues and somatic cells, but commonly expressed in human immortal cells and embryo cells. So the expression of hTERT is thought to be paralleled with telomerase activity and considered as a rate-limiting determinant of enzymatic activity.DNA methylation plays an important role in the regulation of gene transcription. Aberrant DNA methylation is an important alternative mechanism in silencing gene transcription. It is of great interest whether expression of hTERT is also regulated by methylation of the CpG island in the promoter of hTERT gene. We hypothesized that the hTERT CpG island would be unmethylated to permit expression of hTERT in early embryo cells and methylation would occur to silence the transcription of hTERT. To investigate the relationship between DNA methylation of hTERT and cell senescense in senescence modal of human embryo kidney, we studied the length of telomere, telomerase activity, the mRNA level of hTERT and the methylation pattern in hTERT core promoter area. First of all, we established a fast senescence modal of human embryo kidney. Human embryo kidney cells were passaged until no further PDs were achieved (cell number showed little or no increase within a period of 30 days). Phase-contrast photomicrographs showed PD4, PD8, PD12, PD16, PD18 and senescent PD20, which is enlarged and flattened in appearance. Cells at subconfluent density by serial passaging were stained for SA-β-Gal. The cells were photographed under phase-contrast optics. Representative photomicrographs are shown. Senescent cells PD20 display a blue stain, showing very high expression in contrasting with lower levels in younger cells. Southern blot hybridization was performed using telomere specific probe (TTAGGG)4 and genomic DNA extracted from different PDs for measuring telomere length. Representative example of a TRF-Southern blot.Larger variability was found during PD12 and PD20. The telomerase activity in different PDs of human embryo kidney cells was measured by TRAP assay.The telomerase activity disappeared completely in PD16. hTERT mRNA expression was measured by RT-PCR. RT-PCR analysis was performed on total RNA from different PDs.The expression of hTERT was much lower in PD12 and disappeared in PD16. The methylation of hTERT promoter sequence was measure by methylation specific PCR. It was turned out that the core protmoter of hTERT was partly methylated in PD16.Methylation palyed an important part in graduating gene expression, and our research work was confoused on the role of epigenetic modification in the mechanism of cell senescense. All these research may be important in cell cleavage and cell senescence.