Telomerase activity in human cells closely correlates with the expression of its catalytic subunit, telomerase reverse transcriptase (hTERT). Previously, we reported the lack of telomerase activity in normal human oral fibroblasts (NHOF) and the diminution of telomerase activity during senescence in normal human oral keratinocytes (NHOK). To investigate the underlying mechanisms of telomerase regulation in both cell types, we analysed the expression, promoter activity, and methylation status of the hTERT gene. The expression of hTERT mRNA diminished in senescent NHOK, but was not detected in NHOF at any stage of replication. An exogenous hTERT promoter was active in NHOF and in senescing NHOK, indicating that the lack of hTERT gene expression resulted from alteration of the endogenous hTERT promoter. Since methylation is involved in the silencing of numerous genes, we carried out DNA methylation assays. The assay revealed that the hTERT promoter was hypermethylated in NHOF and was gradually methylated during senescence in NHOK. Treatment of NHOF and senescent NHOK with the demethylating agent 5-aza-2 0 -deoxycytidine restored the expression of endogenous hTERT mRNA. Our results suggest that hypermethylation of the hTERT promoter plays a critical role in the negative regulation of telomerase activity in normal human oral cells. (Feng et al, 1995;Harrington et al, 1997;Meyerson et al, 1997;Nakamura et al, 1997). While hTERT is not expressed in most normal human somatic tissues, hTR is expressed constitutively in most human tissues (Kim et al, 1994;Shay and Bacchetti, 1997). In normal human cells and in tumour cells, telomerase activity is positively correlated with the expression of hTERT (Kim et al, 1994;Shay and Bacchetti, 1997). Therefore, regulation of hTERT expression appears to control telomerase activity. The hTERT promoter region has been cloned and characterised Takakura et al, 1999), permitting analysis of the molecular mechanisms involved in its regulation (Cong et al, 1999;Horikawa et al, 1999;Takakura et al, 1999;Wick et al, 1999). Transcription factors c-myc, Sp1, AP2, AP4, and c-myb act as activators and several repressors, such as WT1, p53, MZF-2, and Mad1, have been identified (reviewed by Poole et al, 2001). Analysis of the hTERT promoter region also revealed the presence of CpG islands containing CG-rich methylation sites (Cong et al, 1999;Horikawa et al, 1999;Takakura et al, 1999;Wick et al, 1999). Promoter region hypermethylation associated with transcriptional loss is an alternative mode of gene silencing for several tumour-suppressor genes (Zingg and Jones, 1997;Baylin et al, 1998;Herman, 1999). The hypermethylation status of the hTERT promoter has been examined in a variety of human normal and cancer cells (Devereux et al, 1999;Dessain et al, 2000;Guilleret et al, 2002). In these studies, hTERT promoter methylation was observed in telomerase-negative, as well as in telomerase-positive cells, suggesting different modes of regulation of hTERT expression in methylation-dependent and -independ...

Purpose: From numerous reports on proteins involved in DNA repair and telomere maintenance that physically associate with human telomerase reverse transcriptase (hTERT), we inferred that hTERT/telomerase might play a role in DNA repair. We investigated this possibility in normal human oral fibroblasts (NHOF) with and without ectopic expression of hTERT/telomerase.Experimental Design: To study the effect of hTERT/ telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid.Results: Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase.Conclusions: These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.