Regulation of human telomerase activity: Repression by normal chromosome 3abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-r egulated, thereby removing a critical barrier for unlimited cell proliferat ion. To understand more about hTERT regulation, we measured hTERT RNA level s by quantitative reverse transcription (RT)PCR. Telomerase-positive cell l ines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules wa s below the sensitivity of the assay (<0.004 molecules/cell). Intron-contai ning, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulate d. Microcell transfer of a normal chromosome 3 into the human breast carcin oma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromo some 3 hybrids, even in cells permanently expressing a transfected hTERT cD NA. However, chromosome 3 transfer did not affect the expression of green f luorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investi gated whether chromosome 3 transfer affected c-Myc activity. An at least 30 -fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by c omplex mechanisms involving regulatory elements distant from the 5' flankin g region, and that the putative hTERT repressor on chromosome 3 does not re gulate the expression of hTERT through c-Myc or one of its coregulators.