No abstract

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus‐transformed cell cultures were also negative in the pre‐crisis (i.e. non‐immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase‐negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase‐negative and telomerase‐positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

Telomerase, the enzyme that synthesizes telomeric DNA, is not expressed in most human somatic cells but is activated with in vitro immortalization and during tumorigenesis, and repressed by cell differentiation. Of the two components of the core enzyme, the catalytic protein hTERT is limiting for activity. To investigate mechanisms of hTERT gene regulation, we have cloned genomic sequences encompassing the complete hTERT transcription unit. The hTERT gene consists of 16 exons and 15 introns spanning approximately 35 kb. Transient transfections of immortal human cells with potential regulatory 5' sequences linked to a reporter, combined with deletion analysis of these sequences, indicated that elements responsible for promoter activity are contained within a region extending from 330 bp upstream of the ATG to the second exon of the gene. Assays in different cell types have shown that the hTERT promoter is inactive in normal and in transformed pre-immortal cells, but, like telomerase, it is activated with cell immortalization. Sequence analysis revealed that the hTERT promoter is GC-rich, lacks TATA and CAAT boxes but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggests the possibility that hTERT expression may be subject to multiple levels of control and be regulated by different factors in different cellular contexts.