RECONSTITUTION OF WILD-TYPE OR MUTANT TELOMERASE ACTIVITY IN TELOMERASE-NEGATIVE IMMORTAL HUMAN-CELLS

Telomere shortening in human somatic cells and telomere maintenance in most human immortal cell lines and tumours correlate respectively wit h the absence and presence of telomerase, the enzyme that synthesizes telomeric DNA de novo, However, similar to 30% of in vitro immortalize d human cell lines do not express this enzyme and maintain telomeres b y an alternative pathway (ALT) that may also operate in some tumours, Human telomerase is a reverse transcriptase comprising minimally an RN A subunit (hTER) and a catalytic protein moiety (hTERT), Normal somati c cells retain expression of hTER but not of hTERT, and can be convert ed to a telomerase-positive phenotype by ectopic expression of the cat alytic protein. We similarly have restored enzymatic activity to those ALT cell lines that retain hTER expression. We also report that in th ose ALT cells that are hTER negative, reintroduction of both hTER and hTERT is necessary and sufficient for conversion to telomerase positiv ity. Moreover, transfection of these cells with hTERT in conjunction w ith hTERs with a mutated template results in the expression of an enzy me with altered specificity. Reconstitution of telomerase activity in ALT cells, particularly an activity capable of synthesizing mutant tel omeric DNA, may be exploited for the study of the ALT mechanism and it s interaction with the telomerase-dependent pathway, and for assessing the effects of mutant telomeres on cell viability.