The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit

According to the telomere hypothesis of senescence, the progressiveshortening of telomeres that occurs upon division of normal somatic cellseventually leads to cellular senescence. The immortalisation of human cellsis associated with the acquisition of a telomere maintenance mechanismwhich is usually dependent upon expression of the enzyme telomerase. Aboutone third of in vitro immortalised human cell lines, however, have nodetectable telomerase but contain telomeres that are abnormally long. Thenature of the alternative telomere maintenance mechanism (referred to asALT, for Alternative Lengthening of Telomeres) that must exist in thesetelomerase-negative cells has not been elucidated. It has previously beenshown that abnormal lengthening of yeast telomeres may occur due tomutations in the yeast telomerase RNA gene. That this is not the mechanismof the abnormally long telomeres in ALT cell lines was demonstrated by thefinding that seven of seven ALT lines have wild-type human telomerase RNA(hTR) sequence, including a novel polymorphism that is present in 30% ofnormal individuals. We found that two ALT cell lines have no detectableexpression of the hTR gene. This shows that the ALT mechanism in these celllines is not dependent on hTR. Expression of exogenous hTR via infection ofthese cells with a recombinant hTR-adenovirus vector did not result intelomerase activity, indicating that their lack of telomerase activity isnot due to absence of hTR expression. We conclude that the ALT mechanism isnot dependent on the expression of hTR, and does not involve mutations inthe hTR sequence.