INHIBITION OF HUMAN TELOMERASE BY A RETROVIRUS EXPRESSING TELOMERIC ANTISENSE RNA

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG re peats to chromosome ends, is selectively expressed in immortalised cel ls and most tumours, suggesting a potential role for telomerase inhibi tors in cancer therapy. Replication-deficient retroviruses were used t o determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to in hibit telomerase activity. Telomerase activities measured by the telom eric repeat amplification protocol (TRAP) assay in extracts prepared f rom immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clon es expressing UUAGGG. Telomerase activity was not inhibited by express ion of mRNA containing a transposed sequence, GGGAUU. Telomerase activ ities in vivo were inferred from changes in cellular morphology, proli feration capacity, growth rate and measurement of the content of telom ere DNA. Giant senescent-like cells emerged shortly after cloning mous e PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one cu lture to 2-6% at 50 PD in several other cultures. Giant cells were abs ent in all parental cells and clones expressing GGGAUU. The average ce llular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in v itro and in vivo, but that the effect of inhibition on individual cell s is highly variable. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.