Original Paper Inhibition of Human Telomerase by a Retrovirus Expressing Telomeric Antisense RNA M. BisoY, 1, * A.E. Chakerian, 1, * M.L. Fore, 1 J.E. Bryant, 1 J.P. Hernandez, 1 R.K. Moyzis 3,4 and J.K. GriYth 1,2 1 Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, 915 Carnino de Salud, Albuquerque, 87131-522 New Mexico; 2 Cancer Research and Treatment Center, University of New Mexico School of Medicine; 3 Center for Human Genome Studies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545; and 4 Department of Biological Chemistry, University of California at Irvine, California, U.S.A. Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromo- some ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-de®cient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is suYcient to inhibit telomerase activity. Telomerase activities mea- sured by the telomeric repeat ampli®cation protocol (TRAP) assay in extracts prepared from immortalised mouse ®broblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the ®fth population doubling (PD) in one culture to 2±6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular 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 suYcient to inhibit telomerase in vitro and in vivo, but that the eVect of inhibition on individual cells is highly variable. # 1998 Published by Elsevier Science Ltd. All rights reserved. Key words: telomerase activity, telomerase RNA, retrovirus, antisense RNA, telomerase inhibition, telomere content, cancer Eur J Cancer, Vol. 34, No. 8, pp. 1242±1249, 1998 INTRODUCTION The capacity for unlimited replication of human germ cells, immortalised cell lines and tumour cells is attributed in part to the activity of the enzyme telomerase [1±4]. Normally quiescent in somatic cells, telomerase is a ribonucleoprotein that utilises a short sequence of its integral RNA component, hTR, as a template for synthesising telomeric repeats on to chromosome ends [5, 6]. Telomere synthesis presumably counterbalances truncation of chromosomes which occurs with each replicative cycle, and thereby prevents degradation and deleterious rearrangements of chromosome ends that would otherwise lead to cell death [4, 7]. Initially identi®ed in HeLa cell extracts [8], human telomerase has been detected in immortalised cell lines [1], precrisis and postcrisis cells following retroviral infection [9], and more than 85% of tumours [1]. Low levels of telomerase activity have also been found in activated T cells and normal somatic cells with extended proliferative capacities [10, 11]. European Journal of Cancer, Vol. 34, No. 8, pp. 1242±1249, 1998 # 1998 Published by Elsevier Science Ltd. All rights reserved Pergamon Printed in Great Britain PII: S0959-8049(98)00049-5 0959-8049/98 $19.00+0.00 1242 *M. BisoY and A. Chakerian contributed equally to the present study. Correspondence to J.K. GriYth. Received 2 Oct. 1997; revised 15 Dec. 1997; accepted 18 Dec. 1997.