Cyclic Induction of Senescence with Intermittent AZT Treatment Accelerates both Apoptosis and Telomere Loss

Abstract

3'-azido-2',3'-dideoxythymidine (AZT) is phosphorylated intracellularly to 3'-azido-3'-deoxythymidine-5'-triphosphate (AZT-TP), which is incorporated into telomeric DNA, thereby blocking chain elongation. AZT is also known to inhibit reverse transcriptase, as well as other cellular enzymes including DNA polymerase gamma, thymidine kinase, and telomerase. We induced cancer cell senescence by treating MCF-7 cells with AZT in dosages of IC10 and IC20 for an extended period (about 120 population doublings (PD)). We then investigated the sequential changes in cellular growth, expression of telomerase subunits and transcription factors (c-Myc, Mad1), telomerase activity and telomere length. Senescence, apoptosis, growth delay, inhibition of telomerase activity and shortening of telomere length were all observed in a dose- and time-dependent manner. After the onset of senescence, the apoptosis rate increased slowly during early PDs. In contrast to senescence, the apoptotic rate showed little change after AZT removal, while it increased suddenly and significantly in a dose-dependent manner upon the second introduction of AZT. Continuous shortening of the telomeric length was observed with AZT, and, upon re-exposure to AZT, shortening of the telomere occurred more rapidly than with first exposure. Of the telomerase subunits, telomerase reverse transcriptase (hTERT) and c-Myc were the first to show a reduction in activity after AZT treatment, followed by changes in hTER , Mad1 and hTEP-1. Cyclic treatment with AZT initially suppressed hTERT and c-Myc, followed by suppression of hTER, Mad1 and hTEP-1. Furthermore, the treatment accelerated both telomere loss and apoptosis, even when administered at a senescence-inducing dosage level.