Inhibition of Cell Growth and Telomerase Activity of Breast Cancer Cells in Vitro by 3’-Azido-3’-deoxythymidine1

The effect of zidovudine (3’-azido-3’-deoxythymidine; AZT) was investigated in four breast cancer cell lines, a T4 cell leukemia, and a normal breast cell line in vitro. AZT inhibited the growth of all tumoral cell lines, but it did so in a wide range of concentrations. The growth of a normal breast cell line was also inhibited, although it required a much higher concentration. Furthermore, AZT inhibited colony formation in soft agar and telomerase activity. These results indicated that AZT can be potentially used, alone or in combination, as an anti-breast cancer agent. INTRODUCTION Zidovudine, or AZT,3 is currently used in the treatment of AIDS. AZT is phosphorylated intracellularly to AZT-TP by thymidine kinase and is finally incorporated into viral DNA. blocking chain elongation by RT. AZT-TP can also be incorporated into eukaryotic DNA in place of thymidine, although it has low affinity for DNA polymerases a and 3 (1). Although it was developed as an antineoplastic agent, AZT was not considered as a potential antitumor agent because of its relatively high cytotoxicity in animals when administered in drinking water (2). However, it was found to have very low toxicity when administered as a bolus. Recently, AZT has been used in Phase I and II clinical trials alone or in combination with other drugs in gastrointestinal cancers. Some tumor regression in colorectal cancers has been reported (38). Telomerase, a RNA-dependent DNA polymerase. maintains and elongates tebomeres in human germ lines and stem cells. Reduction of telomeres occurs during differentiation and normal aging. Telomerase activity has been strongly associated with immortalization of cells and cancer and is considered as a Received 6/18/97; revised I 1/18/97: accepted 12/19/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i Supported by grants from The T. J. Martell Foundation for Leukemia. Cancer and AIDS Research and The Chemotherapy Foundation. 2 To whom requests for reprints should be addressed, at Division of Neoplastic Diseases, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-8822; Fax: (212) 860-7186. 3 The abbreviations used are: AZT, 3’-azido-3’-deoxythymidine: TP. triphosphate; MP. monophosphate: RT. reverse transcriptase. target for cancer treatment (9). Recently, the effect of AZT on Chinese hamster ovary cells that display telomerase activity was investigated. AZT was preferentially incorporated into telomeric DNA and Z-DNA-containing regions (2). AZT. alone or in combination with other antimetabolites, also inhibited the growth of human bladder cancer and colon cancer cell lines (10). Furthermore, AZT was shown to cause progressive telomere shortening in immortalized B and T human lymphocytic cell lines ( 1 1 ). Taken together. these results have stimulated further research on the effect of AZT on cancer cells. We have, therefore, investigated the effect of AZT on breast cancer cells that possess tebomerase activity. The results indicated that AZT inhibits breast cancer cell growth. anchorage-independent growth. and telomerase activity. MATERIALS AND METHODS Cells. Four breast cancer lines [MCF-7 ( 12). T47D. (13) and two breast cancer cell lines, developed in our laboratory and designated ED and EK-2]. a T4 lymphocytic leukemia cell line (Jurkat cells), and a normal breast cell line [MCF-1OF (14)] were used. MCF-7 and T47D cells (from American Type Culture Collection) were grown as monolayers in RPMI 1640 supplemented with 100 mg/mI streptomycin. 100 units/mI penicillin. 10% FCS, and 0.2 IU/ml of insulin. Jurkat cells (a gift from Dr. B. Hoyos) were grown in the same medium without insulin. MCF-lOF cells (a gift from Dr. Mira y Lopez, Mount Sinai School of Medicine) were grown in 1 : 1 DMEM:Ham’s F-I 2 Nutrients medium supplemented with penicillin and streptomydin as above, 0.1 mg/mI cholera enterotoxin, 1 p.g/ml insulin. 0.5 p.g/ml hydrocortisone, 2 ng/ml epidermal growth factor, 40 mM CaCI,, and 5% horse serum. ED and EK-2 cells were derived from heparinized pleural effusions from two patients with breast cancer following the procedure of Engel et a!. (15) and maintained in MEGM medium (Clonetics) supplemented with streptomycin and penicillin. as described above, and 10% FCS. ED-R and ED-F are clones of ED. All cells were incubated at 37#{176}C in the presence of 5% CO,. Doubling time was calcubated as previously described (16). Determination of IC50. Cells were grown in the presence of AZT concentrations ranging from I 00 p.M to 2 msi and counted at different times after exposure. The number of cells alive was determined by the trypan blue exclusion method. Telomerase Assays. Cell extracts were prepared according to the conditions described by Kim and co-workers ( 17). The oligonucleotides TS (5’-AATCCGTCGAGCAGAGTT-3 ‘) and CX (5’-CCCTFACCCTTACCCTACCCTAA-3 ‘ ) were purified by high-performance liquid chromatography and dissolved at 50 ng/ml. CX primer, 0-1 p.1, was lyophilized in the reaction tube, and 15 p1 of wax were added on top. The TS primer was 5’ end labeled with [“i-32PIATP by the T4 polynucleotide kinase. purifled through a Sephadex G-25 column, and diluted with nonResearch. on June 7, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Table / Effect of AZT on human cells (, p. passage number. Table 2 Effect of AZT on cloning efficiency of human breast cancer cells Cloning efficiency (%) Control AZT Inhibition (%) 4.70 1.06 77.5 1.60 0.27 83.0 1.12 0.001 99.9 0.20 0.001 99.9 0 I 2345