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Vol. 4, 693-696, Marc/i /998 Clinical Cancer Research 693

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

Stella M. Melana, James F. Holland, and target for cancer treatment (9). Recently, the effect of AZT on Beatriz G-T. Pogo2 Chinese hamster ovary cells that display telomerase activity was investigated. AZT was preferentially incorporated into telomeric Department of Medicine, Division of Neoplastic Diseases [S. M. M.. J. F. H.. B. G-T. P.]. and Department of Microbiology [B. G-T. P.]. DNA and Z-DNA-containing regions (2). AZT. alone or in Mount Sinai School of Medicine, New York, New York 10029 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 te- ABSTRACT lomere shortening in immortalized B and T human lymphocytic The effect of zidovudine (3’-azido-3’-deoxythymidine; cell lines ( 1 1 ). Taken together. these results have stimulated AZT) was investigated in four breast cancer cell lines, a T4 further research on the effect of AZT on cancer cells. We have, cell leukemia, and a normal breast cell line in vitro. AZT therefore, investigated the effect of AZT on breast cancer cells inhibited the growth of all tumoral cell lines, but it did so in that possess tebomerase activity. The results indicated that AZT a wide range of concentrations. The growth of a normal inhibits breast cancer cell growth. anchorage-independent breast cell line was also inhibited, although it required a growth. and telomerase activity. 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 MATERIALS AND METHODS in combination, as an anti-breast cancer agent. Cells. Four breast cancer lines [MCF-7 ( 12). T47D. (13) and two breast cancer cell lines, developed in our laboratory and INTRODUCTION designated ED and EK-2]. a T4 lymphocytic leukemia cell line Zidovudine, or AZT,3 is currently used in the treatment of (Jurkat cells), and a normal breast cell line [MCF-1OF (14)] AIDS. AZT is phosphorylated intracellularly to AZT-TP by were used. thymidine kinase and is finally incorporated into viral DNA. MCF-7 and T47D cells (from American Type Culture blocking chain elongation by RT. AZT-TP can also be incor- Collection) were grown as monolayers in RPMI 1640 supple- porated into eukaryotic DNA in place of thymidine, although it mented with 100 mg/mI streptomycin. 100 units/mI penicillin. has low affinity for DNA polymerases a and 3 (1). Although it 10% FCS, and 0.2 IU/ml of insulin. Jurkat cells (a gift from Dr. was developed as an antineoplastic agent, AZT was not consid- B. Hoyos) were grown in the same medium without insulin. ered as a potential antitumor agent because of its relatively high MCF-lOF cells (a gift from Dr. Mira y Lopez, Mount Sinai cytotoxicity in animals when administered in drinking water (2). School of Medicine) were grown in 1 : 1 DMEM:Ham’s F-I 2 However, it was found to have very low toxicity when admin- Nutrients medium supplemented with penicillin and streptomy- istered as a bolus. Recently, AZT has been used in Phase I and din as above, 0.1 mg/mI cholera enterotoxin, 1 p.g/ml insulin. II clinical trials alone or in combination with other drugs in 0.5 p.g/ml hydrocortisone, 2 ng/ml epidermal growth factor, 40 gastrointestinal cancers. Some tumor regression in colorectal mM CaCI,, and 5% horse serum. ED and EK-2 cells were cancers has been reported (3- 8). derived from heparinized pleural effusions from two patients Telomerase, a RNA-dependent DNA polymerase. main- with breast cancer following the procedure of Engel et a!. (15) tains and elongates tebomeres in human germ lines and stem and maintained in MEGM medium (Clonetics) supplemented cells. Reduction of telomeres occurs during differentiation and with streptomycin and penicillin. as described above, and 10% normal aging. Telomerase activity has been strongly associated FCS. ED-R and ED-F are clones of ED. All cells were incubated with immortalization of cells and cancer and is considered as a at 37#{176}Cinthe presence of 5% CO,. Doubling time was calcu- bated 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

Received 6/18/97; revised I 1/18/97: accepted 12/19/97. counted at different times after exposure. The number of cells The costs of publication of this article were defrayed in part by the alive was determined by the trypan blue exclusion method. payment of page charges. This article must therefore be hereby marked Telomerase Assays. Cell extracts were prepared accord- advertisement in accordance with 18 U.S.C. Section 1734 solely to ing to the conditions described by Kim and co-workers ( 17). The indicate this fact. oligonucleotides TS (5’-AATCCGTCGAGCAGAGTT-3 ‘) and i Supported by grants from The T. J. Martell Foundation for Leukemia. Cancer and AIDS Research and The Chemotherapy Foundation. CX (5’-CCCTFACCCTTACCCTACCCTAA-3 ‘ ) were purified 2 To whom requests for reprints should be addressed, at Division of by high-performance liquid chromatography and dissolved at 50 Neoplastic Diseases, Mount Sinai School of Medicine, One Gustave L. ng/ml. CX primer, 0-1 p.1, was lyophilized in the reaction tube, Levy Place, New York, NY 10029. Phone: (212) 241-8822; Fax: and 15 p1 of wax were added on top. The TS primer was 5’ end (212) 860-7186. labeled with [“i-32PIATP by the T4 polynucleotide kinase. pu- 3 The abbreviations used are: AZT, 3’-azido-3’-deoxythymidine: TP. triphosphate; MP. monophosphate: RT. reverse transcriptase. rifled through a Sephadex G-25 column, and diluted with non-

Table / Effect of AZT on human cells I 2345 Cell line Doubling time (hr) AZT (IC5).

ED-F1” 40.0 0.25 ED-R3, 17.5 0.50 T47D 29.0 0.75 LiI;. I ED2 19.8 1 .(X) EK-2 77.0 I .00 MCF-7 26.7 1.35 Jurkat 12.0 ISO -. MCF-lOF 67.3 1.75

(, p. passage number. ‘,i S Table 2 Effect of AZT on cloning efficiency of human breast cancer cells

Cloning efficiency (%)

Cell line Control AZT Inhibition (%) MCF-7 4.70 1.06 77.5 T47D 1.60 0.27 83.0 ED-R 1.12 0.001 99.9 EK-2 0.20 0.001 99.9 MCF-lOF 0 ‘p.1. Fig. I Telomerase activity in MCF-7 cells measured by the TRAP method using different concentrations of cell extract. Lane 1. 24 p.g; Lane 2, 12 p.g; Lane 3, 6 pg; Lane 4, 3 p.g; Lane 5. 0.3 p.g. Experi- radioactive TS to a specific activity of 5 X 106 cpmlp.g (18). mental conditions were as described in “Materials and Methods.” The TRAP reaction mixture (17) was added, and the reaction was allowed to proceed for 30 mm at 23#{176}C,followed by 27 cycles of 30 s at 94#{176}C,30 s at 50#{176}C,and 90 s at 72#{176}C.One aliquot of the reaction (40 p.1) was analyzed by electrophoresis Effect of AZT on Telomerase Activity. Since the pres-

on 15% polyacrylamide nondenaturing gel, with 0.045 M Tris- ence of an endogenous inhibitor of telomerase activity has been

borate buffer with 0.001 M EDTA and run at 200 V for 12 h. reported. its presence was evaluated in MCF-7 cells using dif- Gels were exposed overnight at 4#{176}C.In some experiments the ferent concentrations of cell extracts ( 17). The results of telom- TRAPeze telomerase detection kit (Oncor, Inc.) was also used. erase activity in MCF-7 cells using the “TRAP reaction” are

Cell Growth in Soft Agar. Cells were resuspended in shown in Fig. 1 . The best activity was detected using either 3 or

0.45% agar (Difco Laboratories) in culture medium and seeded 0.3 p.g of protein of cell extract (Fig. 1 , Lanes 4 and 5). At in 35 X 10-mm dishes containing a base of 0.6% agar in culture higher concentrations of cell extract (Fig. 1, Lanes 1-3), the medium at the concentrations of l0, l0, and l0 cells/dish. activity was less, suggesting the presence of an inhibitor. The The appropriate AZT IC50 for each cell line was incorporated in effect of AZT on telomerase activity was then assessed using 0.3 the medium except for controls. Cells were incubated for 3 jig of protein of cell extract. The activity was partially decreased weeks at 37#{176}Cand 5% CO,. and then colonies were counted. at 1 mi and completely inhibited at 2 mri (Fig. 2). Similar results were obtained with the other breast cancer cell lines. As RESULTS previously reported, tebomerase activity in MCF-lOF cells was Evaluation of Cytotoxicity. The effect of AZT on cell almost undetectable (Fig. 3), and therefore, AZT did not have growth was tested using different concentrations. AZT inhibited any effect on it. the growth of all of the cells tested. However, the IC50 varied from 0.25 to 1 .35 mM as shown in Table 1 for the breast cancer DISCUSSION cell lines, whereas the ICS() for the immortalized normal breast The results reported here indicate that AZT is able to cell line was I .75 mM. To evaluate whether rate of growth was inhibit the growth of breast cells in vitro. The IC50 varies from rebated to the variation in IC50, the doubling time of the cell lines 0.25 to 1 .35 mM for the cancer cell lines, whereas it is higher for was also determined. When the two parameters were compared the immortalized normal breast epithelial cell line MCF-l0. No by correlation analysis, no correlation between the rate of correlation of IC50 with doubling time is seen. Therefore, the growth and 1C5() was found (Table 1). IC50 is not related to the rate of cell growth. Variations in the Cell Growth in Soft Agar. The effect of AZT on IC50 may reflect differences in absorption, excretion, and/or anchorage-independent growth was evaluated by measuring col- phosphorylation of the compound between the cell lines. ony formation in agar. As is shown in Table 2, at the IC50, AZT At the respective IC50 for each cell line, AZT is able to inhibited colony formation of four breast cell lines to different inhibit colony formation in soft agar to different degrees (Table degrees. 2). Although the number of colonies decreased, the number of

I 2345 I 2 34

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Fig. 2 Effect of AZT on telomerase activity. MCF-7 cells were incubated with different AZT concentrations for 72 h, and 0.3 p.g of cell extract was used to perform the TRAP procedure. Lane I. control cells; Lane 2, 0.5 mM; Lane 3, 1.0 mM; Lane 4, 1.5 mM; Lane 5, 2.0 mM AZT. U... single cells did not decrease below the total cell and colony Fig. 3 Telomerase activity in MCF-1OF cells measured by the TRAP number of the untreated cultures, indicating that the drug was method using different concentrations of cell extract. Lane 1. 12 p.g: Lane 2. 6 l.Lg; Lane 3. 3 p.g; Lane 4, 0.3 p.g. Experimental conditions growth inhibitory but not cytocidal for breast cancer cells. were as described in “Materials and Methods.” Finally, AZT inhibited telomerase activity of all of the breast cancer cells that were tested. As a result of telomerase inhibition, we have observed that MCF-7 cells cultured for five passages with AZT at the IC50 showed a marked reduction in the (Ref. 23). Furthermore, Patience et a!. (24) reported that there amount of telomeres.4 is expression of endogenous retrovirab sequences in T47D cells, It was previously reported that when cell extract concen- whereas Wang et a!. (25) showed the presence of mouse mam- trations were high (17), inhibitor(s) of the reaction could be mary tumor virus-env like gene sequences in 38% of human detected in the cell extract. Our results indicate that in the breast breast cancers and in some of the breast cancer cell lines, cancer cell lines tested, an endogenous inhibitor of the telom- including ED, EK-2, and MCF-7. This would imply the erase reaction was also present, consistent with the results of involvement of a retroviral activity as a target for AZT. On others (17). All experiments were conducted at the cell extract the other hand, all of the breast cancer lines tested by us and concentrations below the recognizable endogenous inhibitory the human breast tumors studied by Hiyama et a!. (26) and effect. Bednarek et a!. (27) exhibited telomerase activity. Therefore, The mechanism by which AZT inhibits growth of breast both enzymatic activities can be affected by AZT. But the cancer cells in culture is complex. AZT is known to inhibit viral effect of AZT on other enzymes, such as DNA polymerase -y RT and integrase activities (19), as well as cellular enzymatic and thymidine kinase, which are present in all cell types, activities such as DNA pobymerase -y (1, 20), thymidine kinase cannot be ruled out. However, AZT has low affinity for these (21, 22), and telomerase (1 1) and has also been shown to be enzymes. In this context, it is remarkable that the growth of preferentially incorporated into tebomeric DNA (9). In all cases, MCF-lOF cells, which lack both RT and telomerase activi- AZT-TP is the active molecule. ties, is also inhibited by AZT, although at higher drug con- Breast cancer cell lines T47D, MCF-7, and ED displayed centrations. RT activity in the culture medium, whereas MCF-lOF did not The active form of AZT is AZT-TP, which competes with the endogenous UP. Conversion from the MP form (AZT-MP) to AZT-TP is mediated by thymidylate kinase and is very inefficient in most cells. It has been shown recently that the

4 Unpublished results. interaction between AZT-MP and the enzyme is structurally

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Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3'-azido-3'-deoxythymidine.

S M Melana, J F Holland and B G Pogo

Clin Cancer Res 1998;4:693-696.

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