Amplification of the ATP-Binding Cassette 2 Transporter Gene Is Functionally Linked with Enhanced Efflux of Estramustine in Ovarian Carcinoma Cells1
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[CANCER RESEARCH 58, 1332-1337. April 1. 1998] Advances in Brief Amplification of the ATP-Binding Cassette 2 Transporter Gene Is Functionally Linked with Enhanced Efflux of Estramustine in Ovarian Carcinoma Cells1 Naomi M. Laing, Martin G. Belinsky, Gary D. Kruh, Daphne W. Bell, Jonathan T. Boyd, Linda Barone,2 Joseph R. Testa, and Kenneth D. Tew3 Departments of Pharmacology IN. M. L.. J. T. B.. L B.. K. D. T.I and Medical Oncology ¡M.C. B.. G. D. K.. D. W. B.. J. R. T.]. Fax Chase Cancer Center, Philadelphia, Pennsylvania 19111 Abstract affinity for EM (6) suggests a mechanism by which its overexpression contributes to EM resistance. We have also detected enhanced efflux An estramustine-resistant human ovarian carcinoma cell line, SKIM, of EM in an EM-resistant cell line, suggesting that increased expres was generated to explore resistance mechanisms associated with this sion of a transporter could also contribute to EM resistance. agent. Cytogenetic analysis revealed that SKEM cells have a homoge Although EM can bind to pgp and modulate its transport activity (7, neously staining region (hsr) at chromosome 9q34. Microdissection of the 8), pgp-overexpressing cells do not display cross-resistance to EM. In hsr, followed by fluorescence in situ hybridization to SKEM and normal addition, overexpression of pgp is not detected in any EM-resistant metaphase spreads, confirmed that the amplified region was derived from sequences from 9q34. In situ hybridization with a probe specific lor .I/if?, cell lines studied to date (9). These observations suggest that a a gene located at 9q34 that encodes an ATP-binding cassette 2 (ABC2) transporter distinct from pgp may contribute to EM resistance. transporter, indicated that this gene is amplified (»-foldinthe estramus To explore further EM resistance mechanisms, we characterized tine-resistant cells. Southern analysis confirmed that ABC2 was amplified SKEM in this study, an EM-resistant human ovarian carcinoma cell in SKEM, and Northern analysis indicated that the ABC2 transcript was line (SKOV3). Cytogenetic analysis revealed an hsr in this cell line overexpressed ~5-fold. The ABC1 gene located at 9q22—31was not am located at chromosome 9q34, the location of ABC2, which encodes a plified in the resistant cells, and niRNA levels of several other ABC partially characterized ABC transporter (10). Further analysis re transporter genes were unaltered. Consistent with the concept that in vealed that ABC2 is amplified and overexpressed in SKEM, that creased ABC2 expression contributes to the resistant phenotype, we ob antisense-mediated down-regulation of ABC2 expression sensitized served that the rate of efflux of dansylated estramustine was increased in SKEM compared with control cells. In addition, antisense treatment the cell line to EM, and that SKEM displays enhanced efflux of EM. directed toward ABC2 mRNA sensitized the resistant cells to estramus This study thus suggests that overexpression of ABC2 contributes to tine. Together, these results suggest that amplification and overexpression EM resistance by functioning as an efflux pump of the drug. These of ABC2 contributes to estramustine resistance and provides the first data provide the first evidence for a critical cellular role for this new indication of a potential cellular function for this product. ABC transporter family member. Introduction Materials and Methods EM,4 a conjugate of nor-nitrogen mustard and estradici, is an Development of Estramustine-resistant Cell Lines. SKOV3 ovarian car effective chemotherapeutic agent that is presently used to treat met- cinoma cells were maintained in a-MEM medium and cloned, and then one astatic, hormone-refractory prostate cancer (1). The cytotoxic activity clone was used for all subsequent experiments. Resistant cells were developed of EM relates to its ability to induce metaphase arrest by interfering by exposure to EM at concentrations starting at 1 /J.Mwith a gradual increase with microtubule structure and function (2). Biochemical studies have to 15 /¿Mover a 6-month period. The resulting SKEM cells were maintained shown that EM binds to microtubule-associated proteins and tubulin in 15 /UMEM. (3, 4), the consequence of which is depolymerization of microtubules. Chromosome Microdissection and DNA Amplification. Metaphase tu mor cells of SKEM were harvested by overnight exposure to Colcemid (0.01 To understand mechanisms associated with resistance to this agent, /¿g/ml).Metaphase spreads were prepared on 24 x 60-mm coverslips, air- we have previously characterized EM-resistant tumor cell lines and dried, and banded with trypsin-Giemsa. A single copy of the hsr was dissected studied the effects of EM on MDR cell lines. These studies demon under a Zeiss Axiovert inverted microscope with UV light-sterilized glass strated that alterations in the expression of the ß-subunitof the target needles controlled by an Eppendorf Micromanipulator 5171, as described in protein tubulin are associated with resistance to EM and other anti- detail previously (11). Briefly, needle tips containing dissected chromosomal microtubule agents. In addition, increased expression of the ßn, material were broken off directly into a 0.5-ml thin-walled Eppendorf tube. isotype of tubulin was observed in EM-resistant prostate carcinoma Microdissected DNA was PCR-amplified by the sequence-independent ampli cells (5). The subsequent observation that this isotype has reduced fication method of Bohlander et al. (12). As a positive control, 20 pg of human placenta! DNA was amplified simultaneously. A negative control without DNA template was also included. A 10-ju.l sample of each reaction was Received 12/22/97: accepted 2/16/98. visualized by ethidium bromide staining following electrophoresis through a 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 2.0% agarose gel. 18 U.S.C. Section 1734 solely to indicate this fact. Microdissection-FISH. Metaphase spreads from SKEM cells and phyto- 1This work was supported in part by N1H Grants CA06927 and RR05539. NIH Grant hemagglutin-stimulated lymphocytes from human peripheral blood were pre CA53893 (to K. D. T.), and by an appropriation from the Commonwealth of Pennsylva nia. pared according to the method of Fan et al. (13). Cultures were synchronized 2 Present address: Viro-Pharma. Malvem, PA 19355. by treatment with 5-bromodeoxyuridine (0.18 fig/ml; Sigma Chemical Co.) for 1To whom requests for reprints should be addressed, at Department of Pharmacology, 16 h, followed by release from the block by incubation in fresh medium Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia. PA !91I1. Phone: containing thymidine (2.5 jug/ml) for 6 h. Metaphase cells were harvested, and (215)728-3137; Fax: (215)728-4333. 4 The abbreviations used are: EM. estramustine; MDR. multidrug resistance: pgp, chromosome spreads were prepared according to standard procedures. P-glycoprotein: ABC, ATP-binding cassette: hsr, homogeneously staining region; FISH, A 1-jnl aliquot of the primary PCR was labeled with biotin-16-dUTP (Enzo fluorescence in xilu hybridization; DAPI. diamidino-2-phenylindole. Diagnostics) in a secondary PCR reaction (14), and 200-400 ng (5-10 ¿tl)of 1332 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. ABC2 GENE AMPLIFICATION AND DRUG RESISTANCE this DNA were hybridized to metaphase spreads from both SKEM cells and microscope, and the images were analyzed by ISEE analysis software (Inno- normal blood lymphocytes. vision). Excitation wavelength was 360 nm, and the emission was recorded The ABC2 probe was a 1.75-kb purified PCR product of human ABC2 with a broad spectrum emission filter. cDNA. The primers used to generate the fragment were chosen from the Daunorubicin Fluorescence. Cells on coverslips were treatedwith 10 UM sequence of an expressed sequence tag representing part of the human ABC2 daunorubicin (Sigma) for 2 h. Cells were washed and imaged after various time cDNA (EST0600) and from a 500-bp sequence from the PCR analysis of intervals using the 568-nm excitation line of a Bio-Rad MRC-600 laser ABC2 message levels. The 1.75-kB fragment was gel purified, cloned into the scanning confocal microscope. TA vector (Clontech), and sequenced. The sequence exhibited nearly >90% homology with the murine ABC2 sequence. The purified probe was labeled by Results nick translation with biotin-16-dUTP (Boehringer Mannheim) and mapped to ABC2 Is Amplified in the EM-resistant Cell Line. EM-resistant normal human chromosomes. FISH and detection of immunofluorescence were carried out essentially as ovarian carcinoma cells were generated by exposing sensitive cells to described previously (15). Biotinylated probes were denatured, preannealed gradually increasing concentrations of the drug. The resulting cells with excess Cot-1 DNA, and hybridized overnight at 37°Cto metaphase (designated SKEM) exhibited a 5-6-fold level of resistance to EM spreads on a 24 x 60-mm slide. Hybridization sites were detected with compared with the SKOV3 parental cells. Because drug-resistant cells fluorescein-labeled avidin (Oncor) and amplified by the addition of anti-avidin often display newly acquired chromosomal abnormalities, karyotypic antibody (Oncor) and a second layer of fluorescein-labeled avidin. The chro analysis of parental SKOV3 and SKEM cells was performed. The mosome preparations were counterstained with DAPI and observed with a Giemsa-banded metaphase chromosomes from SKEM cells showed Zeiss Axiophot epifluorescence microscope equipped with a cooled charge the presence of an abnormal chromosome 9 not present in the parental coupled device camera (Photometries, Tucson AZ) operated by a Macintosh cells. This aberrant chromosome 9 contained an hsr located at band computer work station. Digitized images of DAPI staining and fluorescein signals were captured, pseudo-colored, and merged using Oncor version 1.6 9q34 (Fig. la). We used chromosome microdissection techniques to software. characterize the amplicon of the hsr.