Cytokeratin Expression Results in a Drug-resistant Phenotype to Six Different Chemotherapeutic Agents 1

I N T R O D U C T I O N The treatment of epithelial tumors continues to be a major medical challenge due in part to both cellular and noncellular Received 4/19/95; revised 8/23/95; accepted 9/20/95. Supported in part by a research fellowship award from the ASTRO Education and Development Fund, Grants CA56666 and CA23074, and DOE Contract 307190. Financial support does not constitute an endorsement by the DOE of the views expressed in the article. 2 Present address: Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-0146. 3 To whom requests for reprints should be addressed, at Department of Radiation Oncology, 1501 North Campbell, University of Arizona Cancer Center, Tucson, AZ 85724. Phone: (520) 626-7553; Fax: (520) 626-4480; E-mail: [email protected]. mechanisms of drug resistance (1). Noncellular mechanisms of resistance, including vascular perfusion and blood tissue barriers, can be bypassed but cellular mechanisms (acquired and innate) of resistance remain. Examples of the biological mechanism of acquired resistance include the down-regulation of key targets (2), increase in free radical scavengers (3), alteration in cellular pH (4), altered drug activation (5), and increased drug inactivation (3). In contrast, mechanisms of innate resistance have been attributed primarily to the P-glycoprotein MDR 4 pump (3) and drug effiux unrelated to the MDR pump (6). Previously we had shown that a chemotherapeutic agent, mitoxantrone, was associated with the cytokeratin network in human colon carcinoma cells. The association of mitoxantrone to cytokeratin was persistent and suggested that the interaction may either represent a new cellular target (i.e., increased damage) or a mechanism for an intracellular redistribution of the drug (i.e., increased survival; Ref. 7). Recently, work from our laboratory has shown that the addition of a cytokeratin network into a mouse fibroblast cell line conferred a MDR pattern (8). In studies of P-glycoprotein and non-P-glycoprotein mu!tidrug resistance, it has been found that resistant cell lines lose their sensitivity to ionizing radiation and cytotoxic drug-induced apoptosis (9, 10). The purpose of the present study was to determine the universal nature of the cytoskeletal-dependent drug resistance using two different cell lines, six different DNA-damaging agents, and two different assays for cell killing. To investigate the possible cellular mechanism of drug resistance, we compared the apoptotic response of cytokeratin-positive cell lines with cytokeratin-negative cell lines. M A T E R I A L S A N D M E T H O D S Murine L parental and cytokeratin 8/18-transfected cell lines were obtained from Dr. Oshima (La Jolla Cancer Research Foundation, La Jolla, CA) and maintained as previously described (11). NIH 3T3 parental and 8/18-transfected cell lines were courtesy of Dr. B. Lane (University of Dundee, Scotland; Ref. 12). Cytokeratin-positive cells were maintained in DMEM (GIBCO), 10% FBS (Gemini), penicillin, streptomycin (GIBCO), 1 mg/ml G418 (GIBCO), and 200 mg/ml hygromycin (Calbiochem). Parental cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. All cell lines were maintained at 37°C in a humidified incubator containing 5% CO 2. Two subclones of the NIH 3T3 cytokeratin4 The abbreviations used are: MDR, multidrug resistance; FBS, fetal bovine serum; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; FACS, fluorescence-activated cell sorting; IC5o, 50% inhibitory concentration. Research. on January 20, 2018. © 1996 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 98 Cytokeratin and Drug Resistance positive cells were obtained via serial dilution. In brief, 100 cells/100 ml selection medium were diluted with an equal volume of conditioned medium. Two hundred pJ cell suspension were added to each well of a 96-well microtitre plate (Falcon). After 3 weeks of growth, cells from those wells containing single colonies were resuspended with trypsin and maintained in growth medium containing the double selection agents (G418 and hygromycin). For indirect immunofluorescence microscopy studies, cells were grown on 12-mm round coverslips within a 35-mm dish containing the appropriate growth medium for at least 24 h. The medium was removed, and the cells were washed with PBS, and fixed on ice with methanol (-20°C) for 5 rain. Coverslips were washed with 5% BSA/PBS and incubated with a mouse monoclonal antibody directed against cytokeratins 8 and 18 (antibody 10.11; Ref. 13) at a 1:500 dilution for 30 rain at room temperature. A rabbit antimouse rhodamine-conjugated secondary antibody was utilized to demonstrate the cytokeratin filaments. Visualization of filaments was performed using a Zeiss LSM 10 confocal microscope. Images were collected using a HeNe laser and a scan time of 8 s. hnages were transferred onto a Quadra 800 computer and recorded using a film recorder using Kodak TMX 135 film ASA 100. Cell survival was measured using the MTT assay (14) and a colony-forming assay (8). For the MTT assay, 1000-5000 cells in 180 ~1 appropriate growth medium were plated in each well of a 96-well microtiter plate (Falcon) on day 1. The following day, 20 ill of a 10× concentration of the specified drug were added to the appropriate well. After 3 days of incubation, 50 ml of 2 mg/ml MTT were added to each well and incubated for 4 h at 37°C. The medium was subsequently removed and replaced with 100 t~1 DMSO. The plates were shaken, and the optical absorbance was determined at 540 nm. Absorbance values were normalized to the values obtained for the vehicle-treated cells to determine the value for percentage of survival. For determination of cell survival after UV irradiation, cells were plated as described above for the MTT assay. The following day, cells were irradiated with a predominantly UVc germicidal lamp (Sylvania). Prior to irradiation, the energy output (W/cm 2) of the lamp was determined at a source to surface distance of 16 cm with a UV meter. The final UV dose (J/m 2) was achieved for each group of cells by adjusting the total time of exposure. The final doses achieved in creating the dose-response curve were 2, 6, 20, 60, and 200 J/m 2. The remainder of the MTT assay was carried out as described above. The colonyforming assay was performed with the L cells suspended in their appropriate growth medium with or without 0.03 or0.3 IxM mitoxantrone. Cells were plated in 100-mm dishes at cell concentrations of 25, 50, 100, 200, 400, 800, or 1600 cells/dish. After 13 days of undisturbed incubation, the cells were fixed with methanol:acetic acid (3:1), stained with crystal violet, Fig. 1 Immunofluorescent detection of cytokeratin networks. NIH 3T3 cytokeratin 8 + 18 transfectants (clone 1 and clone 2) synthesize an elaborate cytokeratin network extending from the perinuclear zone to the cell surface. LK 8 + 18-transfected ceils synthesize cytokeratins but create a less elaborate network. 4