Protein Kinase C Is a Prosurvival Factor in Human Breast Tumor Cell Lines

Protein kinase C (PKC) promotes cell survival in response to ionizing radiation in a variety of experimental models including human carcinoma, human glioblastoma, and transformed mouse embryo fibroblast cell lines. We have introduced specific antisense oligonucleotides into human mammary tumor cell lines in vitro to analyze the role of individual PKC isoforms in radiation-induced cell death in breast cancer. MDA-MB-231 and MCF-7 cells treated with oligonucleotide directed against the PKC isoform exhibited impaired survival in response to 5.6 Gy radiation as measured by mitochondrial metabolism of tetrazolium dye. The role of PKC in the breast tumor cell lines was of particular interest, because contradictory reports exist in the literature regarding the role of PKC in cell survival and apoptosis. A comparison of the effects of the PKC antisense oligonucleotide and a nucleotide scrambled version of this nucleotide revealed only the antisense oligonucleotide decreased cell survival. The PKC antisense oligonucleotide decreased cell survival after exposure to low (1.5 Gy) radiation doses and in the absence of radiation insult. We found 3 M rottlerin, a selective PKC inhibitor, to reduce MCF-7 and MDAMB-231 cell survival. Furthermore, MCF-7 cells transformed to express a dominant-negative mutant of PKC exhibited reduced survival. Comet analysis showed that PKC oligonucleotide treatment caused an accumulation of cells containing damaged DNA similar to that seen in 1.5 Gy radiation-treated cells. We conclude that PKC acts as a prosurvival factor in human breast tumor cells in vitro. Introduction Malignant breast cell proliferation is attributable in part to the aberrant activity of growth-promoting signal transduction pathways. PKC is implicated in the promotion and progression of breast tumors. PKC overexpression and increased activity were reported in human breast cancers compared with normal mammary tissue (1, 2). Furthermore, a correlation between elevated PKC protein levels and aggressive breast cancer phenotypes, such as those that lack estrogen receptors and exhibit multidrug resistance, has been established (3, 4). PKC is a family of serine/threonine kinase isoforms with wide tissue distribution that mediate intracellular responses to a variety of stimuli including growth factors and hormones that accelerate proliferation in human breast tumor cells (5). As such, PKC is a potential molecular target for pharmacologic intervention in breast tumor promotion as well as tumor cell proliferation. The isoform-specific functions of PKC in mammary epithelium are only partially understood, and with a better definition of the role that specific PKC isoforms play in the promotion of mammary tumor cell growth, more specific therapies could be designed. PKC isoforms are divided into three subfamilies: classical ( , I, II, and ), novel ( , , , , and ), and atypical ( and / ). Classical PKC isoforms possess a calcium-binding domain and two cysteine-rich zinc fingers that bind DAG. Novel PKC isoforms (e.g., PKC ) contain the DAG binding sites, but lack the calcium-binding domain, and differ from the atypical isoforms, which do not use either calcium or DAG for activation. The PKC profile of the MCF-7 human mammary carcinoma cell line includes PKC , , , , , , , and isoforms. MDA-MB-231 human mammary carcinoma cells express a similar PKC profile except that PKC is more highly expressed (6, 7). Overexpression of PKC stimulated MCF-7 breast tumor and C6 glioma cell growth, (7, 8), but overexpression of this same PKC isoform inhibited growth of bovine endothelial cells and rat embryonic smooth muscle cells (9, 10). The mitogenic influence of PKC in the growth of breast cancer is inferred clinically by the success of ISIS 3521, a therapeutic antisense oligonucleotide to PKC , in Phase I and II clinical trials of solid tumors (11). ISIS 3521 is being tested presently in Phase III clinical trails. Experiments from a number of laboratories now support the idea that PKC is a positive regulator of breast cancer growth and metastasis (12–15). In one proposal, PKC activation of the Ras/Erk1/2 pathway is suggested to increase mammary tumor cell growth and metastasis, the latter effect Received 8/30; revised 12/13/02; accepted 12/20/02. 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. 1 Supported by the United States Army Department of Defense Grant DAMD17-99-1-9449 and The Susan G. Komen Breast Cancer Foundation Grant 993249. 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506. Phone: (304) 293-7151; Fax: (304) 293-6854; E-mail: [email protected]. 3 The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; Erk, extracellular signal-regulated kinase; IR, ionizing radiation; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium; RT-PCR, reverse transcription-PCR; Ab, antibody, TM, tail moment. 273 Vol. 2, 273–281, March 2003 Molecular Cancer Therapeutics on May 2, 2017. © 2003 American Association for Cancer Research. mct.aacrjournals.org Downloaded from via an Erk1/2-induced secretion of matrix metalloproteinase (12, 15). However, there is also a collection of literature, largely based on studies with nonmammary cell types, that suggests PKC participates in cell death and growth inhibition (16–20). Such observations prompted us to carefully examine the effect of PKC depletion on growth and survival in two human mammary tumor cell lines, MCF-7 and MDAMB-231, frequently used models for human breast cancer. Our results provide additional evidence that PKC functions as a prosurvival factor in human breast tumor cells. Materials and Methods Cell Culture. MDA-MB-231 and MCF-7 (passage #39–50) human mammary tumor cells were maintained in DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% FBS (Summit Biotechnology, Fort Collins, CO) and 0.04 mg/ml gentamicin in a 7.5% CO2, 37°C, humidified incubator. Cells were passaged weekly at 1:10 or 1:5 ratios, respectively. For clonogenic survival, MCF-7 cells were plated at 1 10 cells/100 mm dish in 10 ml of DMEM/10% FBS and maintained for 7 days in a 7.5% CO2, 37°C, humidified incubator. To visualize colonies, cells were stained with 0.5% crystal violet, 5% formalin, 50% ethanol, and 0.85% NaCl. Colonies were scored using a Nikon Eclipse TS100 (Nikon, Kawasaki, Japan) at 10 magnification with 10 cells 1 colony. For certain experiments, cells were treated with 1.5– 9.0 M rottlerin (Sigma, St. Louis, MO) dissolved in DMSO with a final concentration of 0.1–0.15% DMSO in the cell culture medium. Antisense Oligonucleotides. MDA-MB-231 (1.1 10/ 35-mm dish) or MCF-7 (2.2 10/35-mm dish) cells were treated for 4–5 h with 100–200 nM PKC methoxy-ethoxy modified antisense oligonucleotide (ISIS #13513) or the respective nucleotide scrambled version of the oligonucleotide (ISIS #13514; Isis Pharmaceuticals) in 3 l of Lipofectin (Life Technologies, Inc., Rockville, MD) transfection reagent/1 ml Opti-Mem I reduced serum medium (Life Technologies, Inc.). Transfection was stopped by medium exchange with DMEM/2% FBS. Radiation. Cells were exposed to 1.5–5.6 Gy doses of -IR delivered with a Cs source in a Gammacell 40 (Atomic Energy of Canada Ltd., Ottawa, Ontario, Canada) at 108.7 rads/min or a Gammacell 1000 (Atomic Energy of Canada Ltd.) at 730.0 rads/min under ambient temperature and atmospheric conditions. The clonogenic survival curves generated at these two radiation rates were similar. After radiation medium was replaced with DMEM/10% FBS. MTS Assay. Metabolism of MTS was used as index of cell viability. MDA-MB-231 or MCF-7 cells were plated at 250 or 2500 cells/well, respectively, in a 96-well plate in 225 l of DMEM/10% FBS. After plating (5 or 7 days, respectively), fresh growth medium (100 l DMEM/5% 20 l Cell Titer 96; Promega, Madison, WI) was added. Conversion of MTS reagent to formazan product was measured after a 2-h incubation at 37°C by an increase in absorbance at 490 nm using a Spectra Max 340pc plate reader (Molecular Devices, Sunnyvale, CA). The MTS assay was linear under our assay conditions for 3 h. Mitochondrial metabolism of MTS in MDAMB-231 and MCF-7 as an end point for cell survival showed similar survival curves in response to 0.75–9.5 Gy radiation. PKC RT-PCR. Total cell RNA was isolated using the RNeasy method (Qiagen Inc., Valencia, CA) with DNase I treatment. One-step RT-PCR was performed (Qiagen Inc.) using 25–500 ng of RNA template per reaction and PKC -specific primers (Oxford Biomedical Research, Oxford, MI). Products were analyzed on 4.5% polyacrylamide gels stained with ethidium bromide. A single product corresponding to the expected 351-bp fragment was produced in a primer and template-dependent fashion. Western Blotting. MDA-MB-231 (4.5 10/100-mm dish) or MCF-7 (7.0 10/100-mm dish) cells were rinsed one time with PBS. Total cellular proteins were collected in 100–150 l of boiling lysis buffer [1% SDS and 10 mM Tris (pH 7.4)], boiled for 5 min, then centrifuged at 4°C, 13,000 rpm in an Eppendorf centrifuge 5415 C (Brinkmann Instruments Inc., Westbury, NY). Protease inhibitors were added to the supernatant. Protein samples were resolved on 7.5% acrylamide gels at 100 V, then transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA). Purified PKC , , , , and proteins (#539674 and #539673; Calbiochem, San Diego, CA) were used as standards. The primary Abs used were PKC , , or mouse monoclonal Ab (BD Transduction Laboratories, San Diego, CA), PKC goat polyclonal Ab, PKC rabbit polyclonal Ab, or bcl-2 mouse monoclonal Ab (Santa Cruz, CA). The secondary Abs were horseradish peroxidase-conjugated (Santa Cruz). Signals were detected using Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Autoradiographic signals were quantitated by FluorChem (Alpha Innotech, San Leandro, CA) spot densitometry using automatic background subtraction and normalized to a Mr 50,000 or 200,000 protein band on the Gel Code Blue (Pierce) stained acrylamide gel. Plasmids. PKC dominant-negative plasmid constructs were generously provided by Dr. Jae-Won Soh (Columbia University, New York, NY). MCF-7 cells (1 10/100-mm dish) were transformed with 10 g of the empty vector or 13.7 g of PKC dominant-negative (normalized for the neomycin resistance gene copy number) by the N-[1-(2,3dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate transfection reagent (Roche, Indianapolis, IN) in accordance with the manufacturer’s protocol. After 48 h, medium was exchanged with 10 ml of DMEM/5% FBS. On the following day, medium was exchanged with 10 ml DMEM/10% FBS 400 g/ml of G418 (Stratagene, La Jolla, CA). After 11 days of growth in selection medium, trypan blue excluding viable cells were counted on a hemocytometer, or whole cell protein extracts were prepared. Comet Analysis. Cells were treated with PKC oligonucleotides for 24–48 h. Cells were harvested and resuspended at 1.5 10 cells/ml in ice-cold PBS. Cells were maintained on ice and treated with 1.5 Gy radiation. After radiation, 50 l of PBS cell suspension was mixed with 500 l of 42°C low melting point agarose. Seventy-five l of cell suspension were spread evenly onto a Comet Slide (Trevigen, Gaithersburg, MD) and allowed to dry flat in the refrigerator for 30 min. Slides were then immersed in prechilled 274 PKC Is a Prosurvival Factor in Breast Tumor Cells on May 2, 2017. © 2003 American Association for Cancer Research. mct.aacrjournals.org Downloaded from lysis solution (Trevigen) and incubated on ice for 45 min, then transferred to freshly prepared alkali solution [300 mM NaOH and 1 mM EDTA (pH 8.0)] for 45 min at room temperature, protected from light. Slides aligned equidistant from the electrodes were electrophoresed for 30 min at 1 V/cm and 300 mA. Slides were immersed in 70% ethanol for 5 min and then allowed to air dry overnight at room temperature. Slides were stained with 50 l of SYBR Green stain for 7 min. The comets were visualized using a Nikon Eclipse TS100 microscope with 63 objective and FITC-filter cube. Comet images were captured and analyzed using the LAI Automated Comet Assay Analysis System (Loats Associates, Inc. Westminster, MD). The tail moment (% DNA distance traveled) was used for quantitative analysis of DNA damage for 80 comets per treatment/experiment. Statistical Analysis. Statistically significant differences (P 0.05) were determined using Student’s t test. For multiple comparisons, one-way ANOVA with Tukey’s multiple comparison test was used.