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Is a Prosurvival Factor in Human Breast Tumor Cell Lines1
Department of Biochemistry and Molecular Pharmacology, and Genetics and Developmental Biology Program, West Virginia University, Morgantown, West Virginia 26506 [M. A. M., J. S. S.], and Isis Pharmaceuticals, Carlsbad Research Center, Carlsbad, California 92008 [L. J. M., R. A. M.]
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: jstrobl{at}hsc.wvu.edu
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Abstract |
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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 µM rottlerin, a selective PKC inhibitor, to reduce MCF-7 and MDA-MB-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 |
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PKC isoforms are divided into three subfamilies: classical (
, ßI, ßII, and 
), novel (, 
, 
, 
, and µ), 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 , , , , , , µ, 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 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 MDA-MB-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.
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Materials and Methods |
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10 cells=1 colony. For certain experiments, cells were treated with 1.5–9.0 µM 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 x 105/35-mm2 dish) or MCF-7 (2.2 x 105/35-mm2 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 µl 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 137Cs 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 µl of DMEM/10% FBS. After plating (5 or 7 days, respectively), fresh growth medium (100 µl DMEM/5% + 20 µl 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 MDA-MB-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 x 105/100-mm2 dish) or MCF-7 (7.0 x 105/100-mm2 dish) cells were rinsed one time with PBS. Total cellular proteins were collected in 100–150 µl 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 x 106/100-mm2 dish) were transformed with 10 µg of the empty vector or 13.7 µg of PKC dominant-negative (normalized for the neomycin resistance gene copy number) by the N-[1-(2,3-dioleoyloxyl)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 µg/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 x 105 cells/ml in ice-cold PBS. Cells were maintained on ice and treated with 1.5 Gy radiation. After radiation, 50 µl of PBS cell suspension was mixed with 500 µl of 42°C low melting point agarose. Seventy-five µl 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 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 µl of SYBR Green stain for 7 min. The comets were visualized using a Nikon Eclipse TS100 microscope with 63x 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 x 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.
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Results |
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, , and were expressed differentially in MDA-MB-231 and MCF-7 human mammary tumor cells (Fig. 1). Higher levels of PKC protein were detected in MDA-MB-231 cells than in MCF-7 cells, whereas levels of PKC were higher in MCF-7 cells. Whether these differences in PKC and isoforms reflect alternative or compensatory pathways acting in cell survival is not known. MDA-MB-231 and MCF-7 cells also differed in their expression of the PKC isoform. Whereas MCF-7 cells contained two species of PKC , a faster and slower migrating form, PKC was undetectable in MDA-MB-231 cells. Similar levels of PKC and PKC protein were observed in the two cell lines.
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Oligonucleotide Decreases Survival in Human Breast Tumor Cells. isoform on cell survival after irradiation we introduced oligonucleotides targeting PKC into MDA-MB-231 and MCF-7 cells. PKC oligonucleotide treatment before delivery of 5.6 Gy of radiation caused a 44% decrease in MDA-MB-231 cell survival (P < 0.05) compared with identically irradiated cells that were pretreated with the nucleotide scrambled version of this oligonucleotide (Fig. 2A). Liposome treatment alone or liposome treatment plus the nucleotide-scrambled version of the PKC oligonucleotide had no effect on MDA-MB-231 survival, demonstrating that the oligonucleotide sequence was critical to decreased survival.
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oligonucleotide significantly decreased MCF-7 cell survival after 5.6 Gy of radiation, thus confirming the importance of the PKC in survival in irradiated human breast tumor cell lines.
PKC Oligonucleotide Reduced PKC mRNA Levels.
To provide support for an antisense mechanism of the PKC oligonucleotides, RNA was prepared from MDA-MB-231 cells 16 h after transfection. To show that our PCR protocol was adequate to detect changes in PKC mRNA, we show that the formation of 351-bp DNA product in a 5-µl aliquot of the PCR reaction increased in a linear fashion over a range of RNA template concentrations (25–500 ng; Fig. 3A). In cells transfected with 100 nM PKC oligonucleotide, levels of PCR product were reduced, a result consistent with its action as an antisense molecule. Furthermore, levels of PKC mRNA were not decreased in cells transfected with either 100 nM of the scrambled form of PKC oligonucleotide or 10 nM of the active form of PKC oligonucleotide (Fig. 3B), treatments we found were ineffective in reducing PKC protein levels. We conclude that antisense inhibition of PKC mRNA levels is a probable mechanism contributing to the decreased cell survival caused by PKC oligonucleotides.
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Oligonucleotide Specifically Reduced PKC Protein Levels. protein levels were decreased in cells treated with oligonucleotides that targeted PKC , whole cell extracts were prepared from MCF-7 and MDA-MB-231 cells 52 or 72 h after transfection. No PKC protein was detected in either cell line after 72 h (Fig. 4). Untreated cells (none), cells treated with lipofectin alone (Fig. 4, L) or the inactive nucleotide scrambled (Fig. 4, scr) version of the PKC oligonucleotide showed constant levels of PKC at 52 and 72 h. We conclude that transfection with PKC oligonucleotide reduced PKC protein below the levels detectable by Western blotting. To show that PKC oligonucleotide was selective for the isoform of PKC, protein levels of PKC and PKC were assayed in the same MCF-7 cell extracts (Fig. 4). PKC oligonucleotide had no effect on levels of PKC or PKC . Fig. 4 also shows that the levels of the antiapoptotic protein bcl-2 (21) were unchanged in MCF-7 and MDA-MB-231 cells treated with the PKC oligonucleotide. These data support our hypothesis that selective PKC protein depletion by oligonucleotide treatment participates in the decreased breast tumor cell line survival in response to -radiation.
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Oligonucleotide Decreased Breast Tumor Cell Survival in Response to 1.5 Gy of -Radiation. oligonucleotide would decrease breast tumor cell survival in vitro in this radiation dose range, cells were exposed to 1.5 Gy of radiation 17 h after transfection with oligonucleotides (Fig. 5, 1.5 Gy IR). PKC oligonucleotide treatment without radiation caused a 40% decrease in MDA-MB-231 cell survival measured on day 5 compared with the nucleotide-scrambled oligonucleotide (Fig. 5). This result indicated that PKC was important to cell survival in the absence of radiation. Low-dose radiation (1.5 Gy) decreased survival of the MDA-MB-231 cells by 
20% compared with nonirradiated controls. However, pretreatment of cells with the PKC oligonucleotide before 1.5 Gy irradiation caused a 70% decrease in cell survival (P < 0.05) compared with cells treated identically with the scrambled version of the oligonucleotide. These experiments highlight an important survival role for PKC in MDA-MB-231 human breast tumor cells, and suggest that a regimen combining PKC inhibition with therapeutic radiation doses might improve clinical responses to breast tumor radiotherapy.
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(22) and was used to explore the role of PKC in MCF-7 cells, because the oligonucleotide approach in this cell line was limited by the toxicity of the liposomal reagent. Clonogenic survival of unirradiated MCF-7 cells after a 7-day exposure to 3 µM rottlerin was reduced by 78% compared with solvent-treated control cells (Fig. 6A). Rottlerin (3 µM; 7-day exposure) also reduced survival of unirradiated MCF-7 cells when measured using the MTS assay. The survival of MCF-7 cells exposed to 1.5 or 5.2 Gy irradiation was reduced additionally by 3 µM rottlerin (Fig. 6B). MDA-MB-231 cells were even more sensitive to 3 µM rottlerin (5-day exposure) in the presence (5.2 Gy) or absence of radiation than MCF-7 cells (Fig. 6C). These results support the hypothesis that PKC is a survival factor in breast tumor cells.
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Dominant-Negative Plasmids Impair MCF-7 Cell Survival. into MCF-7 cells using N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N -trimethylammoniummethyl sulfate as the liposomal reagent (23) was another approach taken to examine the role of PKC in MCF-7 cell survival that avoided the acute toxicity of lipofectin. The presence of G418 in the cell growth medium permitted selection for MCF-7 cells with stable expression of the neomycin resistance gene within the empty plasmid vector as well as the plasmid vector containing the dominant-negative PKC protein. The MCF-7 cell population that expressed a dominant-negative PKC mutant protein contained 60% fewer viable cells than parallel cultures of MCF-7 cells that were transformed with the empty vector alone (Fig. 7A). To confirm that the dominant-negative PKC protein was expressed in the transformed MCF-7 cells, Western blot analysis with Abs that recognize the wild-type and the dominant-negative form of PKC were used. Total PKC immunoreactive protein in MCF-7 cells transformed with the PKC dominant-negative plasmid was 3.5-fold higher (n = 3; P < 0.05) than in cells transformed with the empty vector (Fig. 7B). Transformation with the PKC vectors had no effect on bcl-2 protein levels. Thus, interference with PKC function using this dominant-negative plasmid approach reduced MCF-7 cell survival, additionally supporting our hypothesis that PKC is a survival factor in human breast tumor cells.
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Oligonucleotide Treatment and Loss of DNA Integrity. oligonucleotide treatments and radiation. The electrophoretic mobility and relative intensity of fragmented nuclear DNA are incorporated into the tail moment parameter. The frequency of comets in control MDA-MB-231 was highest for tail moments <2, and is indicative of cells with undamaged nuclear DNA (Fig. 8). Treatment of MDA-MB-231 cells with PKC oligonucleotide caused a shift in the comet distribution toward increased tail moments, indicative of damaged DNA, whereas the scrambled oligonucleotide control did not show loss of DNA integrity. MDA-MB-231 cells exposed to 1.5 Gy -radiation caused an increase in the frequency of comets with tail moments >2, similar to the effect of PKC oligonucleotide treatment, and the combination of PKC oligonucleotide and radiation produced an increase in the number of DNA strand breaks. Table 1 summarizes the comet analysis results presented in Fig. 8 for analysis performed at 24 h after treatment of MDA-MB-231 cells with PKC oligonucleotide.
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antisense oligonucleotide treatment resulted in significant DNA damage that was sustained for at least 48 h (PKC TM >2 = 54.6% and > 20=23.3%). Similarly, the DNA damage observed after treatment of MDA-MB-231 cells with PKC oligonucleotide plus radiation also persisted at 48 h (PKC + irradiation TM >2=70.0% and >20=28.3%). The nature of the DNA fragments and the reason for the resistance of PKC oligonucleotide-inducible DNA fragmentation to repair are as yet unknown. However, the persistence of DNA damage is a plausible explanation for the decreased survival of human breast tumor cells treated with PKC oligonucleotides.
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Discussion |
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decreased survival of MDA-MB-231 and MCF-7 human mammary tumor cells. Isoform-selective PKC inhibition in MDA-MB-231 cells was achieved using oligonucleotides that targeted the mRNA. As an alternative method, expression of a dominant-negative form of PKC containing a point mutation in the ATP binding domain (23) decreased survival in MCF-7 cells. Finally, the small molecule inhibitor rottlerin (3 µM), which selectively inhibits PKC (IC50 3–6 µM), inhibited growth in both cell lines and clonogenic survival in MCF-7 cells. Although these later experiments did not preclude a contribution of calmodulin kinase III inhibition by rottlerin (IC50 5.3 µM; Ref. 29), the data support our general conclusion that PKC is a prosurvival factor in human breast tumor cells. Furthermore, because oligonucleotide-mediated PKC depletion in MDA-MB-231 cells caused sustained damage to DNA, we suggest that PKC positively regulated survival in breast tumor cells by facilitating genomic integrity.
The finding that the DNA is fragmented in MDA-MB-231 breast tumor cells selectively depleted of PKC is a novel observation. DNA fragmentation was not dependent on treatment with IR, and DNA fragmentation was not observed after treatment with a nucleotide-scrambled version of the PKC oligonucleotide. Yet, the DNA damage induced by the IR was more significant after PKC depletion, suggesting that PKC inhibition is a possible paradigm for breast tumor radiosensitization.
One potential mechanism for DNA fragmentation in PKC -depleted cells is suggested by the work of Otieno and Kensler (30). These authors showed that PKC is involved in the regulation of ornithine decarboxylase, an essential enzyme in cell replication. Oteino and Kensler (30) found that expression of the PKC dominant-negative mutant in murine papilloma cells attenuated the induction of ornithine decarboxylase in response to oxidative damage by hydrogen peroxide. Because cells in culture are typically under mild chronic oxidative stress, PKC depletion in MDA-MB-231 cells might unmask DNA damage that is produced routinely in the cultured cells. The impairment of an ornithine decarboxylase response to oxidative DNA damage is a potential explanation for the persistent DNA fragmentation observed in the MDA-MB-231 cells treated with PKC antisense oligonucleotides. By the same mechanism, a PKC antisense oligonucleotide-mediated depletion of PKC in the irradiated cells would be expected to augment the DNA damage produced by IR. This model reflects the observations we made in MDA-MB-231 cells.
A role for PKC in promoting mammary tumor cell growth is supported by observations by other laboratories as well. Inhibition of PKC with the dominant-negative mutant or by rottlerin led to the inhibition of estradiol-stimulated Erk activation in MCF- 7 cells (12). The authors concluded that PKC activity stimulated cell growth by increased Ras pathway activation. In MTLn3 mammary tumor cells, expression of the inhibitory PKC regulatory domain (RD) inhibited growth in soft agar, cell motility, and attachment (14). The conclusion drawn from this work was that PKC is critically involved in cytoskeletal processes in these cells. Furthermore, Kiley et al. (13, 31) reported that highly metastatic mammary tumor cells expressed significantly more PKC protein and mRNA than less-metastatic cells. Results from these laboratories as well as our own support the hypothesis that PKC is a potential new molecular target for breast cancer drug development.
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Acknowledgments |
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Footnotes |
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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.
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.
Received 8/30/02; revised 12/13/02; accepted 12/20/02.
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). Controls were treated with radiation alone (IR), radiation plus liposome (L), or radiation plus liposome plus scrambled nucleotide versions of this oligonucleotide (
). After transfection (24 h), cells were irradiated with 5.6 Gy of 
). B, ethidium bromide stained gel of RT-PCR products obtained using 500 ng of RNA template prepared from MDA-MB-231 cells 16 h after transfection with the indicated PKC 
