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Influence of ionizing radiation on induction of apoptotic cell death and cellular redistribution of protein kinase C isozymes in mouse epidermal cells differing in carcinogenesis stages
Mutation Research 426 Ž1999. 41–49
Influence of ionizing radiation on induction of apoptotic cell death and cellular redistribution of protein kinase C isozymes in mouse epidermal cells differing in carcinogenesis stages Su-Jae Lee, Chul-Koo Cho, Seong-Yul Yoo, Tae-Hwan Kim, Yun-Sil Lee
)
Laboratory of Radiation Effect, Korea Cancer Center Hospital, 215-4 Gongneung-Dong, Nowon-Ku, Seoul, 139-706, South Korea Received 27 October 1998; received in revised form 2 March 1999; accepted 2 March 1999
Abstract Although protein kinase C ŽPKC. plays an important role in cellular response to radiation, little is known about the specific role of each isoform in the radiation induced cellular response. In this study, the induction of apoptosis and subcellular distribution of PKC isoforms after g-ray irradiation were examined in three kinds of mouse epidermal cells with different stages of carcinogenesis Žnormal mouse keratinocytes, PK: Õ-ras Ha transfected mouse keratinocytes, ras-PK; and neoplastic cells from mouse skin papilloma, 308 cells.. The induction of apoptosis was different in normal and neoplastic cells; in normal cells after 16 Gy of radiation, apoptosis was 2–10 times higher than that in ras-PK or 308 cells, and was rapidly induced; other cells died more slowly, depending on the stage of carcinogenesis. The responses of each PKC, especially rapid translocation of PKCd and no response of PKC´ by radiation in normal cells may influence the induction of apoptosis by radiation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Normal cell; Õ-ras Ha transformed cell; Papilloma cell; Protein kinase C isozymes; Cellular redistribution; Apoptotic cell death; Gamma ray
1. Introduction Apoptosis is important in normal tissues, carcinogenesis, tumor development, and cancer therapy w1,2x, and this mode of cell death can also be induced in various cell types by insults such as UV, ionizing radiation, and hyperthermia w3,4x. The attenuation of radiation-induced apoptosis by transfected Ha-ras
) Corresponding author. Tel.: q82-2-970-1325; Fax: q82-2977-0381; E-mail: [email protected]
oncogene w5x and radiation resistance is associated with oncogenes that have a direct or indirect involvement of plasma membranes in cellular response to ionizing radiation w6x. Activated ras is also associated with increased protein kinase C ŽPKC. expression w7x and also alters intracellular diacylglycerol ŽDAG. level w8x. The importance of ras oncogene as a causal factor in skin tumor initiation was emphasized when a mutated form of the gene, introduced into normal skin cells, resulted in the formation of benign tumors identical to those produced by initiation and promotion w9x.
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 0 7 8 - 0
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S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
PKC represents a large gene family of isozymes, differing remarkable in their structure and expression in various tissues, in their mode of activation, cofactor requirement, and substrate specificity w10x. Several sets of data have reported that PKC can influence cellular radiosensitivity, internucleosomal DNA fragmentation, and apoptotic cell death w11,12x. Furthermore, PKC plays a crucial role in mediating radiation-induced oncogenic transformation in C3H 10T1r2 cells w13x. Reports describing PKC activation and the induction of apoptosis have been contro-
versial. Certain results have shown that PKC activation blocks glucocorticoid and Ca2q ionophore-induced apoptosis w14x, and that tumor-promoting phorbol esters, which selectively stimulate PKC, also block radiation-induced apoptosis w15x; other data, however, have suggested that PKC inhibition by nonspecific PKC inhibitor prevents DNA fragmentation w16x. All data showed that radiation-induced influence of PKC activation and protein phosphorylation on apoptosis could be mediated by—for example—effects on cell cycles and differentiation.
Fig. 1. Determination of apoptosis by TUNEL assay: primary keratinocytes ŽPK., Õ-ras Ha transfected keratinocytes Žras-PK. and 308 papilloma cells Ž308. were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4, 8 and 16 Gy. After 5, 16, and 48 h, induction of apoptosis was detected by TUNEL assay. For each determination at least 500 cells were scored. All data points represent the result of five counts of 500 cells each. Two separate experiments showed similar results. Ža. Morphology of TUNEL positive cells after 16 h of 8 Gy irradiation arrow apoptotic fragments; Žb. quantitation of TUNEL positive cells.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
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Fig. 2. Cellular redistribution of PKC isozymes by g-rays: primary keratinocytes Ža., Õ-ras Ha transfected primary keratinocytes Žb., and 308 papilloma cells Žc. were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4 and 8 Gy. After the indicated times, cells were lysed and separated into soluble and Triton X-100 soluble particulate fractions, as described in Section 2. Isozyme-specific antibodies for PKCa , -d, -´, -h and -z were used for immunoblotting. We marked ‘U ’ on each PKC isozyme which showed significant alteration in translocation patterns. Similar results were obtained in two separate experiments.
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S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
However, since nonspecific PKC activators or inhibitors were used in the studies, the specific PKC isozymes that involved in these functions are not characterized. Three kinds of mouse epidermal cells at different stages of carcinogenesis Žnormal, ras-initiated, and papilloma tumor cells. were chosen for this study. The extent of apoptosis was greatest in normal cells; after irradiation, the process was rapidly induced. Depending on the stage of carcinogenesis, however, other cells died more slowly, and apoptosis was induced in only a few. We have characterized these
responses, examining the cellular subdistribution of PKC isozymes after exposure to g-rays.
2. Materials and methods 2.1. Cell culture Primary mouse epidermal keratinocytes ŽPK. were isolated from BALBrc mice and were grown in Eagle’s minimal essential medium with 8% Chelextreated fetal calf serum, 0.2% penicillinrstrepto-
Fig. 3. Quantitation of PKC isozymes by densitometry of immunoblots: primary keratinocytes, Õ-ras Ha transfected primary keratinocytes, and 308 papilloma cells were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4 and 8 Gy. After the indicated times, cells were lysed and separated into soluble and Triton X-100 soluble particulate fractions, as described in Section 2. Isozyme-specific antibodies for PKCa , -d, -´, -h and -z were used for immunoblotting. PKC isozymes were quantitated by densitometry of immunoblots and the data are expressed as the percentage particulate fraction relative to the unirradiated control level. Bars, SEs from two separate experiments.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
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Fig. 3 Žcontinued..
mycin solution ŽGibco, BRL, Gaithersburg, MD, USA., and 0.05 mM Ca2q to maintain a basal cell-like population of undifferentiated cells w17x. For Õ-ras Ha infection, primary keratinocytes were infected on day 2 or 3 after plating with a defected retrovirus containing the Õ-ras Ha gene w18x. The Õ-ras Ha keratinocytes Žras-PK. were produced by introducing a replication-defective retroviral vector containing the Õ-ras Ha gene into primary keratinocytes on day 3 of culture, using a high-titer viral supernatant prepared from the pse2 packaging cell line. Papilloma cell line 308 Ž308. was developed from pooled papillomas produced in Balbrc mice by initiation with 7,12-dimethylbenzwaxanthracene and promotion with a phorbol ester. 308 cells have an activated ras Ha gene with an A to T transversion at the second base of codon 61, and they form papillomas when grafted to athymic nude mice w19x.
2.2. Irradiation Cells were plated in sterile 10 cm dishes and incubated at 378C under humidified 5% CO 2 –95% air in 0.05 mM Ca2q containing media. When cells reached 80–90% confluency, they were exposed to gamma rays from 60 Co theraton-780 ŽAtomic Energy of Canada, Canada. at a dose rate of 1.394 Gyrmin. 2.3. TUNEL assay Cells were fixed in 95% ethanol and apoptosis was morphologically analysed using the Apoptag assay kit ŽOncor, Gaithersburg, MD.. DNA strand breaks were detected by 3X end labeling kit containing terminal deoxynucleotidyl transferase ŽTdT. and digoxigenin-11-dUTP according to manufacturer’s instructions. TdT reactions were performed for 1–3 h. After incubation with anti-horseradish pero-
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xidase-conjugated digoxigenin antibody and color development with H 2 O 2 and DAB; this produces a dark brown color. Cells were counter-stained with methyl greens, and for each determination at least 500 cells were scored. All data points represent the result of five counts of 500 cells each. 2.4. Cell fractionation After the treatment indicated, cells were washed twice with ice-cold PBS and scraped into PKC lysis buffer w20 mM Tris–HCl ŽpH 7.5., 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 40 mgrml leupeptinx. After brief sonication, the lysate was centrifuged at 100,000 = g for 1 h, and the supernatant was taken as the soluble fraction. The pellet was resuspended in PKC lysis buffer containing 1% Triton X-100 and centrifuged as before; the supernatant was then removed for use as the particulate fraction w20x. Protein concentration was determined by BioRad protein assay. 2.5. Immunoblotting Proteins were run immediately on 8.5% polyacrylamide gels and transferred electrophoretically to nitrocellulose, and the membranes were blocked in 5% milk. To detect PKC isozymes, the membranes were incubated with antibodies specific for the catalytic subunit of PKCa and -d at a dilution of 2 mgrml ŽSanta Cruz Biotechnology, Santa Cruz, CA, USA., PKC´ at a dilution of 1:1000 ŽGibco., PKCh at 1:100 ŽSanta Cruz Biotechnology., and PKCz at 1:100 ŽTransduction Laboratory, Lexington, KY, USA.. Proteins were detected using the ECL system ŽAmersham, Arlington Hights, IL, USA. with horseradish peroxidase conjugated secondary antibody ŽBio-Rad, Hercules, SA, USA. at a dilution of 1:5000. 2.6. Data analysis and statistics The immunoblots were quantitated using a MCID software program ŽImaging Research, Ontario, Canada.. For evaluation of PKC levels, the rate of change for each isozyme after irradiation was evaluated from three to four replicate experiments using multiple linear regression analysis. A rate of 2 indi-
cates a 2-fold increase. The SEs of each bar was estimated using standard least square methods. 3. Results 3.1. Susceptibility to radiation-induced apoptosis To compare radiosensitivity with different stages of carcinogenesis, the induction of apoptosis was detected by TUNEL assay. The greatest number of fragments was found in PK and the smallest in 308 cells. In PK, apoptosis was induced—at most—5 h after irradiation; in ras-PK, the maximum interval was 16 h, and in 308 cells, the maximum was 48 h ŽFig. 1.. 3.2. Cellular resubdistribution of specific PKC isozymes Changes in the total amounts of PKC isozymes do not indicate the degree to which PKC is activated by radiation; to determine which isozymes were translocated, we therefore analyzed changes in the distribution of PKC isozymes in soluble and particulate fractions of cells at different stages of carcinogenesis. Translocation of PKC to the particulate fraction is considered to be an indicator of cellular PKC activation. PKCa which is usually present in the cytosol fraction, underwent redistribution to the particulate fraction, with the largest translocation occurring 1–30 min after irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b., and 30–90 min after irradiation of 308 cells ŽFig. 2c.; the translocated amount of this isozyme was larger in ras-PK than PK. In the case of PKCd, which was distributed between both soluble and particulate fractions of basal cells w21x, radiation reproducibly caused translocation of PKCd between 1 and 30 min after irradiation of PK ŽFig. 2a., between 30 and 90 min after irradiation of ras-PK ŽFig. 2b., and 90 min after irradiation of 308 cells ŽFig. 2c.; soluble amounts of this isozyme showed selective decreases. PKC´ was distributed approximately equally between soluble and particulate fractions in basal cells of PK, but was usually present in the particulate fraction of ras-PK, suggesting that ras oncogene activation induced PKC´ translocation even without radiation. In 308 cells, PKC´ was abundantly distributed in the cytosol fraction.
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Throughout the experiment, radiation did not induce the redistribution of PKC´ in PK, but did induce translocation to the particulate fraction of ras-PK and 308 cells. The translocation of PKC´ in ras-PK was transient ŽFig. 2b., but in 308 cells was sustained throughout the experiments ŽFig. 2c.. A transient rise in particulate PKCh was caused by irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b.; this phenomenon was more dominant in ras-PK than PK. Increased PKCh was not seen in particulate fraction of 308 cells ŽFig. 2c.. After irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b., PKCz showed no responses; this isozyme was not easily detected in either soluble or particulate fractions of 308 cells. Summarized results are shown in Fig. 3. 4. Discussion Radiation-induced apoptosis does not mark the end of a passive degradation process; as in other cases of apoptosis, active synthesis of RNA and protein is required w21x. Considerable attention has recently focused on the relationship between PKC and apoptosis w22x. The bulk of evidence suggests that increased PKC activity antagonizes apoptosis, while reduced activity facilitates the process. Radiation increased PKC activity, and regulation of PKC mRNA by ionizing radiation, especially by X- and g-rays, has also been shown w23,24x. Even though many reports have described the relationship between PKC activation and apoptosis, the nature of this interaction remains obscure and may differ between cell types. However, one possible explanation is that cellular events by radiation share at least some common steps with the cell activation pathways; cellular activation by radiation may be a consequence of peroxidation at membrane level, which in turn may disturb the homeostasis of the membrane lipid metabolism, and induce hydrolysis of phosphatidylinositol-biphosphate to inositoltriphosphate and diacylglycerol, which are known to be responsible for Ca2q mobilization and PKC activation leading to biological reactions. Furthermore, extremely few reports have detailed the effects specific PKC isozymes on radiation-induced apoptosis, especially the differing responses of normal and neoplastic cells. Radiation caused increased expression of PKCa in Chinese hamster V79 cells w25x, of PKC´
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in lung cancer cells w26x, and of mRNA of PKCb in C3H 10T1r2 cells w27x. The existence of multiple PKC isozymes with distinct cofactor requirements and tissue distributions suggests specific roles for individual PKC isozymes in cellular physiology. Epidermal keratinocytes express at least five PKC isozymes, including those which are Ca2q-dependent ŽPKCa ., Ca2q-independent ŽPKCd, ´, and h., and DAGrphorbol ester-independent ŽPKCz .. In this study, we analyzed the subcellular distribution patterns of each PKC isozyme in irradiating mouse epidermal cells Žnormal, Õ-ras Ha initiated, and papilloma cells. differing in radiosensitivity detected by the induction of apoptosis. Induction varied according to the stage of carcinogenesis; it occurred rapidly in normal keratinocytes, peaking 5 h after irradiation, and in the case of Õ-ras Ha -transformed keratinocytes and 308 cells, peak induction was delayed; it occurred at 16 h in Õ-ras Ha transformed keratinocytes, and at 48 h in 308 cells, suggesting that radioresistance was increased by cell tumorigenesis. Many papers have described the relationship between radioresistance and transformation or tumorigenesis. In particular, radioresistance occurs when ras is overexpressed. ras-PK which are initiated, which showed a proliferative potency higher than that of PK and which caused the development of benign tumors when grafted to athymic nude mice w9x were more radioresistant than its parent cells ŽPK.; this was apparent when survival curves were examined and apoptosis was seen to have been induced ŽFig. 1.. A quantity of 308 cells which have an activated ras Ha gene with an A to T transversion at the second base of codon 61, form papillomas when grafted to athymic nude mice w19x, and these cells were the most radioresistant. These results showed that in mouse epidermal cells, apoptosis decreased and its induction was delayed according to the development of carcinogenesis. To know the relationship between the induction of apoptosis and PKC activation, the cellular subdistribution of PKC isozymes after radiation was examined. Since PKC is activated by radiation within minutes, we analyzed the translocation of PKC from soluble to the particulate fraction at 1, 30, and 90 min after irradiation ŽFigs. 2 and 3.. PKCa in PK and ras-PK was rapidly translocated by radiation; this phenomenon was more rapid in ras-PK and a
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large increase was also seen. Since transfection of Õ-ras Ha increased PKC activity especially Ca2q-dependent ŽPKCa . activity w28x, rapid response of PKCa by radiation in ras-PK may correlate to increased activity of this isozyme. In normal cells, PKCd were rapidly translocated by radiation, but in ras-PK and 308 cells, in which apoptosis was delayed and reduced, the translocation of these isozymes was delayed. These results suggested that decreased and delayed apoptosis might correlate with the responsiveness of PKC to radiation. PKC´ translocation is an interesting phenomenon. This isozymes is translocated only in ras-PK and 308 cells, suggesting that abnormality of these cells alters the responses of PKC´ to radiation and that ras oncogene activation may affect the responsiveness of PKC´ to radiation. The subdistribution of PKCh, on the other hand, was not changed by irradiation of 308 cells, while in PK and ras-PK, this isozyme was translocated. At present, it is not known whether specific PKC isozymes control the radiation-induced apoptosis, nor is it understood why the responsiveness of PKC translocation is different in normal neoplastic cells. It has been reported, however that neoplastic keratinocytes are defective in some of their responses to phorbol ester, and this suggests that functional alterations in PKC responses are characteristic of neoplastic keratinocytes w29x. Together with present results, it appears that in normal and ras-activated neoplastic keratinocytes, the responses of each PKC, especially rapid induction of PKCd or no translocation of PKC´ to radiation, may influence the induction of apoptosis.
Acknowledgements This study was supported by a National Project Grant from the Ministry of Science and Technology. We are grateful to Mr. Kyung-Jung Kim and Miss Sun-Ah Choi for their excellent technical assistance.
References w1x B.W. Stewart, Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators, J. Natl. Cancer Inst. 86 Ž1994. 1286–1296.
w2x J.F.R. Kerr, A.H. Wyllie, A.T. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics, Br. J. Cancer 26 Ž1972. 239–257. w3x R.L. Warters, Radiation-induced apoptosis in a murine T-cell hybridoma, Cancer Res. 52 Ž1992. 883–890. w4x K.S. Sellins, J.J. Cohen, Hyperthermia induces apoptosis in thymocytes, Radiat. Res. 126 Ž1991. 88–95. w5x G.I. Evan, A.H. Wyllie, C.S. Gibert, T.D. Littlewood, H. Land, M. Brooks, C.M. Waters, L.Z. Penn, D.C. Hancock, Induction of apoptosis in fibroblasts by c-myc protein, Cell 69 Ž1992. 119–128. w6x M.D.L. Sklar, The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation, Science 239 Ž1988. 645–647. w7x C. Borner, I.B. Weinstein, Transformation by ras oncogene causes increased expression of protein kinase C, Cell. Growth Differ. 1 Ž1990. 653–660. w8x M. Ruggiero, F. Casamassina, L. Magnelli, S. Pacini, J.H. Pierce, J.S. Greenberger, V.P. Chiarugi, Mitogenic signal transduction: a common target for oncogenes that induce resistance to ionizing radiation, Biochem. Biophys. Res. Commun. 183 Ž1992. 652–658. w9x D.R. Roop, D.R. Lowy, P.E. Tambourin, J.R. Harper, M. Balaschak, E.F. Spangler, S.H. Yuspa, An activated Harvey ras oncogene produces benign tumors on mouse epidermal tissue, Nature 323 Ž1986. 822–824. w10x Y. Nishizuka, Studies and perspectives of protein kinase C, Science 233 Ž1986. 693–698. w11x L.D. Tomei, P. Kanter, C.E. Wenner, Inhibition of radiationinduced apoptosis in vitro by tumor promoter, Biochem. Biophys. Res. Commun. 155 Ž1988. 324–441. w12x D.E. Hallahan, S. Virudachalam, J.L. Schwartz, N. Panje, R. Mustafi, R.R. Weichselbaum, Inhibition of protein kinase sensitizes human cells to ionizing radiation, Radiat. Res. 129 Ž1992. 345–350. w13x C. Borek, A. Ong, V.L. Stevens, E. Wang, A. Merrill, Long chain bases inhibit multistage carcinogenesis in mouse 10T1r2 cells treated with radiation and phorbol-12-myristate 13-acetate, Proc. Natl. Acad. Sci. 88 Ž1991. 1953–1957. w14x D.J. McConkey, P. Hartzell, M. Jondal, S. Orrenius, Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C, J. Biol. Chem. 264 Ž1989. 13399–13402. w15x L.D. Tomei, P. Kanter, C.E. Wenner, Inhibition of radiationinduced apoptosis in vitro by tumor promoters, Biochem. Biophys. Res. Commun. 155 Ž1988. 324–331. w16x F. Ojeda, M.I. Guarda, C. Maldonado, H. Folch, H. Diehl, Role of protein kinase C in thymocyte apoptosis induced by irradiation, Int. J. Radiat. Biol. 61 Ž1992. 663–667. w17x H. Hennings, D. Michael, C. Cheng, P. Steinert, K. Holbrook, S.H. Yuspa, Calcium regulation of growth and differentiation of mouse epidermal cells in culture, Cell 19 Ž1980. 245–254. w18x C. Cheng, A.E. Kilkenny, D. Roop, S.H. Yuspa, The Õ-ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes, Mol. Carcinog. 3 Ž1990. 363–373.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49 w19x J.E. Strickland, E.A. Greenhalgh, A. Koceva-Chyla, H. Hennings, C. Restrepo, M. Balaschak, S.H. Yuspa, Development of murine epidermal cell lines which contain an activated ras Ha oncogene and form papillomas in skin grafts on athymic nude mouse hosts, Cancer Res. 48 Ž1988. 165–169. w20x M.F. Denning, A.A. Dlugosz, E.K. Williams, Z. Szallasi, P.M. Blumberg, S.H. Yuspa, Specific protein kinase C isozymes mediate the induction of keratinocyte differentiation markers by calcium, Cell. Growth Differ. 6 Ž1995. 149–157. w21x K. Sellins, J.J. Cohen, Gene induction of gamma irradiation leads to DNA fragmentation in lymphocytes, J. Immunol. 139 Ž1987. 3199–3206. w22x D.J. McConkey, S. Orrenius, Signal transduction pathways to apoptosis, Trends Cell Biol. 4 Ž1994. 370–374. w23x F. Ojeda, M.I. Guarda, C. Maldonade, H. Folch, H. Diehl, Role of protein kinase C in thymocyte apoptosis induced by irradiation, Int. J. Radiat. Biol. 61 Ž1992. 663–667. w24x G. Woloschak, C.H. Chang-Liu, P. Shearin-Hones, Regulation of protein kinase C by ionizing radiation, Cancer Res. 50 Ž1990. 3963–3967.
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w25x N.H. Hasan, P.J. Parker, G.E. Adams, Induction and phosphorylation of protein kinase C-a and mitogen activated protein kinase by hypoxia and by radiation in Chinese hamster V79 cells, Radiat. Res. 145 Ž1996. 128–133. w26x C.Y. Kim, A.J. Giaccia, B. Strulovici, J.M. Brown, Differential expression of protein kinase C´ protein in lung cancer cell lines by ionizing radiation, Br. J. Cancer 66 Ž1992. 844–849. w27x T.K. Hei, R.S. Krauss, S.X. Liu, E.J. Hall, I.B. Weinstein, Effects of increased expression of protein kinase C on radiation-induced cell transformation, Carcinogenesis 15 Ž1994. 365–370. w28x A.A. Dlugosz, C. Cheng, K.W. Erin, A.G. Dharia, M.F. Denning, S.H. Yuspa, Alteration in murine keratinocyte differentiation induced by activated ras Ha genes are mediated by protein kinase C-a, Cancer Res. 54 Ž1994. 6413–6420. w29x S.H. Yuspa, H. Hennings, R.W. Tucker, The regulation of differentiation in normal and neoplastic keratinocytes, In: P. Nowley, T. Broker ŽEds.., Papillomaviruses, UCLA Symposia and Molecular and Cellular Biology, New Series, Alan R. Liss, New York, 1990, pp. 211–222.
Influence of ionizing radiation on induction of apoptotic cell death and cellular redistribution of protein kinase C isozymes in mouse epidermal cells differing in carcinogenesis stages Su-Jae Lee, Chul-Koo Cho, Seong-Yul Yoo, Tae-Hwan Kim, Yun-Sil Lee
)
Laboratory of Radiation Effect, Korea Cancer Center Hospital, 215-4 Gongneung-Dong, Nowon-Ku, Seoul, 139-706, South Korea Received 27 October 1998; received in revised form 2 March 1999; accepted 2 March 1999
Abstract Although protein kinase C ŽPKC. plays an important role in cellular response to radiation, little is known about the specific role of each isoform in the radiation induced cellular response. In this study, the induction of apoptosis and subcellular distribution of PKC isoforms after g-ray irradiation were examined in three kinds of mouse epidermal cells with different stages of carcinogenesis Žnormal mouse keratinocytes, PK: Õ-ras Ha transfected mouse keratinocytes, ras-PK; and neoplastic cells from mouse skin papilloma, 308 cells.. The induction of apoptosis was different in normal and neoplastic cells; in normal cells after 16 Gy of radiation, apoptosis was 2–10 times higher than that in ras-PK or 308 cells, and was rapidly induced; other cells died more slowly, depending on the stage of carcinogenesis. The responses of each PKC, especially rapid translocation of PKCd and no response of PKC´ by radiation in normal cells may influence the induction of apoptosis by radiation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Normal cell; Õ-ras Ha transformed cell; Papilloma cell; Protein kinase C isozymes; Cellular redistribution; Apoptotic cell death; Gamma ray
1. Introduction Apoptosis is important in normal tissues, carcinogenesis, tumor development, and cancer therapy w1,2x, and this mode of cell death can also be induced in various cell types by insults such as UV, ionizing radiation, and hyperthermia w3,4x. The attenuation of radiation-induced apoptosis by transfected Ha-ras
) Corresponding author. Tel.: q82-2-970-1325; Fax: q82-2977-0381; E-mail: [email protected]
oncogene w5x and radiation resistance is associated with oncogenes that have a direct or indirect involvement of plasma membranes in cellular response to ionizing radiation w6x. Activated ras is also associated with increased protein kinase C ŽPKC. expression w7x and also alters intracellular diacylglycerol ŽDAG. level w8x. The importance of ras oncogene as a causal factor in skin tumor initiation was emphasized when a mutated form of the gene, introduced into normal skin cells, resulted in the formation of benign tumors identical to those produced by initiation and promotion w9x.
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 0 7 8 - 0
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S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
PKC represents a large gene family of isozymes, differing remarkable in their structure and expression in various tissues, in their mode of activation, cofactor requirement, and substrate specificity w10x. Several sets of data have reported that PKC can influence cellular radiosensitivity, internucleosomal DNA fragmentation, and apoptotic cell death w11,12x. Furthermore, PKC plays a crucial role in mediating radiation-induced oncogenic transformation in C3H 10T1r2 cells w13x. Reports describing PKC activation and the induction of apoptosis have been contro-
versial. Certain results have shown that PKC activation blocks glucocorticoid and Ca2q ionophore-induced apoptosis w14x, and that tumor-promoting phorbol esters, which selectively stimulate PKC, also block radiation-induced apoptosis w15x; other data, however, have suggested that PKC inhibition by nonspecific PKC inhibitor prevents DNA fragmentation w16x. All data showed that radiation-induced influence of PKC activation and protein phosphorylation on apoptosis could be mediated by—for example—effects on cell cycles and differentiation.
Fig. 1. Determination of apoptosis by TUNEL assay: primary keratinocytes ŽPK., Õ-ras Ha transfected keratinocytes Žras-PK. and 308 papilloma cells Ž308. were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4, 8 and 16 Gy. After 5, 16, and 48 h, induction of apoptosis was detected by TUNEL assay. For each determination at least 500 cells were scored. All data points represent the result of five counts of 500 cells each. Two separate experiments showed similar results. Ža. Morphology of TUNEL positive cells after 16 h of 8 Gy irradiation arrow apoptotic fragments; Žb. quantitation of TUNEL positive cells.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
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Fig. 2. Cellular redistribution of PKC isozymes by g-rays: primary keratinocytes Ža., Õ-ras Ha transfected primary keratinocytes Žb., and 308 papilloma cells Žc. were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4 and 8 Gy. After the indicated times, cells were lysed and separated into soluble and Triton X-100 soluble particulate fractions, as described in Section 2. Isozyme-specific antibodies for PKCa , -d, -´, -h and -z were used for immunoblotting. We marked ‘U ’ on each PKC isozyme which showed significant alteration in translocation patterns. Similar results were obtained in two separate experiments.
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S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
However, since nonspecific PKC activators or inhibitors were used in the studies, the specific PKC isozymes that involved in these functions are not characterized. Three kinds of mouse epidermal cells at different stages of carcinogenesis Žnormal, ras-initiated, and papilloma tumor cells. were chosen for this study. The extent of apoptosis was greatest in normal cells; after irradiation, the process was rapidly induced. Depending on the stage of carcinogenesis, however, other cells died more slowly, and apoptosis was induced in only a few. We have characterized these
responses, examining the cellular subdistribution of PKC isozymes after exposure to g-rays.
2. Materials and methods 2.1. Cell culture Primary mouse epidermal keratinocytes ŽPK. were isolated from BALBrc mice and were grown in Eagle’s minimal essential medium with 8% Chelextreated fetal calf serum, 0.2% penicillinrstrepto-
Fig. 3. Quantitation of PKC isozymes by densitometry of immunoblots: primary keratinocytes, Õ-ras Ha transfected primary keratinocytes, and 308 papilloma cells were grown in the media of 0.05 Ca2q concentration and exposed to g-rays at doses of 4 and 8 Gy. After the indicated times, cells were lysed and separated into soluble and Triton X-100 soluble particulate fractions, as described in Section 2. Isozyme-specific antibodies for PKCa , -d, -´, -h and -z were used for immunoblotting. PKC isozymes were quantitated by densitometry of immunoblots and the data are expressed as the percentage particulate fraction relative to the unirradiated control level. Bars, SEs from two separate experiments.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
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Fig. 3 Žcontinued..
mycin solution ŽGibco, BRL, Gaithersburg, MD, USA., and 0.05 mM Ca2q to maintain a basal cell-like population of undifferentiated cells w17x. For Õ-ras Ha infection, primary keratinocytes were infected on day 2 or 3 after plating with a defected retrovirus containing the Õ-ras Ha gene w18x. The Õ-ras Ha keratinocytes Žras-PK. were produced by introducing a replication-defective retroviral vector containing the Õ-ras Ha gene into primary keratinocytes on day 3 of culture, using a high-titer viral supernatant prepared from the pse2 packaging cell line. Papilloma cell line 308 Ž308. was developed from pooled papillomas produced in Balbrc mice by initiation with 7,12-dimethylbenzwaxanthracene and promotion with a phorbol ester. 308 cells have an activated ras Ha gene with an A to T transversion at the second base of codon 61, and they form papillomas when grafted to athymic nude mice w19x.
2.2. Irradiation Cells were plated in sterile 10 cm dishes and incubated at 378C under humidified 5% CO 2 –95% air in 0.05 mM Ca2q containing media. When cells reached 80–90% confluency, they were exposed to gamma rays from 60 Co theraton-780 ŽAtomic Energy of Canada, Canada. at a dose rate of 1.394 Gyrmin. 2.3. TUNEL assay Cells were fixed in 95% ethanol and apoptosis was morphologically analysed using the Apoptag assay kit ŽOncor, Gaithersburg, MD.. DNA strand breaks were detected by 3X end labeling kit containing terminal deoxynucleotidyl transferase ŽTdT. and digoxigenin-11-dUTP according to manufacturer’s instructions. TdT reactions were performed for 1–3 h. After incubation with anti-horseradish pero-
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xidase-conjugated digoxigenin antibody and color development with H 2 O 2 and DAB; this produces a dark brown color. Cells were counter-stained with methyl greens, and for each determination at least 500 cells were scored. All data points represent the result of five counts of 500 cells each. 2.4. Cell fractionation After the treatment indicated, cells were washed twice with ice-cold PBS and scraped into PKC lysis buffer w20 mM Tris–HCl ŽpH 7.5., 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 40 mgrml leupeptinx. After brief sonication, the lysate was centrifuged at 100,000 = g for 1 h, and the supernatant was taken as the soluble fraction. The pellet was resuspended in PKC lysis buffer containing 1% Triton X-100 and centrifuged as before; the supernatant was then removed for use as the particulate fraction w20x. Protein concentration was determined by BioRad protein assay. 2.5. Immunoblotting Proteins were run immediately on 8.5% polyacrylamide gels and transferred electrophoretically to nitrocellulose, and the membranes were blocked in 5% milk. To detect PKC isozymes, the membranes were incubated with antibodies specific for the catalytic subunit of PKCa and -d at a dilution of 2 mgrml ŽSanta Cruz Biotechnology, Santa Cruz, CA, USA., PKC´ at a dilution of 1:1000 ŽGibco., PKCh at 1:100 ŽSanta Cruz Biotechnology., and PKCz at 1:100 ŽTransduction Laboratory, Lexington, KY, USA.. Proteins were detected using the ECL system ŽAmersham, Arlington Hights, IL, USA. with horseradish peroxidase conjugated secondary antibody ŽBio-Rad, Hercules, SA, USA. at a dilution of 1:5000. 2.6. Data analysis and statistics The immunoblots were quantitated using a MCID software program ŽImaging Research, Ontario, Canada.. For evaluation of PKC levels, the rate of change for each isozyme after irradiation was evaluated from three to four replicate experiments using multiple linear regression analysis. A rate of 2 indi-
cates a 2-fold increase. The SEs of each bar was estimated using standard least square methods. 3. Results 3.1. Susceptibility to radiation-induced apoptosis To compare radiosensitivity with different stages of carcinogenesis, the induction of apoptosis was detected by TUNEL assay. The greatest number of fragments was found in PK and the smallest in 308 cells. In PK, apoptosis was induced—at most—5 h after irradiation; in ras-PK, the maximum interval was 16 h, and in 308 cells, the maximum was 48 h ŽFig. 1.. 3.2. Cellular resubdistribution of specific PKC isozymes Changes in the total amounts of PKC isozymes do not indicate the degree to which PKC is activated by radiation; to determine which isozymes were translocated, we therefore analyzed changes in the distribution of PKC isozymes in soluble and particulate fractions of cells at different stages of carcinogenesis. Translocation of PKC to the particulate fraction is considered to be an indicator of cellular PKC activation. PKCa which is usually present in the cytosol fraction, underwent redistribution to the particulate fraction, with the largest translocation occurring 1–30 min after irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b., and 30–90 min after irradiation of 308 cells ŽFig. 2c.; the translocated amount of this isozyme was larger in ras-PK than PK. In the case of PKCd, which was distributed between both soluble and particulate fractions of basal cells w21x, radiation reproducibly caused translocation of PKCd between 1 and 30 min after irradiation of PK ŽFig. 2a., between 30 and 90 min after irradiation of ras-PK ŽFig. 2b., and 90 min after irradiation of 308 cells ŽFig. 2c.; soluble amounts of this isozyme showed selective decreases. PKC´ was distributed approximately equally between soluble and particulate fractions in basal cells of PK, but was usually present in the particulate fraction of ras-PK, suggesting that ras oncogene activation induced PKC´ translocation even without radiation. In 308 cells, PKC´ was abundantly distributed in the cytosol fraction.
S.-J. Lee et al.r Mutation Research 426 (1999) 41–49
Throughout the experiment, radiation did not induce the redistribution of PKC´ in PK, but did induce translocation to the particulate fraction of ras-PK and 308 cells. The translocation of PKC´ in ras-PK was transient ŽFig. 2b., but in 308 cells was sustained throughout the experiments ŽFig. 2c.. A transient rise in particulate PKCh was caused by irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b.; this phenomenon was more dominant in ras-PK than PK. Increased PKCh was not seen in particulate fraction of 308 cells ŽFig. 2c.. After irradiation of PK ŽFig. 2a. and ras-PK ŽFig. 2b., PKCz showed no responses; this isozyme was not easily detected in either soluble or particulate fractions of 308 cells. Summarized results are shown in Fig. 3. 4. Discussion Radiation-induced apoptosis does not mark the end of a passive degradation process; as in other cases of apoptosis, active synthesis of RNA and protein is required w21x. Considerable attention has recently focused on the relationship between PKC and apoptosis w22x. The bulk of evidence suggests that increased PKC activity antagonizes apoptosis, while reduced activity facilitates the process. Radiation increased PKC activity, and regulation of PKC mRNA by ionizing radiation, especially by X- and g-rays, has also been shown w23,24x. Even though many reports have described the relationship between PKC activation and apoptosis, the nature of this interaction remains obscure and may differ between cell types. However, one possible explanation is that cellular events by radiation share at least some common steps with the cell activation pathways; cellular activation by radiation may be a consequence of peroxidation at membrane level, which in turn may disturb the homeostasis of the membrane lipid metabolism, and induce hydrolysis of phosphatidylinositol-biphosphate to inositoltriphosphate and diacylglycerol, which are known to be responsible for Ca2q mobilization and PKC activation leading to biological reactions. Furthermore, extremely few reports have detailed the effects specific PKC isozymes on radiation-induced apoptosis, especially the differing responses of normal and neoplastic cells. Radiation caused increased expression of PKCa in Chinese hamster V79 cells w25x, of PKC´
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in lung cancer cells w26x, and of mRNA of PKCb in C3H 10T1r2 cells w27x. The existence of multiple PKC isozymes with distinct cofactor requirements and tissue distributions suggests specific roles for individual PKC isozymes in cellular physiology. Epidermal keratinocytes express at least five PKC isozymes, including those which are Ca2q-dependent ŽPKCa ., Ca2q-independent ŽPKCd, ´, and h., and DAGrphorbol ester-independent ŽPKCz .. In this study, we analyzed the subcellular distribution patterns of each PKC isozyme in irradiating mouse epidermal cells Žnormal, Õ-ras Ha initiated, and papilloma cells. differing in radiosensitivity detected by the induction of apoptosis. Induction varied according to the stage of carcinogenesis; it occurred rapidly in normal keratinocytes, peaking 5 h after irradiation, and in the case of Õ-ras Ha -transformed keratinocytes and 308 cells, peak induction was delayed; it occurred at 16 h in Õ-ras Ha transformed keratinocytes, and at 48 h in 308 cells, suggesting that radioresistance was increased by cell tumorigenesis. Many papers have described the relationship between radioresistance and transformation or tumorigenesis. In particular, radioresistance occurs when ras is overexpressed. ras-PK which are initiated, which showed a proliferative potency higher than that of PK and which caused the development of benign tumors when grafted to athymic nude mice w9x were more radioresistant than its parent cells ŽPK.; this was apparent when survival curves were examined and apoptosis was seen to have been induced ŽFig. 1.. A quantity of 308 cells which have an activated ras Ha gene with an A to T transversion at the second base of codon 61, form papillomas when grafted to athymic nude mice w19x, and these cells were the most radioresistant. These results showed that in mouse epidermal cells, apoptosis decreased and its induction was delayed according to the development of carcinogenesis. To know the relationship between the induction of apoptosis and PKC activation, the cellular subdistribution of PKC isozymes after radiation was examined. Since PKC is activated by radiation within minutes, we analyzed the translocation of PKC from soluble to the particulate fraction at 1, 30, and 90 min after irradiation ŽFigs. 2 and 3.. PKCa in PK and ras-PK was rapidly translocated by radiation; this phenomenon was more rapid in ras-PK and a
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large increase was also seen. Since transfection of Õ-ras Ha increased PKC activity especially Ca2q-dependent ŽPKCa . activity w28x, rapid response of PKCa by radiation in ras-PK may correlate to increased activity of this isozyme. In normal cells, PKCd were rapidly translocated by radiation, but in ras-PK and 308 cells, in which apoptosis was delayed and reduced, the translocation of these isozymes was delayed. These results suggested that decreased and delayed apoptosis might correlate with the responsiveness of PKC to radiation. PKC´ translocation is an interesting phenomenon. This isozymes is translocated only in ras-PK and 308 cells, suggesting that abnormality of these cells alters the responses of PKC´ to radiation and that ras oncogene activation may affect the responsiveness of PKC´ to radiation. The subdistribution of PKCh, on the other hand, was not changed by irradiation of 308 cells, while in PK and ras-PK, this isozyme was translocated. At present, it is not known whether specific PKC isozymes control the radiation-induced apoptosis, nor is it understood why the responsiveness of PKC translocation is different in normal neoplastic cells. It has been reported, however that neoplastic keratinocytes are defective in some of their responses to phorbol ester, and this suggests that functional alterations in PKC responses are characteristic of neoplastic keratinocytes w29x. Together with present results, it appears that in normal and ras-activated neoplastic keratinocytes, the responses of each PKC, especially rapid induction of PKCd or no translocation of PKC´ to radiation, may influence the induction of apoptosis.
Acknowledgements This study was supported by a National Project Grant from the Ministry of Science and Technology. We are grateful to Mr. Kyung-Jung Kim and Miss Sun-Ah Choi for their excellent technical assistance.
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