Abstract
Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14–18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CΘ, Src, Bax and μ-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated μ-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, μ-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells. Cell Death and Differentiation (2000) 7, 531–537
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- ALLN/calpain inhibitor I:
-
N-acetyl-Leu-Leu-norleucinal
- DMSO:
-
dimethylsulfoxide
- MTT:
-
3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide
- SAPK:
-
stress-activated protein kinase
- UV:
-
ultraviolet irradiation
References
Tyrell RM . (1984) Exposure of nondividing populations of primary human fibroblasts to UV (254nm) radiation induces a transient enhancement in capacity to repair potentially lethal cellular damage. Proc. Natl. Acad. Sci. USA 81: 781–784
Tyrrell RM . (1996) Activation of mammalian gene expression by the UV component of sunlight-from models to reality. BioEssays 18: 139–148
Cox LS and Lane DP . (1995) Tumor suppressors, kinases and clamps: How p53 regulates the cell cycle in response to DNA damage. BioEssays 17: 501–508
Devary Y, Gottlieb RA, Smeal T and Karin M . (1992) The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell 71: 1081–1091
Sachsenmaier C, Radler-Pohl A, Zinck R, Nordheim A, Herrlich P and Rahmsdorf HJ . (1994) Involvement of growth factor receptors in the mammalian UVC response. Cell 78: 963–972
Coffer PJ, Burgering BMTh, Peppelenbosch MP, Bos JL and Kruijer W . (1995) UV activation of receptor tyrosine kinase activity. Oncogene 11: 561–569
Schieven GL, Kirihara JM, Burg DL, Geahlen RL and Ledbetter JA . (1993) p72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals. J. Biol. Chem. 268: 16688–16692
Schieven GL, Mittler RS, Nadler SG, Kirihara JM, Bolen JB, Kanner SB and Ledbetter JA . (1994) ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T-cell signal transduction. J. Biol. Chem. 269: 20718–20726
Stein B, Rahmsdorf HJ, Steffen A, Liftin M and Herrlich P . (1989) UV-induced DNA damage is an intermediate step in UV-induced expression of human immunodeficiency virus type 1, collagenase, c-fos, and metallothionein. Mol. Cell. Biol. 9: 5169–5181
Janknecht R, Ernst WH, Pingoud V and Nordheim A . (1993) Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J. 12: 5097–5104
El-Deiry WS, Tokino T, Velculescu VE, Leby DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW and Vogelstein B . (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817–825
Miyashita T and Reed JC . (1995) Tumor suppressor p53 is a direct transcriptional activator of human bax gene. Cell 80: 293–299
Rudoltz MS, Kao G, Blank KR, Muschel RJ and McKenna WG . (1996) Molecular Biology of the cell cycle: potential for therapeutic applications in radiation oncology. Semin. Rad. Oncol. 6: 284–294
Kumar S and Lavin MF . (1996) The ICE family of cysteine proteases as effectors of cell death. Cell Death Differ. 3: 255–267
Oltvai Z, Milliman C and Korsmeyer S . (1993) Bcl-2 heterodimerizes in vivo with a homolog, bax, that accelerates programmed cell death. Cell 74: 609–619
Suzuki K, Imajoh S, Emori Y, Kawasaki H, Minami Y and Ohno S . (1987) Calcium-activated neutral protease and its endogenous inhibitor. FEBS Lett. 220: 271–277
Carafoli E and Molinari M . (1998) Calpain: a protease in search of a function? Biochem. Biophys. Res. Commun. 247: 193–203
Sorimachi H, Imajoh-Ohmi S, Emori Y, Kawasaki H, Ohno S, Minami Y and Suzuki K . (1989) Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and m-types. Specific expression of the mRNA in skeletal muscle. J. Biol. Chem. 264: 20106–20111
Emori Y, Kawasaki H, Imajoh S, Imahori K and Suzuki K . (1987) Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites. Proc. Natl. Acad. Sci. USA 84: 3590–3594
Squier MKT, Miller ACK, Malkinson AM and Cohen JJ . (1994) Calpain activation in apoptosis. J. Cell. Physiol. 159: 229–237
Squier MT and Cohen JJ . (1997) Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158: 3690–3697
Mampuru LJ, Chen SJ, Kalenik JL, Bradley ME and Lee TC . (1996) Analysis of events associated with serum deprivation-induced apoptosis in C3H/Sol8 muscle satellite cells. Exp. Cell Res. 226: 372–380
Nath R, Raser KJ, McGinnis K, Nadimpalli R, Stafford D and Wang KK . (1996a) Effects of ICE-like protease and calpain inhibitors on neuronal apoptosis. Neuroreport 8: 249–255
Gressner AM, Lahme B and Roth S . (1997) Attenuation of TGF-β-induced apoptosis in primary cultures of hepatocytes by calpain inhibitors. Biochem. Biophys. Res. Commun. 231: 457–462
Villa PG, Henzel WJ, Sensenbrenner M, Henderson CE and Pettmann B . (1998) Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis. J. Cell Sci. 111: 713–722
Spinedi A, Oliverio S, Di Sano F and Piacentini M . (1998) Calpain involvement in calphostin C-induced apoptosis. Biochem. Pharmacol. 56: 1492–1498
Debiasi RL, Squier MKT, Pike B, Wynes M, Dermody TS, Cohen JJ and Tyler KL . (1999) Reovirus-induced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors. J. Virol. 73: 695–701
Ruiz-Vela A, de Buitrago GG and Martínez -AC . (1999) Implications of calpain in caspase activation during B cell clonal deletion. EMBO J. 18: 4988–4998
Squier MKT, Sehnert AJ, Sellins KS, Malkinson AM, Takano E and Cohen JJ . (1999) Calpain and calpastatin regulate neutrophil apoptosis. J. Cell. Physiol. 178: 311–319
Wang KKW, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon RA, Talanian RV, Keegan M, Herzog L and Allen H . (1998) Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch. Biochem. Biophys. 356: 187–196
Pörn-Ares MI, Samali A and Orrenius S . (1998) Cleavage of the calpain inhibitor, calpastatin, during apoptosis. Cell Death Differ. 5: 1028–1033
Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner A, Allen H, Talanian RV, Yuen P, Gilbertsen RB and Wang KKW . (1996b) Non-erythroid α-spectrin breakdown by calpain and interleukin 1β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319: 683–690
Wood DE, Thomas A, Devi LA, Berman Y, Beavis RC, Reed JC and Newcomb EW . (1998) Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17: 1069–1078
Lu Q and Mellgren RL . (1996) Calpain inhibitors and serine protease inhibitors can product apoptosis in HL-60 cells. Arch. Biochem. Biophys. 334: 175–181
Zhu W, Murtha PE and Young CYF . (1995) Calpain inhibitor-induced apoptosis in human prostate adenocarcinoma cells. Biochem. Biophys. Res. Commun. 214: 1130–1137
Baghdiguian S, Martin M, Richard I, Pons F, Astier C, Bourg N, Hay RT, Chemaly R, Halaby G, Loiselet J, Anderson LVB, de Munain AL, Fardeau M, Mangeat P, Backmann JS and Lefranc G . (1999) Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IκBa/NF-κB pathway in limb-girdle muscular dystrophy type 2A. Nature Med. 5: 503–511
Atsma DE, Bastiaanse EML, Jerzewski A, Van der Valk LJM and Van der Laarse A . (1995) Role of calcium-activated neutral protease (calpain) in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition. Circ. Res. 76: 1071–1078
Suzuki N and Fuse A . (1981) A UV-sensitive human clonal cell line, RSa, which has low repair activity. Mutat. Res. 84: 133–145
Suzuki N . (1984) A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line. Mutat. Res. 125: 55–63
Suzuki N, Nishimaki J and Kuwata T . (1982) Characterization of a UV-resisitant strain, UVr-10, established from a 4NQQ, MNNG and interferon. Mutat. Res. 106: 357–376
Smith CA, Williams GT, Kingston R, Jenkinson EJ and Owen JJ . (1989) Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymoic culture. Nature 337: 181–184
Nishizuka Y . (1995) Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484–496
Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC and Williams GT . (1996) Apoptosis: molecular regulation of cell death. Eur. J. Biochem. 236: 1–26
Maltzman W and Czyzyk L . (1984) UV-irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell. Biol. 4: 1689–1694
Kishimoto A, Mikawa K, Hashimoto K, Yasuda I, Tanaka S, Tominaga M, Kurada T and Nishizuka Y . (1989) Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). J. Biol. Chem. 264: 4088–4092
Oda A, Druker BJ, Ariyoshi H, Smith M and Salzman EW . (1993) pp60src is an endogenous substrate for calpain in human blood platelets. J. Biol. Chem. 268: 12603–12608
Sorimachi H, Saido TC and Suzuki K . (1994) New era of calpain research; discovery of tissue-specific calpains. FEBS Lett. 343: 1–5
Saido TC, Sorimachi H and Suzuki K . (1994) Calpain: new perspectives in molecular diversity and physiological-pathophysiological involvement. FASEB J. 8: 814–822
Maki M, Bagci H, Hamaguchi K, Ueda M, Murachi T and Hatanaka M . (1989) Inhibition of calpain by a synthetic oligopeptide corresponding to an exon of the human calpastatin gene. J. Biol. Chem. 264: 18866–18869
Gregoriou M, Willis AC, Pearson MA and Crawford C . (1994) The calpain cleavage sites in the epidermal growth factor receptor kinase domain. Eur. J. Biochem. 223: 455–464
Hirai S, Kawasaki H, Yaniv M and Suzuki K . (1991) Degradation of transcription factors, c-Jun and c-Fos, by calpain. FEBS Lett. 287: 57–61
Kubbutat MH and Vousden KH . (1997) Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. Mol. Cell. Biol. 17: 460–468
Glotzer M, Murray AW and Kirschner MW . (1991) Cyclin is degraded by the ubiquitin pathway. Nature 349: 132–138
Treier M, Staszewski L and Bohmann D . (1994) Ubiquitin-dependent c-Jun degradation in vivo is mediated by the d domain. Cell 78: 787–798
Ciechanover A, DiGiuseppe JA, Bercovich B, Orian A, Richter JD, Schwartz AL and Brodeur GM . (1991) Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc. Natl. Acad. Sci. USA 88: 139–143
Scheffner M, Huibregtse JM, Vierstra R and Howley PM . (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75: 495–505
Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbanda S, Robertson M, Ghayur T, Wong WW, Kamen R, Weichselbaum R and Kufe D . (1995) Proteolytic activation of protein kinase by Cδ by an ICE-like protease in apoptotic cells. EMBO J. 14: 6148–6156
Cardone MH, Salvesen GS, Widmann C, Johnson G and Frisch SM . (1997) The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell 90: 315–323
Widmann C, Gibson S and Johnson GL . (1988) Caspase-dependent cleavage of signaling proteins during apoptosis. J. Biol. Chem. 273: 7141–7147
Gervais JLM, Seth P and Zhang H . (1998) Cleavage of CDK inhibitor p21Cip1/Waf1 by caspases is an early event during DNA damage-induced apoptosis. J. Biol. Chem. 273: 19207–19212
Tompa P, Baki A, Schád É and Friedrich P . (1996) The calpain cascade. μ-calpain activates m-calpain. J. Biol. Chem. 271: 33161–33164
Harris KF, Shoji I, Cooper EM, Kumar S, Oda H and Howley PM . (1999) Ubiquitin-mediated degradation of active Src tyrosine kinase. Proc. Natl. Acad. Sci. USA 96: 13738–13743
Ghayur BT, Hugunin M, Talanian RV, Ratnofsky S, Quinla C, Emoto Y, Pandey P, Datta R, Huang Y, Kharbanda S, Allen H, Kame R, Wong W and Kuf D . (1996) Proteolytic activation of protein kinase Cδ by an ICE/CED3-like protease induces characteristics of apoptosis. J. Exp. Med. 184: 2399–2404
Frutos S, Moncat J and Diaz-Meco MT . (1999) Cleavage of ζPKC but not λ/ιPKC by caspase-3 during UV-induced apoptosis. J. Biol. Chem. 274: 10765–10770
Hiwasa T, Sawada T and Sakiyama S . (1990) Cysteine proteinase inhibitors and ras gene products share the same biological activities including transforming activity toward NIH3T3 mouse fibroblasts and the differentiation-inducing activity toward PC12 rat pheochromocytoma cells. Carcinogenesis 11: 75–80
Suzuki N, Suzuki H, Kojima T, Sugita K, Takakubo Y and Okamoto S . (1988) Effects of human interferon on cellular response to UV in UV-sensitive human cell strains. Mutat. Res. 198: 207–214
Mosmann T . (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55–63
Hiwasa T, Tokita H, Sakiyama S and Nakagawara A . (1998) Enhancement of chemosensitivity toward anticancer drugs by high expression of caspase-1 in NIH3T3 cells. Anti-Cancer Drugs 9: 82–87
Kikuchi H and Imajor-Ohmi S . (1995) Activation and possible involvement of calpain, a calcium-activated cysteine protease, in down-regulation of apoptosis of human monoblast U937 cells. Cell Death Differ. 2: 195–199
Hiwasa T and Sakiyama S . (1996) Nuclear localization of procathepsin L/MEP in ras-transformed mouse fibroblasts. Cancer Lett 99: 87–91
Hiwasa T, Ma J, Ike Y, Katunuma N and Sakiyama S . (1995) Increase of cyclin B by overexpression of cystatin α. Cell Biochem. Func. 13: 293–296
Hitomi K, Yokoyama A and Maki M . (1998) Expression of biologically active human calpastatin in baculovirus-infected insect cells and in Escherichia coli. Biosci. Biotechnol. Biochem. 62: 136–141
Acknowledgements
This work was supported in part by a Grant-in-Aid from Tokyo Biochemical Research Foundation, grants from the Smoking Research Foundation, ‘Ground Research for Space Utilization’ promoted by NASDA and Japan Space Forum, and a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by S Kumar
Rights and permissions
About this article
Cite this article
Hiwasa, T., Arase, Y., Kikuno, K. et al. Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells. Cell Death Differ 7, 531–537 (2000). https://doi.org/10.1038/sj.cdd.4400685
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4400685
Keywords
This article is cited by
-
Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells
Archives of Dermatological Research (2004)
