The alkylating carcinogen N-methyl-N’-nitro-N-nitrosoguanidine activates the plasminogen activator inhibitor-1 gene through sequential phosphorylation of p53 by ATM and ATR kinases

Summary

The alkylating agent MNNG is an environmental carcinogen that causes DNA lesions leading to cell death. We previously demonstrated that MNNG induced the transcriptional activity of the plasminogen activator inhibitor-1 (PAI-1) gene in a p53-dependent manner. However, the mechanism(s) linking external MNNG stimulation and PAI-1 gene induction remained to be elucidated. Here, we show that ATM and ATR kinases, but not DNA-PK, which participate in DNA damage-activated checkpoints, regulate the phosphorylation of p53 at serine 15 in response to MNNG cell treatment. Using ATM-deficient cells, ATM was shown to be required for early phosphorylation of serine 15 in response to MNNG, whereas catalytically inactive ATR selectively interfered with late phase serine 15 phosphorylation. In contrast, DNA-PK-deficient cells showed no change in the MNNG-induced serine 15 phosphorylation pattern. In agreement with this, sequential activation of ATM and ATR kinases was also required for adequate induction of the endogenous PAI-1 gene by MNNG. Finally, we showed that cells derived from PAI-1-deficient mice were more resistant to MNNG-induced cell death than normal cells, suggesting that p53-dependent PAI-1 expression partially mediated this effect. Since PAI-1 is involved in the control of tumor invasiveness, our finding that MNNG induces PAI-1 gene expression via ATM/ATR-mediated phosphorylation of p53 sheds new insight on the role of these DNA damage-induced cell cycle checkpoint kinases.

Keywords

Gene expression - proteases / inhibitors - signal transduction

^* Both authors have equally contributed to this work and should therefore be considered first authors.

• References

• 1 Adamson AW, Kim WJ, Shangary S. et al. ATM is activated in response to N-methyl-N'-nitro-N-nitrosoguanidine- induced DNA alkylation. J Biol Chem 2002; 277: 38222-9.
• 2 Appella E, Anderson CW. Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 2001; 268: 2764-72.
• 3 Banin S, Moyal L, Shieh S. et al. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998; 281: 1674-7.
• 4 Block WD, Merkle D, Meek K. et al. Selective inhibition of the DNA-dependent protein kinase (DNAPK) by the radiosensitizing agent caffeine. Nucleic Acids Res 2004; 32: 1967-72.
• 5 Brown KD, Ziv Y, Sadanandan SN. et al. The ataxiatelangiectasia gene product, a constitutively expressed nuclear protein that is not up-regulated following genome damage. Proc Natl Acad Sci U S A 1997; 94: 1840-5.
• 6 Canman CE, Lim DS, Cimprich KA. et al. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998; 281: 1677-9.
• 7 Carmeliet P, Kieckens L, Schoonjans L. et al. Plasminogen activator inhibitor-1 gene-deficient mice. J Clin Invest 1993; 92: 2746-55.
• 8 Dunkern T, Roos W, Kaina B. Apoptosis induced by MNNG in human TK6 lymphoblastoid cells is p53 and Fas/CD95/Apo-1 related. Mutat Res 2003; 544: 167-72.
• 9 Eadie JS, Conrad M, Toorchen D. et al. Mechanism of mutagenesis by O6-methylguanine. Nature 1984; 308: 201-3.
• 10 Frisch SM, Screaton RA. Anoikis mechanisms. Curr Opin Cell Biol 2001; 13: 555-62.
• 11 Giaccia AJ, Kastan MB. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 1998; 12: 2973-83.
• 12 Griswold DP, Corbett TH. A colon tumor model for anticancer agent evaluation. Cancer 1975; 36: 2441-4.
• 13 Irigoyen JP, Munoz-Canoves P, Montero L. et al. The plasminogen activator system: biology and regulation. Cell Mol Life Sci 1999; 56: 104-32.
• 14 Jimenez GS, Bryntesson F, Torres-Arzayus I M. et al. DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage. Nature 1999; 400: 81-3.
• 15 Kaina B, Fritz G, Mitra S. et al. Transfection and expression of human O6-methylguanine-DNAmethyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents. Carcinogenesis 1991; 12: 1857-67.
• 16 Keegan KS, Holtzman DA, Plug AW. et al. The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes. Genes Dev 1996; 10: 2423-37.
• 17 Kleihues P, Margison GP. Carcinogenicity of N-methyl-N-nitrosourea: possible role of excision of O6-methylguanine from DNA. J Natl Cancer Inst 1974; 53: 1839-41.
• 18 Lees-Miller SP, Sakaguchi K, Ullrich SJ. et al. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol 1992; 12: 5041-9.
• 19 Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323-31.
• 20 Lindstrom CG, Rosengren JE, Ekberg O. Experimental colonic tumours in the rat. III. Induction time, distribution and appearance of induced tumours. Acta Radiol Diagn 1978; 19: 799-816.
• 21 Loveless A. Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature 1969; 223: 206-7.
• 22 Munoz-Canoves P, Miralles F, Baiget M. et al. Inhibition of urokinase-type plasminogen activator (uPA) abrogates myogenesis in vitro. Thromb Haemost 1997; 77: 526-34.
• 23 Nghiem P, Park PK, Kim YS. et al. ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint. J Biol Chem 2002; 277: 4428-34.
• 24 Oren M. Regulation of the p53 tumor suppressor protein. J Biol Chem 1999; 274: 36031-4.
• 25 Parra M, Jardi M, Koziczak M. et al. p53 Phosphorylation at serine 15 is required for transcriptional induction of the plasminogen activator inhibitor-1 (PAI-1) gene by the alkylating agent N-methyl- N'-nitro-N-nitrosoguanidine. J Biol Chem 2001; 276: 36303-10.
• 26 Peto R, Gray R, Brantom R. et al. N-nitroso compounds: occurrence, biological effects and relevance to human cancer. IARC Scientific Publication nº 57. International Agency for Research on Cancer, Lyon 1985; 627-55.
• 27 Prives C, Hall PA. The p53 pathway. J Pathol 1999; 187: 112-26.
• 28 Rotman G, Shiloh Y. ATM: a mediator of multiple responses to genotoxic stress. Oncogene 1999; 18: 6135-44.
• 29 Saffhill R, Margison GP, O'Connor PJ. Mechanisms of carcinogenesis induced by alkylating agents. Biochimica et Biophysica Acta 1985; 823: 111-45.
• 30 Saito S, Goodarzi AA, Higashimoto Y. et al. ATM mediates phosphorylation at multiple p53 sites, including Ser(46), in response to ionizing radiation. J Biol Chem 2002; 277: 12491-4.
• 31 Sakaguchi K, Herrera JE, Saito S. et al. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 1998; 12: 2831-41.
• 32 Sanchez-Prieto R, Rojas JM, Taya Y et al. A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemotherapeutic agents. Cancer Res 2000; 60: 2464-72.
• 33 Sarkaria JN, Busby EC, Tibbetts RS. et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 1999; 59: 4375-82.
• 34 Shulman LN. The biology of alkylating-agent cellular injury. Hematol Oncol Clin North Am 1993; 7: 325-35.
• 35 Siliciano JD, Cancan CE, Taya Y. et al. DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev 1997; 11: 3471-81.
• 36 Singer B. The chemical effects of nucleic acids alkylation and their relation to mutagenesis and carcinogenesis. Prog Nucleic Acid Res 1975; 15: 219-84.
• 37 Singer B, Grunberger D. Molecular biology of mutagens and carcinogens. Plenum Press; NewYork: 1983
• 38 Thomale J, Huh NH, Nehls P. et al. Repair of O6-ethylguanine in DNA protects rat 208F cells fom tumorigenic conversion by N-ethyl-N-nitrosourea. Proc Natl Acad Sci U S A 1997; 87: 9883-7.
• 39 Tibbetts RS, Brumbaugh KM, Williams JM. et al. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999; 13: 152-7.
• 40 Tsujii M, Kawano S, Tsuji S. et al. Mechanism for ammonia-induced promotion of gastric carcinogenesis in rats. Carcinogenesis 1995; 16: 563-6.
• 41 Wesierska-Gadek J, Gueorguieva M, Schloffer D. et al. Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). J Cell Biochem 2003; 89: 1222-34.
• 42 Ye R, Bodero A, Zhou BB. et al. The plant isoflavenoid, genistein, activates p53 and Chk2 in an ATM-dependent manner. J Biol Chem 2000; 28: 28
• 43 Yih LH, Lee TC. Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res 2000; 60: 6346-52.
• 44 Zhang Y, Ma WY, Kaji A. et al. Requirement of ATM in UVA-induced signaling and apoptosis. J Biol Chem 2002; 277: 3124-31.