Apoptosis

, Volume 16, Issue 5, pp 449–459 | Cite as

Chk1 has an essential role in the survival of differentiated cortical neurons in the absence of DNA damage

Original Paper

Abstract

Neuronal death in the central nervous system contributes to the development of age-related neurodegeneration. The ATR/Chk1 pathway appears to function neuroprotectively to prevent DNA damage induced by cytotoxic agents. Here, we examine the function of Chk1 on cell viability of cortical neurons in the absence of additional DNA damaging stimuli. The Chk1-specific inhibitor, UCN-01, and the ATR inhibitor, Caffeine, cause neuronal apoptosis in differentiated neurons in the absence of additional treatment, whereas inhibition of ATM or Chk2, does not. UCN-01 treatment increased the detection of γ-H2AX phosphorylation, DNA strand breaks, and an activated p53-dependent DNA damage response (DDR), suggesting that Chk1 normally helps to maintain genomic stability. UCN-01 treatment also enhanced the apoptosis seen in neurons treated with DNA damaging agents, such as camptothecin (CPT). Our results indicate that Chk1 is essential for neuronal survival, and perturbation of this pathway increases a cell’s sensitivity to naturally accumulating DNA damage.

Keywords

Chk1 ATR p53 Apoptosis DNA damage response Neurons 

References

  1. 1.
    Cha RS, Kleckner N (2002) ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones. Science 297:602–606PubMedCrossRefGoogle Scholar
  2. 2.
    Ward IM, Minn K, Chen J (2004) UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress. J Biol Chem 279:9677–9680PubMedCrossRefGoogle Scholar
  3. 3.
    Martin LJ, Liu Z, Pipino J, Chestnut B, Landek MA (2009) Molecular regulation of DNA damage-induced apoptosis in neurons of cerebral cortex. Cereb Cortex 19:1273–1293PubMedCrossRefGoogle Scholar
  4. 4.
    Zachos G, Rainey MD, Gillespie DA (2003) Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects. EMBO J 22:713–723PubMedCrossRefGoogle Scholar
  5. 5.
    Syljuasen RG, Sorensen CS, Hansen LT et al (2005) Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25:3553–3562PubMedCrossRefGoogle Scholar
  6. 6.
    Brown EJ, Baltimore D (2000) ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 14:397–402PubMedGoogle Scholar
  7. 7.
    Takai H, Tominaga K, Motoyama N et al (2000) Aberrant cell cycle checkpoint function and early embryonic death in Chk1(−/−) mice. Genes Dev 14:1439–1447PubMedGoogle Scholar
  8. 8.
    Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates fragile site stability. Cell 111:779–789PubMedCrossRefGoogle Scholar
  9. 9.
    Durkin SG, Arlt MF, Howlett NG, Glover TW (2006) Depletion of CHK1, but not CHK2, induces chromosomal instability and breaks at common fragile sites. Oncogene 25:4381–4388PubMedCrossRefGoogle Scholar
  10. 10.
    Davis ST, Benson BG, Bramson HN et al (2001) Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors. Science 291:134–137PubMedCrossRefGoogle Scholar
  11. 11.
    Kubo A, Nakagawa K, Varma RK et al (1999) The p16 status of tumor cell lines identifies small molecule inhibitors specific for cyclin-dependent kinase 4. Clin Cancer Res 5:4279–4286PubMedGoogle Scholar
  12. 12.
    Ye W, Blain SW (2010) S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons. Brain 133:2295–2312PubMedGoogle Scholar
  13. 13.
    Crouch SP, Kozlowski R, Slater KJ, Fletcher J (1993) The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160:81–88PubMedCrossRefGoogle Scholar
  14. 14.
    James MK, Ray A, Leznova D, Blain SW (2008) Differential modification of p27Kip1 controls its cyclin D-cdk4 inhibitory activity. Mol Cell Biol 28:498–510PubMedCrossRefGoogle Scholar
  15. 15.
    Tice RR, Agurell E, Anderson D et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221PubMedCrossRefGoogle Scholar
  16. 16.
    Ye W, Zhang L (2004) Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells. Biochem Biophys Res Commun 319:1065–1071PubMedCrossRefGoogle Scholar
  17. 17.
    Martinez G, Di Giacomo C, Carnazza ML et al (1997) MAP2, synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion. Dev Neurosci 19:457–464PubMedCrossRefGoogle Scholar
  18. 18.
    Sarkaria JN, Busby EC, Tibbetts RS et al (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59:4375–4382PubMedGoogle Scholar
  19. 19.
    Zhao B, Bower MJ, McDevitt PJ et al (2002) Structural basis for Chk1 inhibition by UCN-01. J Biol Chem 277:46609–46615PubMedCrossRefGoogle Scholar
  20. 20.
    Celeste A, Fernandez-Capetillo O, Kruhlak MJ et al (2003) Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 5:675–679PubMedCrossRefGoogle Scholar
  21. 21.
    Sionov RV, Haupt Y (1999) The cellular response to p53: the decision between life and death. Oncogene 18:6145–6157PubMedCrossRefGoogle Scholar
  22. 22.
    Murphy PJ, Galigniana MD, Morishima Y et al (2004) Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation. J Biol Chem 279:30195–30201PubMedCrossRefGoogle Scholar
  23. 23.
    Kondratov RV, Komarov PG, Becker Y, Ewenson A, Gudkov AV (2001) Small molecules that dramatically alter multidrug resistance phenotype by modulating the substrate specificity of P-glycoprotein. Proc Natl Acad Sci USA 98:14078–14083PubMedCrossRefGoogle Scholar
  24. 24.
    Strom E, Sathe S, Komarov PG et al (2006) Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol 2:474–479PubMedCrossRefGoogle Scholar
  25. 25.
    Morris EJ, Geller HM (1996) Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. J Cell Biol 134:757–770PubMedCrossRefGoogle Scholar
  26. 26.
    Park DS, Morris EJ, Greene LA, Geller HM (1997) G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. J Neurosci 17:1256–1270PubMedGoogle Scholar
  27. 27.
    Seshadri S, Beiser A, Selhub J et al (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 346:476–483PubMedCrossRefGoogle Scholar
  28. 28.
    Mizrahi EH, Bowirrat A, Jacobsen DW et al (2004) Plasma homocysteine, vitamin B12 and folate in Alzheimer’s patients and healthy Arabs in Israel. J Neurol Sci 227:109–113PubMedCrossRefGoogle Scholar
  29. 29.
    Agnati LF, Genedani S, Rasio G et al (2005) Studies on homocysteine plasma levels in Alzheimer’s patients. Relevance for neurodegeneration. J Neural Transm 112:163–169PubMedCrossRefGoogle Scholar
  30. 30.
    Ruzankina Y, Pinzon-Guzman C, Asare A, Ong T, Pontano L, Cotsarelis G, Zediak VP, Velez M, Bhandoola A, Brown EJ (2007) Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1:113–126PubMedCrossRefGoogle Scholar
  31. 31.
    Pauklin S, Kristjuhan A, Maimets T, Jaks V (2005) ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress. Biochem Biophys Res Commun 334:386–394PubMedCrossRefGoogle Scholar
  32. 32.
    Liu Q, Guntuku S, Cui XS et al (2000) Chk1 is an essential kinase that is regulated by ATR and required for the G(2)/M DNA damage checkpoint. Genes Dev 14:1448–1459PubMedCrossRefGoogle Scholar
  33. 33.
    Takai H, Naka K, Okada Y et al (2002) Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription. EMBO J 21:5195–5205PubMedCrossRefGoogle Scholar
  34. 34.
    Friedberg EC (2005) Suffering in silence: the tolerance of DNA damage. Nat Rev Mol Cell Biol 6:943–953PubMedCrossRefGoogle Scholar
  35. 35.
    Zhang Y, Qu D, Morris EJ et al (2006) The Chk1/Cdc25A pathway as activators of the cell cycle in neuronal death induced by camptothecin. J Neurosci 26:8819–8828PubMedCrossRefGoogle Scholar
  36. 36.
    Wohlbold L, Fisher RP (2009) Behind the wheel and under the hood: functions of cyclin-dependent kinases in response to DNA damage. DNA Repair (Amst) 8:1018–1024CrossRefGoogle Scholar
  37. 37.
    Satyanarayana A, Berthet C, Lopez-Molina J, Coppola V, Tessarollo L, Kaldis P (2008) Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2. Development 135:3389–3400PubMedCrossRefGoogle Scholar
  38. 38.
    Vogel C, Hager C, Bastians H (2007) Mechanisms of mitotic cell death induced by chemotherapy-mediated G2 checkpoint abrogation. Cancer Res 67:339–345PubMedCrossRefGoogle Scholar
  39. 39.
    Tse AN, Schwartz GK (2004) Potentiation of cytotoxicity of topoisomerase I poison by concurrent and sequential treatment with the checkpoint inhibitor UCN-01 involves disparate mechanisms resulting in either p53-independent clonogenic suppression or p53-dependent mitotic catastrophe. Cancer Res 64:6635–6644PubMedCrossRefGoogle Scholar
  40. 40.
    Myers K, Gagou ME, Zuazua-Villar P, Rodriguez R, Meuth M (2009) ATR and Chk1 suppress a caspase-3-dependent apoptotic response following DNA replication stress. PLoS Genet 5:e1000324PubMedCrossRefGoogle Scholar
  41. 41.
    Evans TA, Raina AK, Delacourte A et al (2007) BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease. Int J Med Sci 4:140–145PubMedGoogle Scholar
  42. 42.
    Thakur A, Siedlak SL, James SL et al (2008) Retinoblastoma protein phosphorylation at multiple sites is associated with neurofibrillary pathology in Alzheimer disease. Int J Clin Exp Pathol 1:134–146PubMedGoogle Scholar
  43. 43.
    Lanni C, Racchi M, Stanga S et al (2010) Unfolded p53 in blood as a predictive signature signature of the transition from mild cognitive impairment to Alzheimer’s disease. J Alzheimers Dis 20:97–104PubMedGoogle Scholar
  44. 44.
    Wilson C, Henry S, Smith MA, Bowser R (2004) The p53 homologue p73 accumulates in the nucleus and localizes to neurites and neurofibrillary tangles in Alzheimer disease brain. Neuropathol Appl Neurobiol 30:19–29PubMedCrossRefGoogle Scholar
  45. 45.
    Zhu X, Raina AK, Boux H, Simmons ZL, Takeda A, Smith MA (2000) Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int J Dev Neurosci 18:433–437PubMedCrossRefGoogle Scholar
  46. 46.
    McShea A, Wahl AF, Smith MA (1999) Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease. Med Hypotheses 52:525–527PubMedCrossRefGoogle Scholar
  47. 47.
    Jordan-Sciutto KL, Malaiyandi LM, Bowser R (2002) Altered distribution of cell cycle transcriptional regulators during Alzheimer disease. J Neuropathol Exp Neurol 61:358–367PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PediatricsState University of New York, Downstate Medical CenterBrooklynUSA
  2. 2.Department of Cell BiologyState University of New York, Downstate Medical CenterBrooklynUSA
  3. 3.Department of ResearchHackensack University Medical CenterHackensackUSA

Personalised recommendations