Skip to main content
SpringerLink
Log in

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco-2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco-2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF-α/CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

Abbreviations

IEC-6:

Intestinal epithelial cells

p21Cip1:

p21Waf1/Cip1

DFMO:

α-difluromethylornithine

TNF-α:

Tumor necrosis factor-α

CPT:

Camptothecin

ODC:

Ornithine decarboxylase

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

dFBS:

Dialyzed FBS

ECL:

Enhanced chemiluminescence

DPBS:

Dulbecco’s PBS

Caco-2:

Colon carcinoma cells

References

  1. Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine I columnar cell. Am J Anat 141:461–479

    Article  CAS  PubMed  Google Scholar 

  2. Hall PA, Coates PJ, Ansari B, Hopwood D (1994) Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107:3569–3577

    CAS  PubMed  Google Scholar 

  3. Potten CS, Grant HK (1998) The relationship between ionizing radiation-induced apoptosis and stem cells in the small and large intestine. Br J Cancer 78:993–1003

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Vermeulen K, Van Bockstaele DR, Berneman ZN (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 36:131–149

    Article  CAS  PubMed  Google Scholar 

  5. Arellano M, Moreno S (1997) Regulation of CDK/cyclin complexes during the cell cycle. Int J Biochem Cell Biol 29:559–573

    Article  CAS  PubMed  Google Scholar 

  6. Johnsen SA (2012) Cdk9 and H2B monoubiquitination: a well-choreographed dance. PLoS Genet 8:e1002860

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Lee MH, Reynisdottir I, Massague J (1995) Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev 9:639–649

    Article  CAS  PubMed  Google Scholar 

  8. Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang P, Dobrowolski S, Bai C, Connell-Crowley L, Swindell E et al (1995) Inhibition of cyclin-dependent kinases by p21. Mol Biol Cell 6:387–400

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Simone C, Bagella L, Bellan C, Giordano A (2002) Physical interaction between pRb and cdk9/cyclinT2 complex. Oncogene 21:4158–4165

    Article  CAS  PubMed  Google Scholar 

  10. Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH (1991) The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 67:293–302

    Article  CAS  PubMed  Google Scholar 

  11. Purvis JE, Karhohs KW, Mock C, Batchelor E, Loewer A, Lahav G (2012) p53 dynamics control cell fate. Science 336:1440–1444

    Article  CAS  PubMed  Google Scholar 

  12. Bhattacharya S, Ray RM, Johnson LR (2009) Role of polyamines in p53-dependent apoptosis of intestinal epithelial cells. Cell Signal 21:509–522

    Article  CAS  PubMed  Google Scholar 

  13. Fragkos M, Jurvansuu J, Beard P (2009) H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol Cell Biol 29:2828–2840

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Morris EJ, Keramaris E, Rideout HJ, Slack RS, Dyson NJ, Stefanis L, Park DS (2001) Cyclin-dependent kinases and p53 pathways are activated independently and mediate Bax activation in neurons after DNA damage. J Neurosci 21:5017–5026

    CAS  PubMed  Google Scholar 

  15. Chung JH, Bunz F (2010) Cdk2 is required for p53-independent G2/M checkpoint control. PLoS Genet 6:e1000863

    Article  PubMed Central  PubMed  Google Scholar 

  16. Johnson N, Shapiro GI (2010) Cyclin-dependent kinases (Cdks) and the DNA damage response: rationale for cdk inhibitor-chemotherapy combinations as an anticancer strategy for solid tumors. Expert Opin Ther Targets 14:1199–1212

    Article  CAS  PubMed  Google Scholar 

  17. Golsteyn RM (2005) Cdk1 and Cdk2 complexes (cyclin dependent kinases) in apoptosis: a role beyond the cell cycle. Cancer Lett 217:129–138

    Article  CAS  PubMed  Google Scholar 

  18. Ferguson M, Luciani MG, Finlan L, Rankin EM, Ibbotson S, Fersht A, Hupp TR (2004) The development of a Cdk2-docking site peptide that inhibits p53 and sensitizes cells to death. Cell Cycle 3:80–89

    Article  CAS  PubMed  Google Scholar 

  19. Wang Y, Prives C (1995) Increased and altered DNA binding of human p53 by S and G2/M but not G1 cyclin-dependent kinases. Nature 376:88–91

    Article  CAS  PubMed  Google Scholar 

  20. Bates S, Phillips AC, Clark PA, Stott F, Peters G, Ludwig RL, Vousden KH (1998) p14ARF links the tumor suppressors RB and p53. Nature 395:124–125

    Article  CAS  PubMed  Google Scholar 

  21. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI (1994) p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013–1023

    Article  CAS  PubMed  Google Scholar 

  22. Elias BC, Bhattacharya S, Ray RM, Johnson LR (2010) Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation. Cell Adh Migr 4:419–430

    Article  PubMed  Google Scholar 

  23. Bhattacharya S, Ray RM, Johnson LR (2006) Integrin β3-mediated Src activation regulates apoptosis in IEC-6 cells via Akt and STAT3. Biochem J 397:437–447

    Article  CAS  PubMed  Google Scholar 

  24. Deng W, Viar MJ, Johnson LR (2005) Polyamine depletion inhibits irradiation-induced apoptosis in intestinal epithelia. Am J Physiol Gastrointest Liver Physiol 289:G599–G606

    Article  CAS  PubMed  Google Scholar 

  25. Bhattacharya S, Ray RM, Johnson LR (2007) Basic helix-loop-helix protein E47-mediated p21Waf1/Cip1 gene expression regulates apoptosis of intestinal epithelial cells. Biochem J 407:243–254

    Article  CAS  PubMed  Google Scholar 

  26. Bhattacharya S, Ray RM, Johnson LR (2005) Decreased apoptosis in polyamine depleted IEC-6 cells depends on Akt-mediated NF-κB activation but not GSK3β activity. Apoptosis 10:759–776

    Article  CAS  PubMed  Google Scholar 

  27. Ray RM, Zimmerman BJ, McCormack SA, Patel TB, Johnson LR (1999) Polyamine depletion arrests cell cycle and induces inhibitors p21(Waf1/Cip1), p27(Kip1), and p53 in IEC-6 cells. Am J Physiol 276:C684–C691

    CAS  PubMed  Google Scholar 

  28. Quaroni A, Wands J, Trelstad R, Isselbacher KJ (1979) Epithelial cell cultures from rat small intestine. characterization by morphologic and immunologic criteria. J Cell Biol 80:248–265

    Article  CAS  PubMed  Google Scholar 

  29. McCormack SA, Viar MJ, Johnson LR (1993) Polyamines are necessary for cell migration by a small intestinal crypt cell line. Am J Physiol 264:G367–G374

    CAS  PubMed  Google Scholar 

  30. Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nature Med 10:789–799

    Article  CAS  PubMed  Google Scholar 

  31. Ray RM, Bhattacharya S, Johnson LR (2011) Mdm2 inhibition induces apoptosis in p53-defiecient human colon cancer cells by activating p73-and E2F1-mediated expression of PUMA and Siva-1. Apoptosis 16:35–44

    Article  CAS  PubMed  Google Scholar 

  32. Chassot AA, Lossaint G, Turchi L, Meneguzzi G, Fisher D, Ponzio G, Dulic V (2008) Confluence-induced cell cycle exit involves pre-mitotic Cdk inhibition by p27(Kip1) and cyclin D1 downregulation. Cell Cycle 7:2038–2046

    Article  CAS  PubMed  Google Scholar 

  33. Knudsen ES, Wang JY (1996) Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites. J Biol Chem 271:8313–8320

    Article  CAS  PubMed  Google Scholar 

  34. Ray RM, Jin S, Bavaria MN, Johnson LR (2011) Regulation of JNK activity in the apoptotic response of intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 300:G761–G770

    Article  CAS  PubMed  Google Scholar 

  35. Kim SG, Kim SN, Jong HS, Kim NK, Hong SH, Kim SJ, Bang YJ (2001) Caspase-mediated Cdk2 activation is a critical step to execute transforming growth factor-β1-induced apoptosis in human gastric cancer cells. Oncogene 20:1254–1265

    Article  CAS  PubMed  Google Scholar 

  36. Pucci B, Kasten M, Giordano A (2000) Cell cycle and apoptosis. Neoplasia 2:291–299

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Morgan DO (1997) Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13:261–291

    Article  CAS  PubMed  Google Scholar 

  38. Ortega S, Prieto I, Odajima J, Martin A, Dubus P, Sotillo R, Barbero JL, Malumbres M, Barbacid M (2003) Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat Genet 35:25–31

    Article  CAS  PubMed  Google Scholar 

  39. Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P (2003) Cdk2 knockout mice are viable. Curr Biol 13:1775–1785

    Article  CAS  PubMed  Google Scholar 

  40. Maude SL, Enders GH (2005) Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response. Cancer Res 65:780–786

    CAS  PubMed  Google Scholar 

  41. Firsanov DV, Solovjeva LV, Svetlova MP (2011) H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues. Clin Epigenetics 2:283–297

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Xu N, Libertini S, Zhang Y, Gillespie DA (2011) Cdk phosphorylation of Chk1 regulates efficient Chk1 activation and multiple checkpoint proficiency. Biochem Biophys Res Commun 413:465–470

    Article  CAS  PubMed  Google Scholar 

  43. Zhu Y, Alvarez C, Doll R, Kurata H, Schebye XM, Parry D, Lees E (2004) Intra-S-phase checkpoint activation by direct Cdk2 inhibition. Mol Cell Biol 24:6268–6277

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Freeman AK, Monteiro AN (2010) Phosphatases in the cellular response to DNA damage. Cell Commun Signal 8:27

    Article  PubMed Central  PubMed  Google Scholar 

  45. Donzelli M, Draetta GF (2003) Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Rep 4:671–677

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Kotala V, Uldrijan S, Horky M, Trbusek M, Strnad M, Vojtesek B (2001) Potent induction of wild-type p53-dependent transcription in tumor cells by a synthetic inhibitor of cyclin-dependent kinases. Cell Mol Life Sci 58:1333–1339

    Article  CAS  PubMed  Google Scholar 

  47. Carvajal LA, Manfredi JJ (2013) Another fork in the road: life or death decisions by the tumor suppressor p53. EMBO Rep 14:414–421

    Article  CAS  PubMed  Google Scholar 

  48. Garner E, Raj K (2008) Protective mechanisms of p53-p21-pRb proteins against DNA damage-induced cell death. Cell Cycle 7:277–282

    Article  CAS  PubMed  Google Scholar 

  49. Wallick CJ, Gamper I, Thorne M, Feith DJ, Takasaki KY, Wilson SM, Seki JA, Pegg AE, Byus CV, Bachmann AS (2005) Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine-inhibitor induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells. Oncogene 24:5606–5618

    Article  CAS  PubMed  Google Scholar 

  50. Park DS, Farinelli SE, Greene LA (1996) Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons. J Biol Chem 271:8161–8169

    Article  PubMed  Google Scholar 

  51. Appert-Collin A, Hugel B, Levy R, Niederhoffer N, Coupin G, Lombard Y, Andre P, Poindron P, Gies JP (2006) Cyclin-dependent kinase inhibitors prevent apoptosis of postmitotic mouse motorneurons. Life Sci 79:484–490

    Article  CAS  PubMed  Google Scholar 

  52. Wu J, Kharebava G, Piao C, Stoica BA, Dinizo M, Sabirzhanov B, Hanscom M, Guanciale K, Faden AI (2012) Inhibition of E2F1/Cdk1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo. PLoS One 7:e42129

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Knockaert M, Greengard P, Meijer L (2002) Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci 23:417–425

    Article  CAS  PubMed  Google Scholar 

  54. MacCallum DE, Melville J, Frame S, Watt K, Anderson S, Gianella-Borradori A, Lane DP, Green SR (2005) Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1. Cancer Res 65:5399–5407

    Article  CAS  PubMed  Google Scholar 

  55. Jin YH, Yoo KJ, Lee YH, Lee SK (2000) Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells. J Biol Chem 275:30256–30263

    Article  CAS  PubMed  Google Scholar 

  56. Gervais JL, Seth P, Zhang H (1998) Cleavage of Cdk inhibitor p21(Cip1/Waf1) by caspases is an early event during DNA damage-induced apoptosis. J Biol Chem 273:19207–19212

    Article  CAS  PubMed  Google Scholar 

  57. Shangary S, Wang S (2008) Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res 14:5318–5324

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Arisan ED, Coker A, Palavan-Unsal N (2012) Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells. Amino Acids 42:655–665

    Article  CAS  PubMed  Google Scholar 

  59. Deng Y, Lin Y, Wu X (2002) TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO. Genes Dev 16:33–45

    Article  CAS  PubMed  Google Scholar 

  60. Kashkar H, Haefs C, Shin H, Hamilton-Dutoit SJ, Salvesen GS, Kronke M, Jurgensmeler JM (2003) XIAP-mediated caspase inhibition in Hodgkin’s lymphoma-derived B cells. J Exp Med 198:341–347

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  61. Gatsinzi T, Ivanova EV, Iverfeldt K (2012) TRAIL resistance in human neurobasltoma SK-N-AS cells is dependent on protein kinase C and involves inhibition of caspase-3 proteolytic processing. J Neuro-Oncol 109:503–512

    Article  CAS  Google Scholar 

  62. Squires MS, Feltell RE, Wallis NG, Lewis EJ, Smith DM, Cross DM, Lyons JF, Thompson NT (2009) Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines. Mol Cancer Ther 8:324–332

    Article  CAS  PubMed  Google Scholar 

  63. Vesely J, Havlicek L, Strnad M, Blow JJ, Donella-Deana A, Pinna L, Letham DS, Kato J, Detivaud L, Leclerc S et al (1994) Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem 224:771–786

    Article  CAS  PubMed  Google Scholar 

  64. Waldman T, Zhang Y, Dillehay L, Yu J, Kinzler K, Vogelstein B, Williams J (1997) Cell-cycle arrest versus cell death in cancer therapy. Nat Med 3:1034–1036

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) grant DK-16505, and by the Thomas A. Gerwin Endowment. We gratefully acknowledge Mary Jane Viar for technical assistance.

Author information

Authors and Affiliations

  1. Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Nash Building, Memphis, TN, 38163, USA

    Sujoy Bhattacharya, Ramesh M. Ray & Leonard R. Johnson

Authors
  1. Sujoy Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ramesh M. Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leonard R. Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sujoy Bhattacharya.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bhattacharya, S., Ray, R.M. & Johnson, L.R. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells. Apoptosis 19, 451–466 (2014). https://doi.org/10.1007/s10495-013-0942-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-013-0942-3

Keywords

Search

Navigation