Skip to main content

Advertisement

SpringerLink
Log in

Widdrol activates DNA damage checkpoint through the signaling Chk2–p53–Cdc25A–p21–MCM4 pathway in HT29 cells

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Widdrol is an odorant compound isolated from Juniperus chinensis. We previously reported that widdrol induces Gap 1 (G1) phase cell cycle arrest and leads to apoptosis in human colon adenocarcinoma HT29 cells. It was also reported that this cell cycle arrest is associated with the induction of checkpoint kinase 2 (Chk2), p53 phosphorylation and cyclin dependent kinase (Cdk) inhibitor p21 expression. In this paper, we investigated the molecular mechanisms of widdrol on the activation of G1 DNA damage checkpoint at early phase when DNA damages occurred in HT29 cells. First of all, we examined that widdrol breaks DNA directly or not. As the results of DNA electrophoresis and formation of phosphorylated histone H2AX (γH2AX) foci in HT29 cells, widdrol generates DNA double-strand breaks directly within 0.5 h both in vitro and in vivo. Based on this result, the change of proteins related in checkpoint pathway was examined over a time course of 0.5–24 h. Treatment of HT29 cells with widdrol elicits the following: (1) phosphorylation of Chk2 and p53, (2) reduction of cell division cycle 25A (Cdc25A) expression, (3) increase of Cdk inhibitor p21 expression, and (4) decrease of the levels of Cdk2 and cyclin E expression in a time-dependent manner. Moreover, only the expression level of mini-chromosome maintenance 4 (MCM4) protein, a subunit of the eukaryotic DNA replicative helicase, is rapidly down-regulated in HT29 cells treated with widdrol over the same time course, but those of the other MCM proteins are unchanged. Overall, our results indicated that widdrol breaks DNA directly in HT29 cells, and this DNA damage results in checkpoint activation via Chk2–p53–Cdc25A–p21–MCM4 pathway and finally cells go to G1-phase cell cycle arrest and apoptosis.

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

Similar content being viewed by others

References

  1. Sherr CJ (2000) The pezcoller lecture: cancer cell cycles revisited. Cancer Res 60:3689–3695

    PubMed  CAS  Google Scholar 

  2. Sherr CJ, Roberts JM (2004) Living with or without cyclins and cyclin-dependent kinases. Genes Dev 18:2699–2711

    Article  PubMed  CAS  Google Scholar 

  3. Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27:247–254

    Article  PubMed  CAS  Google Scholar 

  4. Dotto GP (2000) p21WAF1=Cip1 : more than a break to the cell cycle? Biochim Biophys Acta 1471:43–56

    Google Scholar 

  5. Forsburg SL (2004) Eukaryotic MCM proteins: beyond replication initiation. Microbiol Mol Biol Rev 68:109–131

    Article  PubMed  CAS  Google Scholar 

  6. Liang DT, Hodson JA, Forsburg SL (1999) Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay. J Cell Sci 112:559–567

    PubMed  CAS  Google Scholar 

  7. Cortez D, Glick G, Elledge SJ (2004) Mini-chromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci USA 101:10078–10083

    Article  PubMed  CAS  Google Scholar 

  8. Ishimi Y, Kohno YK, Kwon HJ, Yamada K, Nakanishi M (2003) Identification of MCM4 as a target of the DNA replication block checkpoint system. J Biol Chem 278:24644–24650

    Article  PubMed  CAS  Google Scholar 

  9. Schories B, Engel K, Dörken B, Gossen M, Bommert K (2004) Characterization of apoptosis-induced Mcm3 and Cdc6 cleavage reveals a proapoptotic effect for one Mcm3 fragment. Cell Death Differ 11:940–942

    Article  PubMed  CAS  Google Scholar 

  10. Feng D, Tu Z, Wu W, Liang C (2003) Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. Cancer Res 63:7356–7364

    PubMed  CAS  Google Scholar 

  11. Kwon HJ, Hong YK, Park C, Choi YH, Yun HJ, Lee EW, Kim BW (2010) Widdrol induces cell cycle arrest, associated with MCM down-regulation, in human colon adenocarcinoma cells. Cancer Lett 290:96–103

    Article  PubMed  CAS  Google Scholar 

  12. Giono LE, Manfredi JJ (2006) The p53 tumor suppressor participates in multiple cell cycle checkpoints. J Cell Physiol 209:13–20

    Article  PubMed  CAS  Google Scholar 

  13. Matsuoka S, Huang M, Elledge SJ (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282:1893–1897

    Article  PubMed  CAS  Google Scholar 

  14. Kwon HJ, Lee EW, Hong YK, Yun HJ, Kim BW (2010) Widdrol from Juniperus chinensis induces apoptosis in human colon adenocarcinoma HT29 cells. Biotechnol Bioprocess Eng 15:167–172

    Article  CAS  Google Scholar 

  15. Zhou C, Li Z, Diao H, Yu Y, Zhu W, Dai Y, Chen FF, Yang J (2006) DNA damage evaluated by γH2AX foci formation by a selective group of chemical/physical stressors. Mutat Res 604:8–18

    PubMed  CAS  Google Scholar 

  16. Riches LC, Lynch AM, Gooderham NJ (2008) Early events in the mammalian response to DNA double-strand breaks. Mutagenesis 23(5):331–339

    Article  PubMed  CAS  Google Scholar 

  17. Yuan J, Adamski R, Chen J (2010) Focus on histone variant H2AX: To be or not to be. FEBS Lett 584:3717–3724

    Article  PubMed  CAS  Google Scholar 

  18. Bartek J, Lukas J (2001) Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol 13:738–747

    Article  PubMed  CAS  Google Scholar 

  19. Brugarolas J, Moberg K, Boyd SD, Taya Y, Jacks T, Lees JA (1999) Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after γ-irradiation. Proc Natl Acad Sci USA 96:1002–1007

    Article  PubMed  CAS  Google Scholar 

  20. Dasika GK, Lin SC, Zhao S, Sung P, Tomkinson A, Lee EY (1999) DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene 18:7883–7899

    Article  PubMed  CAS  Google Scholar 

  21. Yang J, Yu Y, Hamrick HE, Duerksen-Hughes PJ (2003) ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 24:1571–1580

    Article  PubMed  CAS  Google Scholar 

  22. Delia D, Fontanella E, Ferrario C, Chessa L, Mizutani S (2003) DNA damage-induced cell-cycle phase regulation of p53 and p21waf1 in normal and ATM-defective cells. Oncogene 22:7866–7869

    Article  PubMed  CAS  Google Scholar 

  23. Costanzi-Strauss E, Strauss BE, Naviaux RK, Haas M (1998) Restoration of growth arrest by p16INK4, p21WAF1, pRB, and p53 is dependent on the integrity of the endogenous cell-cycle control pathways in human glioblastoma cell lines. Exp Cell Res 238:51–62

    Article  PubMed  CAS  Google Scholar 

  24. Sørensen CS, Syljuasen RG, Falck J, Schroeder T, Ronnstrand L, Khanna KK, Zhou BB, Bartek J, Lukas J (2003) Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 3:247–258

    Article  PubMed  Google Scholar 

  25. Hoffmann I, Draetta G, Karsenti E (1994) Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J 13:4302–4310

    PubMed  CAS  Google Scholar 

  26. Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF (2000) Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep 1:71–79

    Article  PubMed  CAS  Google Scholar 

  27. Costanzo V, Robertson K, Ying CY, Kim E, Avvedimento E, Gottesman M, Grieco D, Gautier J (2000) Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. Mol Cell 6:649–659

    Article  PubMed  CAS  Google Scholar 

  28. Braun KA, Breeden LL (2007) Nascent transcription of MCM2–7 is important for nuclear localization of the mini-chromosome maintenance complex in G1. Mol Biol Cell 18:1447–1456

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No.2010-0004993) and Blue-Bio Industry Regional Innovation Center (RIC08-06-07) at Dongeui University as a RIC program of KIAT under Ministry of Knowledge Economy.

Author information

Authors and Affiliations

  1. Department of Biomaterial Control (BK21 Program), Dongeui University Graduate School, Busan, Korea

    Hee Jung Yun, Byung Woo Kim & Hyun Ju Kwon

  2. Blue-Bio Industry RIC, 995 Eomgwangno BusanJin-Gu, Busan, Korea

    Sook Kyung Hyun, Byung Woo Kim & Hyun Ju Kwon

  3. Department of Life Science and Biotechnology, College of Natural Science, Dongeui University, 995 Eomgwangno, Busanjin-gu, Busan, 614-714, Korea

    Byung Woo Kim & Hyun Ju Kwon

  4. Department of Food Nutrition, College of Human Ecology, Dongeui University, Busan, Korea

    Sook Kyung Hyun

  5. Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, Tokyo University, Tokyo, Japan

    Jung Ha Park

Authors
  1. Hee Jung Yun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sook Kyung Hyun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jung Ha Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Byung Woo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hyun Ju Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyun Ju Kwon.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yun, H.J., Hyun, S.K., Park, J.H. et al. Widdrol activates DNA damage checkpoint through the signaling Chk2–p53–Cdc25A–p21–MCM4 pathway in HT29 cells. Mol Cell Biochem 363, 281–289 (2012). https://doi.org/10.1007/s11010-011-1180-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-011-1180-z

Keywords

Search

Navigation