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p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine

Oncogene volume 25, pages 2839–2849 (2006)Cite this article

Abstract

This study provides evidence for the importance of p21CDKN1A for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21CDKN1A and p53 enhance camptothecin-induced histone H2AX phosphorylation (γH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, γH2AX reverses after camptothecin removal. By contrast, γH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53−/−) or p21CDKN1A (p21−/−) as the cells reach the late-S and G2 phases. Since p21−/− cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced γH2AX formation in p21−/− cells, we conclude that enhanced γH2AX formation in p21−/− cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced γH2AX formation and inhibits camptothecin-induced p21CDKN1A upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that γH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

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Abbreviations

BSA:

bovine serum albumin

FBS:

fetal bovine serum

DMSO:

dimethylsulfoxide

DSB:

DNA-double strand break

IR:

ionizing radiation

LSC:

laser scanning cytometry

p53−/−:

deficient for p53

p21−/−:

deficient for p21CDKN1A

PBS:

phosphate-buffered saline

PVDF:

polyvynilidene difluoride

TBS:

Tris-buffered saline

TBS-T:

Tris-buffered saline with Tween 20

TCA:

trichloroacetic acid

TUNEL:

terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling

UCN-01:

7-hydroxystaurosporine

wt:

wild type

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Acknowledgements

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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Authors and Affiliations

  1. Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    T Furuta, R L Hayward, L-H Meng, H Takemura, G J Aune, W M Bonner, M I Aladjem, K W Kohn & Y Pommier

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  1. T Furuta
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  2. R L Hayward
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  3. L-H Meng
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Furuta, T., Hayward, R., Meng, LH. et al. p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine. Oncogene 25, 2839–2849 (2006). https://doi.org/10.1038/sj.onc.1209313

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