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Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1

Oncogene volume 21, pages 5912–5922 (2002)Cite this article

Abstract

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polβ) or DNA polymerase β (polβ) gene-knockout (MEFpolβKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G2/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polβ-dependent pathway and by blocking both FEN1 and DNA ligase activity in polβ-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polβ-dependent and -independent LP-BER pathways in MEF cells.

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Abbreviations

AP:

apurinic/apyrimidinic

APE:

AP endonuclease

bp:

base pair

BER:

base excision repair

cccDNA:

covalently closed circular DNA

dRP:

2′-deoxyribose 5′-phosphate

dRPase:

2′-deoxyribose 5′-phosphatase

FEN1:

flap endonuclease 1

LP:

long-patch

MEF:

mouse embryonic fibroblast cell line

PAGE:

polyacrylamide gel electrophoresis

polβ:

DNA polymerase β

polβKO:

polβ-gene knocked out

PCNA:

proliferating cell nuclear antigen

SP:

short-patch

UDG:

uracil-DNA glycosylase

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Acknowledgements

We would like to thank Drs Rajendra Prasad and Samuel H Wilson from NIEHS, Research Triangle Park, NC, for providing the neutralizing anti-polβ antibody and the mouse embryonic fibroblast (MEF) cell lines with wild-type and polβ-gene-knockout clones; Dr Arun Fotedar from the Cellular and Molecular Biology Program, Sidney Kimmel Cancer Center (San Diego, CA, USA), for providing us the wild-type and mutant GST-p21 protein overexpression vectors. We thank Jessica A Salinas for technical assistance and Nirupama Gupta for editorial comments. The FACS analysis was performed in the FCC Laboratory of the ICBR of the University of Florida. These studies were supported in part by the grants awarded to S Narayan from the National Cancer Institute, NIH (CA77721); the 2001-Research Opportunity Fund by the University of Florida (Gainesville, FL, USA); and the 2001-Ralph E Powe Junior Faculty Enhancement Award by the Oak Ridge Associated Universities (Oak Ridge, TN, USA). The work of LB Bloom was supported by NSF grant MCB-9722356.

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, College of Medicine, The University of Florida, Gainesville, 32610, Florida, FL, USA

    Aruna S Jaiswal & Satya Narayan

  2. UF Shands Cancer Center, College of Medicine, The University of Florida, Gainesville, 32610, Florida, FL, USA

    Aruna S Jaiswal & Satya Narayan

  3. Department of Biochemistry and Molecular Biology, College of Medicine, The University of Florida, Gainesville, 32610, Florida, FL, USA

    Linda B Bloom

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  1. Aruna S Jaiswal
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Correspondence to Satya Narayan.

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Jaiswal, A., Bloom, L. & Narayan, S. Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1. Oncogene 21, 5912–5922 (2002). https://doi.org/10.1038/sj.onc.1205789

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