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Phorbol ester abrogates up-regulation of DNA polymerase beta by DNA-alkylating agents in Chinese hamster ovary cells

D K Srivastava1, T Y Rawson, S D Showalter

  • 1Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068, USA.

The Journal of Biological Chemistry
|July 7, 1995
PubMed

Insights

DNA alkylating agents and TPA both increase DNA polymerase beta (beta-pol) protein levels in cells. However, these two pathways negatively interfere with each other, suggesting complex regulation of this DNA repair enzyme.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Mammalian DNA polymerase beta (beta-pol) is a DNA repair enzyme constitutively expressed in cells.
  • Previous studies indicated that DNA alkylating agents up-regulate beta-pol mRNA levels.
  • The protein-level response and its regulatory mechanisms remained largely uncharacterized.

Purpose of the Study:

  • To investigate the effect of DNA alkylating agents on beta-pol protein levels.
  • To explore the relationship between DNA alkylation-induced beta-pol expression and TPA-induced expression.
  • To characterize the regulatory pathways controlling beta-pol protein expression.

Main Methods:

  • Monoclonal antibodies were generated against beta-pol.
  • Immunoblotting was used to quantify beta-pol protein levels in cell extracts.
  • Chinese hamster ovary (CHO) cells, including a genetically altered cell line overexpressing O6-methylguanine-methyltransferase, were treated with DNA alkylating agents (MNNG, methyl methanesulfonate) and/or TPA.

Main Results:

  • Treatment with DNA alkylating agents (MNNG or methyl methanesulfonate) resulted in a 5-10 fold increase in beta-pol protein levels in CHO cells.
  • This induction was dependent on DNA alkylation, as it was not observed in cells overexpressing O6-methylguanine-methyltransferase.
  • TPA treatment also increased beta-pol protein expression approximately 10-fold.
  • Co-treatment with MNNG and TPA showed cross-antagonistic effects: MNNG-induced upregulation was abrogated by TPA, and TPA-induced upregulation was abrogated by MNNG.

Conclusions:

  • DNA alkylating agents induce beta-pol protein expression in a manner secondary to DNA alkylation.
  • TPA also induces beta-pol protein expression through a distinct pathway.
  • The DNA alkylation and TPA signaling pathways for beta-pol induction exhibit mutual antagonism, indicating complex regulatory crosstalk.

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