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Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain

Simeon Santourlidis1, Fumihiro Kimura, Johannes Fischer

  • 1Urologische Klinik Heinrich Heine Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany. santourlidis@itz.uni-duesseldorf.de

Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
|October 23, 2004
PubMed

Insights

Overexpression of DNA methyltransferase 1 (Dnmt1) reduces cell survival by impairing clonogenicity. This effect is linked to its N-terminal domain interacting with proliferating cell nuclear antigen (PCNA), suggesting a protective mechanism against cancer.

Area of Science:

  • Epigenetics and DNA Methylation
  • Cell Biology
  • Cancer Biology

Background:

  • Aberrant DNA methylation is implicated in cancer development.
  • DNA methyltransferase 1 (Dnmt1) overexpression is hypothesized to cause cytotoxicity through gene hypermethylation.
  • The precise mechanisms and cellular consequences of Dnmt1 overexpression require further elucidation.

Purpose of the Study:

  • To investigate the cytotoxic effects of DNA methyltransferase 1 (Dnmt1) overexpression.
  • To identify the specific domains and interactions responsible for Dnmt1-mediated cytotoxicity.
  • To explore the potential role of Dnmt1 overexpression in cellular defense against carcinogenicity.

Main Methods:

  • Overexpression of mouse and human Dnmt1 in murine and human cell lines.
  • Utilized frame-shift and deletion constructs to map the functional domain of Dnmt1.
  • Investigated the role of proliferating cell nuclear antigen (PCNA) interaction via mutation of the binding site.
  • Assessed effects on clonogenicity, apoptosis, replication, and global DNA methylation.
  • Examined Dnmt1 overexpression effects in cell lines with altered p53, p21(CIP1), or p16(INK4A) status.

Main Results:

  • Dnmt1 overexpression significantly decreased cell clonogenicity in various cell lines.
  • The cytotoxic effect was localized to the N-terminal 124 amino acid domain of Dnmt1, mediating PCNA interaction.
  • Mutation of the PCNA-binding site abolished the suppressive effect on clonogenicity.
  • Overexpression of Dnmt3A and Dnmt3B, which do not bind PCNA, had weaker effects.
  • No significant impact on apoptosis, replication, or overall DNA methylation was observed within 3 days.
  • Dnmt1-mediated suppression of clonogenicity occurred independently of wild-type p53, p21(CIP1), or p16(INK4A).

Conclusions:

  • Dnmt1 overexpression induces cytotoxicity by suppressing cell clonogenicity, primarily through its PCNA-interacting N-terminal domain.
  • This mechanism appears to function independently of major cell cycle regulators like p53.
  • Dnmt1's ability to suppress clonogenicity may serve as a safeguard against the oncogenic potential of its sustained overexpression.

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