Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain.
Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has been hypothesized to result in aberrant hypermethylation of genes required for cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and human cell lines decreased clonogenicity. By frame-shift and deletion constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino acid domain, which mediates interaction with proliferating cell nuclear antigen (PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies. Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded weaker effects on clonogenicity. Following introduction of the toxic domain, no significant effects on apoptosis, replication, or overall DNA methylation were observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed in cell lines lacking wild-type p53, p21(CIP1), or p16(INK4A). Suppression of clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against carcinogenicity of sustained Dnmt1 overexpression.
Santourlidis
Simeon
S
Urologische Klinik Heinrich Heine Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany. santourlidis@itz.uni-duesseldorf.de
Kimura
Fumihiro
F
Fischer
Johannes
J
Schulz
Wolfgang A
WA
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004261:DNA Replication / Q000502:physiology*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000097584:DNA Methyltransferase 3B
D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53; D000097584:DNA Methyltransferase 3B; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse