Activation of the maternally preset program of apoptosis by microinjection of 5-aza-2′-deoxycytidine and 5-methyl-2′-deoxycytidine-5′-triphosphate in Xenopus laevis embryos

Chikara Kaito, Masatake Kai, Takayasu Higo, Eiji Takayama, Hiroshi Fukamachi, Kazuhisa Sekimizu, Koichiro Shiokawa

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5-aza-2′-deoxycytidine (5-Aza-CdR), which induces hypomethylation of DNA, and 5-methyl-2′-deoxycytidine-5′-triphosphate (5-methyl-dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid-blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end-labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a 'ladder' on electrophoresis. Furthermore, the induction of cell dissociation by 5-Aza-CdR and 5-methyl-dCTP was postponed by 2-3 h by co-injection of Bcl-2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5-methyl-cytosine, we confirmed that 5-Aza-CdR induces hypomethylation, whereas 5-methyl-dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5-Aza-CdR-injected and 5-methyl-dCTP-injected embryos. These results show that 5-Aza-CdR and 5-methyl-dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.

Original languageEnglish
Pages (from-to)383-390
Number of pages8
JournalDevelopment Growth and Differentiation
Volume43
Issue number4
DOIs
Publication statusPublished - 2001

Keywords

  • 5-Aza-2′-deoxycytidine
  • 5-Methyl-2′-deoxycytidine-5′- triphosphate
  • DNA methylation
  • Maternal apoptosis program
  • Mid-blastula transition

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

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