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 journalArticle

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 - Sep 26 2001
Externally publishedYes

Fingerprint

decitabine
Xenopus laevis
Microinjections
Embryonic Structures
Apoptosis
DNA
Blastula
Gastrula
Digoxigenin
DNA Nucleotidylexotransferase
Zygote
Cytosine
DNA Methylation
DNA Replication
Chromatin
Embryonic Development
Electrophoresis
5-methyldeoxycytidine triphosphate
RNA
Messenger RNA

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

Cite this

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

In: Development Growth and Differentiation, Vol. 43, No. 4, 26.09.2001, p. 383-390.

Research output: Contribution to journalArticle

Kaito, Chikara ; Kai, Masatake ; Higo, Takayasu ; Takayama, Eiji ; Fukamachi, Hiroshi ; Sekimizu, Kazuhisa ; Shiokawa, Koichiro. / 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. In: Development Growth and Differentiation. 2001 ; Vol. 43, No. 4. pp. 383-390.
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AU - Kaito, Chikara

AU - Kai, Masatake

AU - Higo, Takayasu

AU - Takayama, Eiji

AU - Fukamachi, Hiroshi

AU - Sekimizu, Kazuhisa

AU - Shiokawa, Koichiro

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AB - 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.

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KW - Maternal apoptosis program

KW - Mid-blastula transition

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