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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
Author(s) -
Kaito Chikara,
Kai Masatake,
Higo Takayasu,
Takayama Eiji,
Fukamachi Hiroshi,
Sekimizu Kazuhisa,
Shiokawa Koichiro
Publication year - 2001
Publication title -
development, growth and differentiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 66
eISSN - 1440-169X
pISSN - 0012-1592
DOI - 10.1046/j.1440-169x.2001.00579.x
Subject(s) - microbiology and biotechnology , deoxycytidine , biology , xenopus , blastula , chromatin , dna , dna replication , methylation , dna methylation , embryo , biochemistry , gastrulation , embryogenesis , genetics , gene expression , chemotherapy , gemcitabine , gene
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.