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.
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.
Kaito
C
C
Laboratory of Molecular Embryology, Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Kai
M
M
Higo
T
T
Takayama
E
E
Fukamachi
H
H
Sekimizu
K
K
Shiokawa
K
K
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D053938:DNA Fragmentation; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005260:Female; D020287:In Situ Nick-End Labeling; D008845:Microinjections; D009865:Oocytes / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D019253:Proto-Oncogene Proteins c-bcl-2; D003841:Deoxycytidine; D000077209:Decitabine; C016569:5-methyldeoxycytidine; D015254:DNA Modification Methylases; D001374:Azacitidine