Proc Natl Acad Sci U S A
January 30, 2001;
The midblastula transition in Xenopus embryos activates multiple pathways to prevent apoptosis in response to DNA damage.
Apoptosis is controlled by a complex interplay between regulatory proteins. Previous work has shown that Xenopus embryos remove damaged cells by apoptosis when irradiated before, but not after, the midblastula transition (MBT). Here we demonstrate that Akt
/protein kinase B
is activated and mediates an antiapoptotic signal only in embryos irradiated after the MBT. In addition, an increase in xBcl-2
oligomerization and a decrease in xBax
homodimerization promote a protective effect against apoptosis only after the MBT. The post-MBT survival mechanism arrests cells in G(1) phase by increasing expression of the cyclin-dependent kinase inhibitor p27
) associates with cyclin D/Cdk4
and cyclin A/Cdk2
complexes to cause G(1)/S arrest, perhaps allowing more time for DNA repair. Taken together, the results define the DNA damage response as an element of the MBT and indicate that multiple mechanisms prevent apoptosis after the MBT.
Proc Natl Acad Sci U S A
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Phosphorylation of Akt in response to ionizing radiation. (A) Embryos were not irradiated (control) or were exposed to γ-IR at either stage 6 or stage 9, collected at different times, and analyzed as described in Materials and Methods. The blots were incubated with anti-P-Ser 473 Akt antibody (Upper) or anti-Akt antibody (Lower) and visualized by enhanced chemiluminescence. The arrows on the right denote the Akt protein and the phosphorylated isoforms. Molecular mass markers (in kDa) are indicated on the left. (B) (Upper) Embryos were injected at the one-cell stage with β-galactosidase, WT-, DN-, or CA-Akt mRNA and allowed to develop until they reached stage 6 (Top row). At that stage, embryos were not irradiated (first column on left) or were treated with ionizing radiation (γ-IR) and allowed to progress through early developmental stages. Embryos were collected and photographed at the time of irradiation (Top row, stage 6), at the MBT (Middle row, stage 8), and 7 h after the MBT (Bottom row), when the morphological changes of apoptosis were conspicuous (scale bar, 1 mm). (Lower) At the indicated stages, levels of the indicated expressed proteins were analyzed by Western blotting.
(A) Interaction between xBcl-2 and xBax changes at the MBT. Embryos were injected at the one-cell stage with a mixture of FLAG-tagged xBcl-2 and c-myc-tagged xBax mRNAs. The embryos were not irradiated (control) or were irradiated 3 h (stage 6) or 7 h (stage 9) after fertilization, collected at different times, and frozen. Samples equivalent to five embryos were immunoprecipitated with anti-FLAG M2-agarose beads, and the immunocomplexes were subjected to Western blot analysis with anti-c-myc antibody. (B) Analysis of xBax homodimerization. Embryos were injected at the one-cell stage with FLAG-tagged and c-myc-tagged xBax mRNAs, were not irradiated (control) or were irradiated at either stage 6 (I) or stage 9 (II), and were collected at different times. Samples equivalent to five embryos were immunoprecipitated with anti-c-myc-agarose beads, and the immunocomplexes were blotted with anti-FLAG M2 antibody (I, Top, and II, Top). Total FLAG-xBcl-2, FLAG-xBax, and c-myc-xBax expression levels were assessed at all stages by Western blotting with specific anti-tag antibodies (A and B, I and II, Middle and Bottom). Arrows on the right denote the FLAG-tagged or c-myc-tagged protein.
(A) Analysis of the level of p27Xic1 in embryos irradiated either pre- or post-MBT. Embryos were not irradiated (control) or were irradiated (γ-IR) at the indicated stages, and samples were collected at the indicated times and analyzed by Western blotting with anti-p27Xic1 antibody. One embryo equivalent was loaded per lane. (B) Cell cycle profile of irradiated embryos. Embryos were irradiated at stage 9 and collected 2 and 6 h after irradiation. Embryonic nuclei were prepared from a single sibling group and analyzed by flow cytometry as described in Materials and Methods. Stages of the cell cycle are defined by DNA content, as measured by fluorescence intensity.
Targets for p27Xic1 in vivo. Embryos were not irradiated (control) or were irradiated after the MBT (stage 9 γ-IR), collected at different times, and frozen. (A) The total level of p27Xic1 at the indicated times. (B) Samples equivalent to five embryos were precipitated with p13suc-1 beads, and the bound proteins were analyzed by Western blotting with anti-p27Xic1 antibody. (C) Association of p27Xic1 with cyclin A2 complexes was detected by immunoprecipitation with cyclin A2 antibody and analysis of the precipitates by Western blotting with anti-p27Xic1 antibody. (D) Samples from embryos irradiated after the MBT were immunoprecipitated with anti-Cdk4 antibody and blotted with anti-p27Xic1 antibody.
Inhibition of cyclin D/Cdk4 and cyclin A2/Cdk2 activity by p27Xic1. Purified cyclin D/Cdk4 or cyclin A2/Cdk2 complexes were preincubated with the indicated amounts of GST-p27Xic1 or GST alone (control), and the kinase reaction was performed with either the C-terminal half of human retinoblastoma protein (hRb) as substrate for Cdk4 or histone H1 for Cdk2, as described in Materials and Methods.
Model of apoptosis regulation in Xenopus embryos. Ionizing radiation promotes apoptosis in embryos irradiated before the MBT by triggering different proapoptotic factors, including the homodimerization of xBax, and caspase cascade activation. Apoptosis is prevented in embryos irradiated after the MBT by the coordinated action of antiapoptotic stimuli. Survival signaling pathways act at at least three levels: by promotion of cell cycle delay by increasing the amount of p27Xic1 and its association with the complexes involved in the G1/S transition, by inactivation of proapoptotic molecules by heterodimerization, and by activation of antiapoptotic pathways like Akt.