Anderson JA et al. (1997), Ionizing radiation induces apoptosis and elevat...

XB-ART-16298

Mol Biol Cell 1997 Jul 01;87:1195-206.

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Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus.

Anderson JA , Lewellyn AL , Maller JL .

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After the twelfth cell division in Xenopus embryos, zygotic gene transcription is activated, cells become motile, and cell division becomes asynchronous. This developmental change is termed the midblastula transition. High doses of gamma-irradiation (gamma-IR) before the midblastula transition induced apoptotic cell death and increased the levels of cyclin A1 and cyclin A1-Cdk2 activity. The addition of recombinant cyclin A1-Cdk2 induced the formation of apoptotic nuclei in Xenopus egg extracts, suggesting a role for cyclin A1-Cdk2 in apoptosis. Hallmarks of apoptosis, such as internucleosomal DNA fragmentation, pyknotic and uniformly condensed nuclei, and loss of intercellular attachments, were evident in embryos exposed to gamma-IR before the midblastula transition. Apoptotic cells accumulated in the blastocoel, suggesting that before the midblastula transition Xenopus embryos use apoptosis to eliminate cells containing damaged DNA. However, embryos treated with the same dose of gamma-IR after the midblastula transition developed normally and exhibited no signs of apoptosis, no change in cyclin A1 level, and no increase in cyclin A1-Cdk2 activity. These results indicate that there is a change in the response to DNA damage at the midblastula transition in Xenopus embryos.

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Species referenced: Xenopus
Genes referenced: adm ccna1 cdk1 cdk2
GO keywords: DNA damage checkpoint [+]

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	Figure 1. Exposure to y-IR before but not after the MBT results in arrested abnormal embryonic development. Embryos treated with y-IR received 20 Gy (2000 rad) from a 60Co source. Control [stage (St) 8 and St 91 and 20-Gy-treated embryos (St 8 and St 9) were collected and photographed 4, 6, and 8 h after treatment.
	Figure 2. Internucleosomal DNA fragmentation occurs in preMBT embryos exposed to y-IR. Embryos were untreated (-) or treated at the indicated stages with 20 Gy of y-IR (+). Genomic DNA was isolated 8 h after exposure and electrophoresed on a 1% agarose gel. Arrows indicate intemucleosomal fragments in multiples of 180-200 bp. MW markers indicate HindIII-digested A DNA.
	Figure 3. -y-IR induces apoptosis in pre-MBT embryonic cells. DAPI staining of sections of control embryos collected 8 h after stage 7 (A), stage 8 (C), and stage 9 (E). DAPI staining of sections of y-IR-treated embryos collected 8 h after treatment at stage 7 (B) and stage 8 (D) indicates apoptotic bodies (condensed or fragmented DNA, indicated by arrows) in the nuclei, but intact nuclei lacking apoptotic bodies in embryos treated at stage 9 (F). Bars, 10 ,Am (A, C, and E, bar in A; B, D, and F, bar in B).
	Figure 4. Apoptotic cells accumulate in the blastocoel of embryos treated with -y-IR before the MBT. Phase-contrast photomicrographs of embryo sections showing the blastocoel from an untreated embryo (A) and embryos treated at stage 7 (B), stage 8 (C), and stage 9 (D). Bar, 50 ,um.
	Figure 5. Ionizing radiation does not affect the onset of zygotic transcription. Embryos loaded with [3H]uridine were untreated (LI) or treated with 20 Gy of lIR at stage (St) 8 (A) or 9 (0). Embryos were collected at the indicated stages and the incorporation of [3H]uridine into RNA was analyzed by trichloroacetic acid precipitation. Similar results were obtained in two separate experiments.
	Figure 6. Level of cyclin Al and its associated kinase activity increase in y-IR-induced apoptosis. Embryos were exposed to 0 Gy or 20 Gy of y-IR at the indicated stages and frozen 8 h after treatment. (A) Cyclin immunoblots reveal that only cyclin Al level increases significantly in extracts of apoptotic embryos [stages (St) 7 and 81 but not in untreated embryos or embryos treated after the MBT (stage 9). (B) Samples were assayed for Hi kinase activity in cyclin immunoprecipitates. Results similar to those presented were observed in three separate experiments. Open bars, activity in control embryo extracts; solid bars, activity in extracts from embryos treated with 20 Gy.
	Figure 7. Level of cyclin Al and its associated kinase activity increase during a-amanitin-induced apoptosis. Embryos were injected at the one-cell stage with buffer or 1 mg/ml a-amanitin and 9 h after fertilization the embryos were frozen for analysis. (A) Cyclin immunoblots reveal that only cyclin Al increases significantly in a-amanitin-injected embryos (+ a) but not in embryos injected with buffer (- a). (B) Samples were assayed for HI kinase activity in cyclin and Cdc2 immunoprecipitates. Results similar to those presented were observed in three separate experiments. Open bars, activity in buffer-injected embryo extracts; solid bars, activity in extracts from embryos injected with a-amanitin.
	Figure 8. Cyclin Al associates with Cdk2 in apoptotic embryos. Embryos were exposed to 0 Gy or 20 Gy of -y-IR at the indicated stages, and 8 h after treatment the embryos were frozen for analysis. Cyclin Al and A2 immunoprecipitates (IP) blotted for Cdk2 reveal an increase in cyclin Al-Cdk2 complexes in y-IR-treated apoptotic embryos [stages (St) 7 and St 8] but not in untreated embryos or embryos treated after the MBT (St 9 and St 10); the level of cyclin A2-Cdk2 complexes remain constant. Similar results were obtained in two separate experiments.
	Figure 9. Recombinant cyclin Al-Cdk2 induces apoptosis-like nuclear morphology in egg extracts. Equal volumes of buffer, kinasedead cyclin Al-Cdk2 (DN) or WT cyclin Al-Cdk2 were added to CSF-released egg extracts 60 min after CaCl2 addition. Aliquots were fixed, stained with DAPI, and photographed at 60, 80, and 90 min after CaCl2 addition. Nuclear morphology consistent with apoptosis is observed at 90 min in extracts containing recombinant WT cyclin Al-Cdk2. Results similar to those presented were observed in three separate experiments.

References [+] :

Arends, Apoptosis. The role of the endonuclease. 1990, Pubmed