DNA Damage-Induced Activation of p53 by the Checkpoint Kinase Chk2
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An 11-kb genomic Chk2 fragment was isolated from a mouse genomic 129/J library. A 5-kb sequence containing exons encoding the kinase domain of Chk2 was replaced by a neomycin cassette inserted in the antisense orientation. E14K ES cells were electroporated with the linearized construct and G418-resistant ES colonies were identified by polymerase chain reaction and Southern blotting. Chk2 −/− ES cell lines were generated by culturing G418-resistant Chk2 +/ − ES clones in G418 (3.6 mg/ml; Gibco).
Protein immunoblots were performed with antibodies to p53 (CM5 Novocastra) mouse Chk2 Bax (Santa Cruz) or β-actin (Sigma) followed by incubation with antibody to rabbit immunoglobulin conjugated to horseradish peroxidase–coupled antibody (Amersham). Proteins were visualized with ECL (Amersham). Rabbit polyclonal antibodies to mouse Chk2 were raised against glutathione S-transferase (GST)–Chk2.
Mitotic indices were determined by the nocodazole trapping method (8). ES cells were treated with 10 Gy of γ irradiation followed by the addition of nocodazole (0.2 μg/ml; Calbiochem). The mitotic index was calculated as the percentage of total cells that were in mitosis.
Freshly isolated thymocytes from Chk2 +/+ /Rag1 −/− and Chk2 −/− /Rag1 −/− somatic chimeras and p53 −/− mice (Jackson Labs) and their control littermates (all 10 to 16 weeks old) were stimulated with γ irradiation (1 to 4 Gy) adriamycin (0.3 to 1.0 μg/ml; Sigma) etoposide (0.1 to 1.0 μM; Sigma) UV irradiation (30 to 90 J/m 2 ; Stratalinker Stratagene) dexamethasone (3 to 10 nM; Sigma) recombinant CD95-CD8 fusion protein (FasL 3 to 10 μg/ml) or staurosporine (2 to 10 μM; Calbiochem) for 24 or 48 hours followed by staining with antibody to CD4-PE antibody to CD8-fluorescein isothiocyanate (FITC) (PharMingen) and 7-amino-actinomycin D (7-AAD; Sigma) or Annexin V (R&D Systems) plus PI to identify apoptotic cells.
For generation of primary MEFs Chk2 −/− ES clones and control ES clones (Chk2 +/+ OPGL +/ − ) (28) were injected into 3.5-day C57BL/6 blastocysts and transferred to pseudopregnant foster mothers. MEFs were derived from embryonic day 14.5 embryos and selected in G418 (400 μg/ml) for 10 days. Loss of Chk2 protein in Chk2 −/− MEFs was confirmed by Western blotting. Cells up to passage 6 were used as primary MEFs.
For retroviral infections a retrovirus (G. P. Nolan Stanford University School of Medicine) carrying either the mouse Chk2 or GFP cDNA was packaged with the øNX-Eco cell line and Chk2 −/− MEFs were infected as previously described (29). The infection efficiency of GFP was about 60%.
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Y. Sanchez and S. J. Elledge unpublished results.
Bargonetti J., Chicas A., White D., Prives C., Cell. Mol. Biol. 43, 935 (1997).
We thank the members of the Amgen Institute for helpful discussions; V. Stambolic L. Harrington and S. Pownall for critical reading of the manuscript; D. Bouchard for technical support; M. Saunders for scientific editing; G. P. Nolan for retroviral vectors and øNX cells; R. Jaenisch for p53 −/− ES cells; K. Tamai and Y. Taya for antibody to phosphorylated Ser 20 antibodies; and C. Prives for pGST-p53(1-80). Supported by grants from the National Cancer Institute of Canada and Medical Research Council of Canada (T.W.M.) and from the NIH (grant GM44664 to S.J.E.). S.J.E. is an Investigator with the Howard Hughes Medical Institute.