An anti-apoptotic role for the p53 family member, p73, during developmental neuron death

Science

Volumn 289, Issue 5477, 2000, Pages 304-306

  Pozniak, Christine D ^a   Radinovic, Stevo ^b   Yang, Annie ^c   McKeon, Frank ^c   Kaplan, David R ^a,b   Miller, Freda D ^a  

^a MCGILL UNIVERSITY   (Canada)

^b MCGILL UNIVERSITY   (Canada)

^c HARVARD MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN P53; PROTEIN P73;

APOPTOSIS; ARTICLE; CELL MATURATION; CONTROLLED STUDY; GENE OVEREXPRESSION; HUMAN; HUMAN CELL; NERVE CELL NECROSIS; PRIORITY JOURNAL; PROTEIN FAMILY; PROTEIN STRUCTURE; SYMPATHETIC NERVE;

ADENOVIRIDAE; ANIMALS; APOPTOSIS; CELLS, CULTURED; DNA-BINDING PROTEINS; ESCHERICHIA COLI; GENES, TUMOR SUPPRESSOR; HUMANS; MICE; MICE, INBRED BALB C; NERVE GROWTH FACTOR; NEURONS; NUCLEAR PROTEINS; PROTEIN ISOFORMS; RECOMBINANT PROTEINS; SYMPATHETIC NERVOUS SYSTEM; TUMOR SUPPRESSOR PROTEIN P53; TUMOR SUPPRESSOR PROTEINS;

EID: 0034647718     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5477.304     Document Type: Article

References (26)

1. A. J. Levine, Cell 88, 323 (1997).
2. R. S. Morrison et al., J. Neurosci. 16, 1337 (1996).
3. R. S. Aloyz, S. X. Bamji, C. D. Pozniak, D. R. Kaplan, F. D. Miller, J. Cell Biol. 143, 1691 (1998).
4. R. S. Slack et al., J. Cell Biol. 135, 1085 (1996).
5. M. Kaghad et al., Cell 90, 809 (1997).
6. C. A. Jost, M. C. Marin, W. G. Kaelin Jr., Nature 389, 191 (1997).
7. A. Yang et al., Nature 404, 99 (2000).
8. W. G. Kaelin Jr., Oncogene 18, 7701 (1999).
9. V. De Laurenzi et al., J. Exp. Med. 188, 1763 (1998).
10. A. Yang et al., Mol. Cell 2, 305 (1998).
11. A. Yang et al., Nature 398, 714 (1999).
12. Total RNA was prepared and RT-PCR performed as described in [X.-M. Yang et al., J. Neurosci. 18, 8369 (1998)]. Primers specific to truncated p73 were 5′ATGGGCCCTGTGTATGAATCCTTG3′ and 5′GGTATTGGAAGGGATGACAGGCG3′. Primers specific to full-length p73 were 5′GAGCACCTGTGGAGTTCTCTAGAG3′ and 5′GGTATTGGAAGGGATGACAGGCG3′.
13. C. D. Pozniak et al., data not shown.
14. Whole brain and sympathetic neurons were lysed and 1D Western blot analysis performed as described using antibody to p73 (1;200) (ER-15; Neomarkers, Union City, CA) (3). Two-dimensional gels were run as per manufacturer's instructions (Amersham Pharmacia Biotech) using lysates of sympathetic neurons infected with adenoviruses expressing full-length human p73α, and mouse ΔN-p73α and ΔN-p73β to standardize the system.
15. Mice heterozygous for a targeted mutation in the p73 gene (7) were maintained in a c129-Balb/c background. Progeny from p73 heterozygote crosses were screened for the mutant and wild-type alleles with PCR.
16. Purified cultures of sympathetic neurons were cultured and then withdrawn from NGF as described (3, 19).
17. Recombinant adenoviruses expressing ΔN-p73α or -β were generated, purified over CsCl, and titered as described (23, 24).
18. Sympathetic neurons were cultured for 4 to 5 days in 50 ng/ml NGF and then infected with recombinant adenovirus as previously described (3). Three days later, neurons were withdrawn from NGF or were maintained in 10 ng/ml NGF, and MTT survival assays and TUNEL were performed after 2 days as described (25).
19. S. X. Bamji et al, J. Cell Biol. 140, 911 (1998).
20. A. M. Fagan et al., J. Neurosci. 16, 6208 (1996).
21. For morphometric analysis, the SCGs were prepared and analyzed as previously (3, 19). Sections were stained with toluidine blue or cresyl violet and were analyzed with a computer-based image analysis system that counted every third section.
22. 2-terminus of ΔN-p73α or ΔN-p73β, and proteins were produced in Escherichia coli. To assess interactions, lysates of 293 cells infected with p53 adenovirus (4) were incubated with the CST fusion proteins for 4 hours at 4°C and were precipitated with glutathione-agarose. Precipitated proteins were analyzed by Western blot analysis with antibody to p53 (DO-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
23. T. C. He et al., Proc. Natl. Acad. Sci. U.S.A. 95, 2509 (1998).
24. I. E. Mazzoni, F. A. Said, R. Aloyz, F. D. Miller, D. Kaplan, J. Neurosci. 19, 9716 (1999).
25. A. Vaillant et al., J. Cell Biol. 146, 955 (1999).
26. We thank R. Aloyz, A. Boudreau, F. Arab-Said, and S. Morris for their advice and assistance. C.D.P. and S.R. are supported by Canadian Medical Research Council (MRC) and National Sciences Engineering Research Council of Canada studentships, respectively; D.R.K. is an National Cancer Institute of Canada Scientist; and F.D.M. is an MRC Senior Scientist and Killam Scholar. Supported in part by grants from the MRC to F.D.M. and D.R.K.