Requirement for p53 and p21 to sustain G2 arrest after DNA damage

Science

Volumn 282, Issue 5393, 1998, Pages 1497-1501

  Bunz, F ^a   Dutriaux, A ^b   Lengauer, C ^a   Waldman, T ^a   Zhou, S ^a   Brown, J P ^b   Sedivy, J M ^b   Kinzler, K W ^a   Vogelstein, B ^a  

^a JOHNS HOPKINS UNIVERSITY   (United States)

^b BROWN UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

NOCODAZOLE; PROTEIN P21; PROTEIN P53;

ARTICLE; CELL CYCLE G2 PHASE; CELL CYCLE S PHASE; CYTOKINESIS; DNA CONTENT; DNA DAMAGE; DNA SYNTHESIS; ENZYME ACTIVATION; GAMMA RADIATION; GENE DISRUPTION; HUMAN; HUMAN CELL; MITOSIS; PRIORITY JOURNAL; PROTEIN EXPRESSION;

EID: 0032553485     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5393.1497     Document Type: Article

References (60)

1. M. B. Kastan, O. Onyekwere, D. Sidransky, B. Vogelstein, R. W. Craig, Cancer Res. 51, 6304 (1991); A. Maity, W. G. McKenna, R. J. Muschel, Radiother. Oncol. 31, 1 (1994); L. S. Cox and D. P. Lane, Bioessays 17, 501 (1995).
2. M. B. Kastan, O. Onyekwere, D. Sidransky, B. Vogelstein, R. W. Craig, Cancer Res. 51, 6304 (1991); A. Maity, W. G. McKenna, R. J. Muschel, Radiother. Oncol. 31, 1 (1994); L. S. Cox and D. P. Lane, Bioessays 17, 501 (1995).
3. M. B. Kastan, O. Onyekwere, D. Sidransky, B. Vogelstein, R. W. Craig, Cancer Res. 51, 6304 (1991); A. Maity, W. G. McKenna, R. J. Muschel, Radiother. Oncol. 31, 1 (1994); L. S. Cox and D. P. Lane, Bioessays 17, 501 (1995).
4. L. H. Hartwell and M. B. Kastan, Science 266, 1821 (1994); A. B. Niculescu et al., Mol. Cell. Biol. 18, 629 (1998).
5. L. H. Hartwell and M. B. Kastan, Science 266, 1821 (1994); A. B. Niculescu et al., Mol. Cell. Biol. 18, 629 (1998).
6. C. Deng, P. Zhang, J. W. Harper, S. J. Elledge, P. Leder, Cell 82, 675 (1995); J. Brugarolas et al., Nature 377, 552 (1995).
7. C. Deng, P. Zhang, J. W. Harper, S. J. Elledge, P. Leder, Cell 82, 675 (1995); J. Brugarolas et al., Nature 377, 552 (1995).
8. T. Waldman, K. W. Kinzler, B. Vogelstein, Cancer Res. 55, 5187 (1995).
9. J. P. Brown, W. Wei, J. M. Sedivy, Science 277, 831 (1997).
10. S. N. Powell et al., Cancer Res. 55, 1643 (1995); B. B. Wang, K. J. Hayenga, D. G. Payan, J. M. Fisher, Biochem. J. 314, 313 (1996); S. Fan et al., Cancer Res. 55, 1649 (1995); K. J. Russell et al., ibid. 55, 1639 (1995).
11. S. N. Powell et al., Cancer Res. 55, 1643 (1995); B. B. Wang, K. J. Hayenga, D. G. Payan, J. M. Fisher, Biochem. J. 314, 313 (1996); S. Fan et al., Cancer Res. 55, 1649 (1995); K. J. Russell et al., ibid. 55, 1639 (1995).
12. S. N. Powell et al., Cancer Res. 55, 1643 (1995); B. B. Wang, K. J. Hayenga, D. G. Payan, J. M. Fisher, Biochem. J. 314, 313 (1996); S. Fan et al., Cancer Res. 55, 1649 (1995); K. J. Russell et al., ibid. 55, 1639 (1995).
13. S. N. Powell et al., Cancer Res. 55, 1643 (1995); B. B. Wang, K. J. Hayenga, D. G. Payan, J. M. Fisher, Biochem. J. 314, 313 (1996); S. Fan et al., Cancer Res. 55, 1649 (1995); K. J. Russell et al., ibid. 55, 1639 (1995).
14. Y. Peng et al. Science 277, 1501 (1997); Y. Sanchez et al., ibid., p. 1497; P. Nurse, Cell 91, 865 (1997).
15. Y. Peng et al. Science 277, 1501 (1997); Y. Sanchez et al., ibid., p. 1497; P. Nurse, Cell 91, 865 (1997).
16. Y. Peng et al. Science 277, 1501 (1997); Y. Sanchez et al., ibid., p. 1497; P. Nurse, Cell 91, 865 (1997).
17. M. J. Solomon, T. Lee, M. W. Kirschner, Mol. Biol. Cell. 3, 13 (1992); C. Smythe and J. W. Newport, Cell 68, 787 (1992); C. Jessus and D. Beach, ibid. p. 323; P. Nurse, Nature 344, 503 (1990).
18. M. J. Solomon, T. Lee, M. W. Kirschner, Mol. Biol. Cell. 3, 13 (1992); C. Smythe and J. W. Newport, Cell 68, 787 (1992); C. Jessus and D. Beach, ibid. p. 323; P. Nurse, Nature 344, 503 (1990).
19. M. J. Solomon, T. Lee, M. W. Kirschner, Mol. Biol. Cell. 3, 13 (1992); C. Smythe and J. W. Newport, Cell 68, 787 (1992); C. Jessus and D. Beach, ibid. p. 323; P. Nurse, Nature 344, 503 (1990).
20. M. J. Solomon, T. Lee, M. W. Kirschner, Mol. Biol. Cell. 3, 13 (1992); C. Smythe and J. W. Newport, Cell 68, 787 (1992); C. Jessus and D. Beach, ibid. p. 323; P. Nurse, Nature 344, 503 (1990).
21. T. Waldman, C. Lengauer, K. W. Kinzler, B. Vogelstein, Nature 381, 713 (1996).
22. P. L. Olive, J. P. Banath, R. E. Durand, Radiat. Res. 146, 595 (1996).
23. S. H. Khan and G. M. Wahl, Cancer Res. 58, 396 (1998); J. S. Lanni and T. Jacks, Mol. Cell. Biol. 18, 1055 (1998).
24. S. H. Khan and G. M. Wahl, Cancer Res. 58, 396 (1998); J. S. Lanni and T. Jacks, Mol. Cell. Biol. 18, 1055 (1998).
25. T. J. McGarry and M. W. Kirschner, Cell 93, 1043 (1998).
26. T. T. Su, P. J. Follette, P. H. O'Farrell, ibid. 81, 825 (1995); R. A. Laskey, D. Gorlich, M. A. Madine, J. P. S. Makkerh, P. Romanowski, Exp. Cell. Res. 229, 204 (1996).
27. T. T. Su, P. J. Follette, P. H. O'Farrell, ibid. 81, 825 (1995); R. A. Laskey, D. Gorlich, M. A. Madine, J. P. S. Makkerh, P. Romanowski, Exp. Cell. Res. 229, 204 (1996).
28. F. Bunz, C. Lengauer, K. W. Kinzler, B. Vogelstein, unpublished data.
29. D. P. Cahill et al., Nature 392, 300 (1998).
30. The targeting vectors used in this experiment were constructed so that the first codons of the drug resistance markers replaced the first codon of the p53 gene within exon 2. Transfection was done with LipofectAmine (Life Technologies) and subconfluent monolayers of cells. The LipofectAmine-DNA mixture was removed 3 hours after transfection and cells were grown in complete medium for 18 hours before they were replated in selective medium. After transfection with the neomycin-resistance vector, resistant colonies were grown in the presence of geneticin (0.4 mg/ml) (Gibco). One geneticin-resistant clone of 600 assayed was found to harbor a homologous recombinant, and this clone was expanded and transfected with the hygromycin B-resistance vector. Colonies were selected in hygromycin B (0.1 mg/ml). One clone of 940 assayed had a homologous recombinant of the second p53 allele.
31. A. J. Levine, Cell 88, 323 (1997); L. J. Ko and C. Prives, Genes Dev. 10, 1054 (1996); R. Hansen and M. Oren, Curr. Opin. Genet. Dev. 7, 461 (1997).
32. A. J. Levine, Cell 88, 323 (1997); L. J. Ko and C. Prives, Genes Dev. 10, 1054 (1996); R. Hansen and M. Oren, Curr. Opin. Genet. Dev. 7, 461 (1997).
33. A. J. Levine, Cell 88, 323 (1997); L. J. Ko and C. Prives, Genes Dev. 10, 1054 (1996); R. Hansen and M. Oren, Curr. Opin. Genet. Dev. 7, 461 (1997).
34. H. Hermeking et al., Mol. Cell 1, 3 (1997).
35. S. Zhou et al., Proc. Natl. Acad. Sci. U.S.A. 95, 2412 (1998).
36. P. Michieli et al., Cancer Res. 54, 3391 (1994); K. F. Macleod et al., Genes Dev. 9, 935 (1995); A. Moustakas and D. Kardassis, Proc. Natl. Acad. Sci. U.S.A. 95, 6733 (1998).
37. P. Michieli et al., Cancer Res. 54, 3391 (1994); K. F. Macleod et al., Genes Dev. 9, 935 (1995); A. Moustakas and D. Kardassis, Proc. Natl. Acad. Sci. U.S.A. 95, 6733 (1998).
38. P. Michieli et al., Cancer Res. 54, 3391 (1994); K. F. Macleod et al., Genes Dev. 9, 935 (1995); A. Moustakas and D. Kardassis, Proc. Natl. Acad. Sci. U.S.A. 95, 6733 (1998).
39. Y. Xiong et al., Nature 366, 701 (1993); J. W. Harper et al., Mol. Biol. Cell 6, 387 (1995).
40. Y. Xiong et al., Nature 366, 701 (1993); J. W. Harper et al., Mol. Biol. Cell 6, 387 (1995).
41. Lung-derived human fetal fibroblasts (cell strain LF-1) were electroporated with the neomycin-resistant p53 targeting vector (Fig. 2A) and grown under G418 selection (5). Southern blotting (DNA) analysis revealed that several resultant clones had undergone the desired recombination event. In three independent experiments, the efficiency of targeting was 5, 6, and 31%, respectively. Subclones from heterozygous cells were then serially cultured; 30% of them had an increased life span (an additional 30 population doublings before senescence). Southern blotting indicated that all such long-lived clones had sustained a loss of the remaining wild-type p53 gene. The p53-deficient clones used for nocodazole trapping experiments were in the early phases of life span extension.
42. -/- cells were transfected with a vector, pBOSH2BGFP-N1, that encodes H2B/GFP (T. Kanda, K. F. Sullivan, G. M. Wahl, Curr. Biol. 8, 377 [1998]). Colonies with stable expression of this fusion protein were identified by fluorescence microscopy and expanded into cell lines. Normal mitoses were observed by time-lapse videomicroscopy and both fluorescent and bright-field images were acquired every 6 min with the MetaMorph software package (Universal Imaging).
43. K. Fukasawa, T. Choi, R. Kuriyama, S. Rulong, G. F. Vande Woude, Science 271, 1744 (1996).
44. A. Paoletti and M. Bornens, Prog. Cell Cycle Res. 3, 285 (1997).
45. R. S. Paules et al., Cancer Res 55, 1763 (1995); T. M. Guadagno and J.W. Newport, Cell 84, 73 (1996); M. L. Agarwal, A. Agarwal, W. R. Taylor, G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 92, 8493 (1995); N. Stewart, G. G. Hicks, F. Paraskeva, M. Mowat, Oncogene 10, 109 (1995).
46. R. S. Paules et al., Cancer Res 55, 1763 (1995); T. M. Guadagno and J.W. Newport, Cell 84, 73 (1996); M. L. Agarwal, A. Agarwal, W. R. Taylor, G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 92, 8493 (1995); N. Stewart, G. G. Hicks, F. Paraskeva, M. Mowat, Oncogene 10, 109 (1995).
47. R. S. Paules et al., Cancer Res 55, 1763 (1995); T. M. Guadagno and J.W. Newport, Cell 84, 73 (1996); M. L. Agarwal, A. Agarwal, W. R. Taylor, G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 92, 8493 (1995); N. Stewart, G. G. Hicks, F. Paraskeva, M. Mowat, Oncogene 10, 109 (1995).
48. R. S. Paules et al., Cancer Res 55, 1763 (1995); T. M. Guadagno and J.W. Newport, Cell 84, 73 (1996); M. L. Agarwal, A. Agarwal, W. R. Taylor, G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 92, 8493 (1995); N. Stewart, G. G. Hicks, F. Paraskeva, M. Mowat, Oncogene 10, 109 (1995).
49. Y. Li, C. W. Jenkins, M. A. Nichols, Y. Xiong, Oncogene 9, 2261 (1994).
50. V. Dulic, G. H. Stein, D. F. Far, S. I. Reed, Mol. Cell. Biol. 18, 546 (1998); S. Bates, K. M. Ryan, A. C. Phillips, K. H. Vousden, Oncogene 17, 1691 (1998).
51. V. Dulic, G. H. Stein, D. F. Far, S. I. Reed, Mol. Cell. Biol. 18, 546 (1998); S. Bates, K. M. Ryan, A. C. Phillips, K. H. Vousden, Oncogene 17, 1691 (1998).
52. A. B. Niculescu et al., Mol. Cell. Biol. 18, 629 (1998).
53. L. Muhua, N. R. Adames, M. D. Murphy, C. R. Shields, J. A. Cooper, Nature 393, 487 (1998).
54. L. Hartwell, P. Szankasi, C. J. Roberts, A. W. Murray, S. H. Friend, Science 278, 1064 (1997).
55. 137Cs source (GammaCell 40) for a total dose of 12 Gy delivered over 14.5 min. Where indicated, cells were exposed to lower doses for proportionally shorter time periods. It was important that the irradiation of cells occurred when they were ∼70% confluent, as very sparse or completely confluent cells responded differently than subconfluent cells to γ radiation. Mitotic trapping experiments were done by adding nocodazole to the culture medium (0.2 μg/ml). Cells were collected by incubation with trypsin containing EDTA, centrifuged, and fixed in a solution containing 3.7% formaldehyde, 0.5% Nonidet P-40, and Hoescht 33258 (10 μ/ml) in phosphate-buffered saline. Nuclei were visualized by fluorescence microscopy. Nuclei with condensed, evenly staining chromosomes were scored as mitotic. At least 300 cells were counted for each determination.
56. Genomic DNA was purified from cell lysates with the QiaAMP spin blood kit (Qiagen) and used as a substrate for polymerase chain reaction and Southern blot assessment of targeting vector integration.
57. Equal numbers of cells were collected, lysed in Laemmli sample buffer, and subjected to electrophoresis and protein immunoblotting. Filters were probed with antibodies to p53 (pAb 1801) and p21 {EA10 [W. S. El-Deiry et al., Cancer Res. 55, 2910 (1995)]}. Signals were visualized with enhanced chemilunescence (Pierce).
58. 32P-labeled histone H1 was visualized by autoradiography.
59. Cells were fixed in methanol at -80°C and stained with antibody to γ-tubulin (Sigma) and a fluorescently labeled secondary antibody to mouse immunoglobulin G (Molecular Probes).
60. Supported by the Clayton Fund and NIH grants CA 43460, CA 57345, CA 62924, and GM 41690. We thank G. Wahl for the H2B-GFP fusion vector.