Rapid Destruction of Human Cdc25A in Response to DNA Damage
10.1126/science.288.5470.1425
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G 1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.

Bibliography

Mailand, N., Falck, J., Lukas, C., Syljuåsen, R. G., Welcker, M., Bartek, J., & Lukas, J. (2000). Rapid Destruction of Human Cdc25A in Response to DNA Damage. Science, 288(5470), 1425–1429.

Authors 7
  1. Niels Mailand (first)
  2. Jacob Falck (additional)
  3. Claudia Lukas (additional)
  4. Randi G. Syljuåsen (additional)
  5. Markus Welcker (additional)
  6. Jiri Bartek (additional)
  7. Jiri Lukas (additional)
References 55 Referenced 618
  1. 10.1126/science.7997877
  2. 10.1126/science.274.5293.1664
  3. Nasmyth K., Science 274, 1643 (1996); (10.1126/science.274.5293.1643) / Science by Nasmyth K. (1996)
  4. Weinert T., Cell 94, 555 (1998). (10.1016/S0092-8674(00)81597-4) / Cell by Weinert T. (1998)
  5. 10.1101/gad.10.9.1054
  6. 10.1016/S0092-8674(00)81871-1
  7. 10.1038/25292
  8. J. A. D'Anna
  9. Valdez J. G., Habbersett R. C., Crissman H. A., Radiat. Res. 148, 260 (1997); (10.2307/3579611) / Radiat. Res. by Valdez J. G. (1997)
  10. Lee H., Larner J. M., Hamlin J. L., Proc. Natl. Acad. Sci. U.S.A. 94, 526 (1997); (10.1073/pnas.94.2.526) / Proc. Natl. Acad. Sci. U.S.A. by Lee H. (1997)
  11. ; G. Xie et al. Oncogene 16 721 (1998). (10.1038/sj.onc.1201793)
  12. J. Brugarolas et al. Nature 377 552 (1995); (10.1038/377552a0)
  13. 10.1016/0092-8674(95)90039-X
  14. 10.1126/science.277.5331.1495
  15. ; C. Y. Peng et al. S cience 277 1501 (1997); Y. Sanchez et al. Science 277 1497 (1997); (10.1126/science.277.5331.1497)
  16. 10.1126/science.277.5331.1450
  17. S. Jinno et al. EMBO J. 13 1549 (1994); (10.1002/j.1460-2075.1994.tb06417.x)
  18. Hoffmann I., Draetta G., Karsenti E., EMBO J. 13, 4302 (1994); (10.1002/j.1460-2075.1994.tb06750.x) / EMBO J. by Hoffmann I. (1994)
  19. ; V. Sexl et al. Oncogene 18 573 (1999). (10.1038/sj.onc.1202362)
  20. N. Mailand J. Falck J. Bartek J. Lukas unpublished data.
  21. Treier M., Staszewski L. M., Bohmann D., Cell 78, 787 (1994). (10.1016/S0092-8674(94)90502-9) / Cell by Treier M. (1994)
  22. 10.1016/S0955-0674(96)80080-9
  23. Draetta G., Eckstein J., Biochim. Biophys. Acta 1332, M53 (1997); / Biochim. Biophys. Acta by Draetta G. (1997)
  24. Rhind N., Russell P., Curr. Opin. Cell Biol. 10, 749 (1998). (10.1016/S0955-0674(98)80118-X) / Curr. Opin. Cell Biol. by Rhind N. (1998)
  25. Our assay was as described by Blasina et al. [
  26. 10.1016/S0960-9822(99)80041-4
  27. ] except for monitoring phosphorylation of Tyr 15 on Cdk2 instead of Cdk1.
  28. Shaulian E., Zauberman A., Ginsberg D., Oren M., Mol. Cell Biol. 12, 5581 (1992). / Mol. Cell Biol. by Shaulian E. (1992)
  29. Kumagai A., Guo Z., Emami K. H., Wang S. X., Dunphy W. G., J. Cell Biol. 142, 1559 (1998). (10.1083/jcb.142.6.1559) / J. Cell Biol. by Kumagai A. (1998)
  30. J. N. Sarkaria et al. Cancer Res. 59 4375 (1999). UCN-01 was a gift from R. J. Schultz (Drug Synthesis & Chemistry Branch National Cancer Institute).
  31. DNA single-strand breaks were measured by alkaline elution with H 2 O 2 -treated L1210 cells as internal standard exactly as described [
  32. Sehested M., et al., Cancer Res. 58, 1460 (1998); / Cancer Res. by Sehested M. (1998)
  33. ]. Relative DNA single-strand break yields were calculated with the SAS statistical software package.
  34. Nelson W. G., Kastan M. B., Mol. Cell Biol. 14, 1815 (1994). / Mol. Cell Biol. by Nelson W. G. (1994)
  35. 10.1016/0092-8674(93)90496-D
  36. Poon R. Y., Chau M. S., Yamashita K., Hunter T., Cancer Res 57, 5168 (1997); / Cancer Res by Poon R. Y. (1997)
  37. Freedman D. A., Levine A. J., Cancer Res. 59, 1 (1999); / Cancer Res. by Freedman D. A. (1999)
  38. Blattner C., Sparks A., Lane D., Mol. Cell Biol. 19, 3704 (1999) . (10.1128/MCB.19.5.3704) / Mol. Cell Biol. by Blattner C. (1999)
  39. 10.1038/2181064a0
  40. 10.1101/gad.8.21.2540
  41. K. Galaktionov et al. Science 269 1575 (1995) (10.1126/science.7667636)
  42. D. Gasparotto et al. Cancer Res. 57 2366 (1997);
  43. Wu W., Fan Y. H., Kemp B. L., Walsh G., Mao L., Cancer Res. 58, 4082 (1998). / Cancer Res. by Wu W. (1998)
  44. UV-C (254 nm) irradiation was done in a UV Stratalinker 1800 (Stratagene); the ionizing radiation was delivered by x-ray generator (RT100 Philips Medico).
  45. Cdc25 phosphatase activity was measured as activation of cyclin B1–Cdc2. U-2-OS cells were treated with doxorubicin (Calbiochem) (0.2 μg/ml) for 24 hours to induce inhibitory phosphorylation of Cdc2 on Thr 14 and Tyr 15 [R. Y. C Poon
  46. Jiang W., Toyoshima H., Hunter T., J. Biol. Chem. 271, 132831 (1996); / J. Biol. Chem. by Jiang W. (1996)
  47. ]. Inactive cyclin B1–Cdc2 was immunoprecipitated with antibodies to cyclin B1 (200 μg of cell lysate per reaction). Cdc25 was immunoprecipitated in parallel from 1 mg of cell extracts treated as indicated. The beads from the cyclin B and Cdc25 immunoprecipitates were mixed incubated in a volume of 50 μl of a phosphatase buffer [20 mM tris-HCl (pH 8.3) 150 mM NaCl 2 mM EDTA 0.1% Triton X-100 5 mM dithiothreitol] at 30°C for 1 hour and the reaction was stopped by addition of kinase assay buffer. The activity of cyclin B-Cdc2 was assayed as described [
  48. Lukas J., Draetta G., Bartek J., Eur. J. Biochem. 207, 169 (1992)]. (10.1111/j.1432-1033.1992.tb17034.x) / Eur. J. Biochem. by Lukas J. (1992)
  49. Mouse monoclonal antibodies to Cdc25A (DCS-122 DCS-124) Cdc25B (DCS-162 DCS-164) and Cdc25C (DCS-193) were generated by standard hybridoma technology. Monoclonal antibody F-6 to Cdc25A was from Santa Cruz and C23420 to cyclin B1 was from Transduction Laboratories. Rabbit antisera to Cdc25A (SC-7157) and Cdc25C (SC-327) were from Santa Cruz Biotechnology antiserum to Cdk2 Tyr 15 (219440) was purchased from Calbiochem and antiserum to Chk1 was provided by S. Elledge. DO-1 monoclonal antibody to human p53 was from Oncogene Science. Mouse monoclonal antibodies 12CA5 to the hemagglutinin epitope DCS-60 and DCS-61 to p21 5D4 to cyclin D1 and D2 HE12 and HE172 against cyclin E as well as immunoblotting immunoprecipitation immunostaining and in vitro kinase assays were described [J. Lukas et al. Genes Dev. 11 1479 (1997)
  50. M. Thullberg et al. Hybridoma 19 63 (2000)]. (10.1089/027245700315806)
  51. U-2-OS sublines conditionally expressing HA-Cdc25A (U-2-OS/B3C4) or p53DD (U-2-OS/C6) were generated by transfection with the respective transgenes in pBI plasmids as described [
  52. Lukas J., Storgaard Sørensen C., Lukas C., Santoni-Rugiu E., Bartek J., Oncogene 18, 3930 (1999); (10.1038/sj.onc.1202777) / Oncogene by Lukas J. (1999)
  53. ]. U-2-OS/C6 cells were microinjected with the Mdm2-Luc reporter plasmid (25 μg/ml) and nonimmune mouse IgG as a microinjection marker. After 18 hours the cells were fixed and processed for combined anti-mouse immunoglobulin G and anti-luciferase immunostaining as described [J. Lukas et al. Genes Dev. 11 1479 (1997)].
  54. Conditions for single- and multiple-parameter flow cytometry with a FACSCalibur cytometer (Becton Dickinson) and CellQuest and ModFit software were described [J. Lukas et al. Oncogene 9 2159 (1994)].
  55. Supported by grants from the Danish Cancer Society the Human Frontier Science Program the Danish Medical Research Council and the Alfred Benzons Fund. We thank B. Ducommun S. Elledge M. Oren S. Reed and C. Ward for plasmids and antibodies and R. J. Schultz (Drug Synthesis & Chemistry Branch NCI) for UCN-01.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:50 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:49 a.m.)
Indexed 4 weeks ago (July 30, 2025, 11:20 a.m.)
Issued 25 years, 3 months ago (May 26, 2000)
Published 25 years, 3 months ago (May 26, 2000)
Published Print 25 years, 3 months ago (May 26, 2000)
Funders 0

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@article{Mailand_2000, title={Rapid Destruction of Human Cdc25A in Response to DNA Damage}, volume={288}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.288.5470.1425}, DOI={10.1126/science.288.5470.1425}, number={5470}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Mailand, Niels and Falck, Jacob and Lukas, Claudia and Syljuåsen, Randi G. and Welcker, Markus and Bartek, Jiri and Lukas, Jiri}, year={2000}, month=may, pages={1425–1429} }