Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle

Science

Volumn 272, Issue 5263, 1996, Pages 877-880

  Coats, Steve ^a   Flanagan, W Michael ^b   Nourse, Jamison ^c   Roberts, James M ^a  

^a Fred Hutchinson Cancer Research Center   (United States)

^b GILEAD SCIENCES   (United States)

^c STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MURINAE;

CYCLIN DEPENDENT KINASE; MITOGENIC AGENT; PROTEIN KINASE INHIBITOR; ANTISENSE OLIGONUCLEOTIDE; CDKN1B PROTEIN, MOUSE; CELL CYCLE PROTEIN; CYCLIN DEPENDENT KINASE INHIBITOR 1B; CYCLINE; ENZYME INHIBITOR; EPIDERMAL GROWTH FACTOR; MICROTUBULE ASSOCIATED PROTEIN; PLATELET DERIVED GROWTH FACTOR; PLATELET DERIVED GROWTH FACTOR BB; PLATELET-DERIVED GROWTH FACTOR BB; SOMATOMEDIN C; TUMOR SUPPRESSOR PROTEIN;

ANIMAL CELL; ARTICLE; CELL CYCLE; CELL CYCLE G1 PHASE; CELL STRAIN 3T3; CONTROLLED STUDY; FIBROBLAST; MITOSIS; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROTEIN EXPRESSION; AMINO ACID SEQUENCE; ANIMAL; BIOSYNTHESIS; CULTURE MEDIUM; DOWN REGULATION; DRUG ANTAGONISM; DRUG EFFECT; GENE EXPRESSION; GENETICS; METABOLISM; MOLECULAR GENETICS; NUCLEOTIDE SEQUENCE;

3T3 CELLS; AMINO ACID SEQUENCE; ANIMALS; BASE SEQUENCE; CELL CYCLE PROTEINS; CULTURE MEDIA; CYCLIN-DEPENDENT KINASE INHIBITOR P27; CYCLIN-DEPENDENT KINASES; CYCLINS; DOWN-REGULATION; ENZYME INHIBITORS; EPIDERMAL GROWTH FACTOR; G1 PHASE; GENE EXPRESSION; INSULIN-LIKE GROWTH FACTOR I; MICE; MICROTUBULE-ASSOCIATED PROTEINS; MITOGENS; MOLECULAR SEQUENCE DATA; OLIGONUCLEOTIDES, ANTISENSE; PLATELET-DERIVED GROWTH FACTOR; TUMOR SUPPRESSOR PROTEINS;

EID: 0029664461     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5263.877     Document Type: Article

References (58)

1. H. Temin, J. Cell. Physiol. 78, 161 (1971).
2. A. Pardee, Proc. Natl. Acad. Sci. U.S.A. 71, 1286 (1974).
3. O. Larsson, C. Latham, P. Zickert, A. Zetterberg, J. Cell. Physiol. 139, 477 (1989); A. Zetterberg and O. Larsson, Proc. Natl. Acad. Sci. U.S.A. 82, 5365 (1985).
4. O. Larsson, C. Latham, P. Zickert, A. Zetterberg, J. Cell. Physiol. 139, 477 (1989); A. Zetterberg and O. Larsson, Proc. Natl. Acad. Sci. U.S.A. 82, 5365 (1985).
5. C. Sherr, Cell 79, 551 (1994).
6. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
7. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
8. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
9. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
10. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
11. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
12. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
13. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
14. M. Serrano, G. Hannon, D. Beach, Nature 366, 704 (1993); G. Hannon and D. Beach, ibid. 371, 257 (1994); W. El-Deiry et al., Cell 75, 89 (1993); Y. Xiong et al., Nature 366, 701 (1993); K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67; M. Lee, I. Reynisdottir, J. Massaugue, Genes Dev. 9, 639 (1995) ; S. Matsuoka et al., ibid., p. 650; A. Koff et al., Science 260, 536 (1993).
15. K. Gould, S. Morreno, D. Owen, S. Sazer, P. Nurse, EMBO J. 10, 3297 (1991); M. Solomon, J. Harper, J. Shutlleworth, ibid. 12, 3133 (1993); M. Solomon, T. Lee, M. Kirschner, Mol. Biol. Cell 3, 13 (1992); P. Jeffrey et al., Nature 376, 313 (1995); D. Morgan, ibid. 374, 131 (1995).
16. K. Gould, S. Morreno, D. Owen, S. Sazer, P. Nurse, EMBO J. 10, 3297 (1991); M. Solomon, J. Harper, J. Shutlleworth, ibid. 12, 3133 (1993); M. Solomon, T. Lee, M. Kirschner, Mol. Biol. Cell 3, 13 (1992); P. Jeffrey et al., Nature 376, 313 (1995); D. Morgan, ibid. 374, 131 (1995).
17. K. Gould, S. Morreno, D. Owen, S. Sazer, P. Nurse, EMBO J. 10, 3297 (1991); M. Solomon, J. Harper, J. Shutlleworth, ibid. 12, 3133 (1993); M. Solomon, T. Lee, M. Kirschner, Mol. Biol. Cell 3, 13 (1992); P. Jeffrey et al., Nature 376, 313 (1995); D. Morgan, ibid. 374, 131 (1995).
18. K. Gould, S. Morreno, D. Owen, S. Sazer, P. Nurse, EMBO J. 10, 3297 (1991); M. Solomon, J. Harper, J. Shutlleworth, ibid. 12, 3133 (1993); M. Solomon, T. Lee, M. Kirschner, Mol. Biol. Cell 3, 13 (1992); P. Jeffrey et al., Nature 376, 313 (1995); D. Morgan, ibid. 374, 131 (1995).
19. K. Gould, S. Morreno, D. Owen, S. Sazer, P. Nurse, EMBO J. 10, 3297 (1991); M. Solomon, J. Harper, J. Shutlleworth, ibid. 12, 3133 (1993); M. Solomon, T. Lee, M. Kirschner, Mol. Biol. Cell 3, 13 (1992); P. Jeffrey et al., Nature 376, 313 (1995); D. Morgan, ibid. 374, 131 (1995).
20. K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67.
21. K. Polyak et al., Cell 78, 59 (1994); H. Toyashima and T. Hunter, ibid., p. 67.
22. J, Nourse et al., Nature 372, 570 (1994); J. Kato et al., Cell 79, 487 (1994).
23. J, Nourse et al., Nature 372, 570 (1994); J. Kato et al., Cell 79, 487 (1994).
24. Proliferating Balb/c-3T3 fibroblasts were rinsed in serum-free medium and refed with medium containing 0.1% serum. p27 protein immunoblots (ECL. Amersham) were performed on cells harvested at 4, 8, 12, 16, and 24 hours after refeeding. Histone H1 kinase assays (8) were done on cyclin A, cyclin E, and Cdk2 (23) immunoprecipitated from Balb/c-3T3 extracts made from proliferating and serum-starved cells. Serum-starved cells lipofected with p27 antisense oligonucleotides contained increased amounts of cyclin E- and cyclin A-associated histone H1 kinase activity as compared with serum-slarved cells. For experiments done with proliferating cells, proliferating cells were lipofected with either p27 mismatch or p27 antisense oligonucleotides and analyzed 24 hours later by flow cytometry and p27 immunoblots. The amount of p27 was three to five times less in cells treated with p27 antisense oligonucleotides than in proliferating cells and proliferating cells lipofected with p27 mismatch oligonucleotides.
25. Experiments were done as described (Fig. 1 C), with the exception that cyclin A and p27 immunoblots were done on extracts depleted in p27. All of the cyclin A was bound to p27 in extracts from serum-starved cells, whereas only a small fraction (5%) of cyclin A was associated with p27 in proliferating cells.
26. W. Pledger, C. Stiles, H. Antoniades, C. Scherr, Proc. Natl. Acad. Sci. U.S.A. 74, 4481 (1977); E. Leof, J. Van Wyk, E. O'Keefe, W. Pledger, Exp. Cell Res. 147, 202 (1983).
27. W. Pledger, C. Stiles, H. Antoniades, C. Scherr, Proc. Natl. Acad. Sci. U.S.A. 74, 4481 (1977); E. Leof, J. Van Wyk, E. O'Keefe, W. Pledger, Exp. Cell Res. 147, 202 (1983).
28. Density-arrested Balb/c-3T3 fibroblasts were rinsed in serum-free medium and refed with medium containing 0.1 % serum and 10 ng per milliliter of medium of POGF-BB, IGF-1, EGF, or IGF-1 and EGF, or all three growth factors. Cells were harvested 24 hours later and analyzed by flow cytometry for DNA content and p27 immunoblots. A combination of all three growth factors was required to stimulate 70% of the cells to enter the cell cycle and decrease p27 amounts by 10 times.
29. K. Raviprakash et al., J. Virol. 69, 69 (1995).
30. The oligonucleotides were synthesized on an automated synthesizer (model 8750, Milligen Bioresearch, Bedford, MA) as described (24). The antisense oligonucleotide sequence used in these experiments was 5′-UGG CUC UCC UGC GCC-3′ (targets base pairs 306 to 320 of murine Kip1) and the mismatch sequence was 5′-UCC CUU UGG CGC GCC-3′. For the lipofection procedure, 30nM oligonucleotides were mixed with GS2888 cytofectin (2.5 μg/ ml) (25) (Gilead Scientific, Foster City, CA) in serum-free medium and incubated for 10 min at 37°C. Proliferating Balb/c-3T3 fibroblasts were rinsed once in serum-free medium and refed with oligonucleotide-cytofectin solution in medium containing 0.1% serum. Cells were then incubated for 24 hours in humidified incubators at 37°C with 5% CO,. The percentage of cells that were positive for uptake of fluorescein isothiocyanate-labeled oligonucleotides was determined by ultraviolet fluorescence microscopy. Microinjection, immunofluorescence staining, and fluorescence microscopy were carried out as described (26). For costaining of β-Gal and BrdU, the cells were fixed and stained as previously described (26, 27).
31. Proliferating Balb/c-3T3 fibroblasts were lipofected with antisense and mismatch oligonucleotides (14). The amount of p21 was increased in proliferating cells as compared with that in serum-starved cells (23). Cells lipofected With either p27 mismatch or antisense oligonucleotides expressed slightly larger amounts of p21 as compared with amounts in serum-starved control cells.
32. 3H]thymidine incorporated into asynchronously proliferating cells.
33. R. Wagner, Nature Med. 1, 1116 (1995).
34. C. Sherr and J. Roberts, Genes Dev. 9, 1149 (1995).
35. M. Ohtsubo and J. Roberts, Science 259 1908 (1993); D. Quelle et al., Genes Dev. 7, 1559 (1993); D Resnitzky and S. Reed, Mol. Cell. Biol. 15, 3463 (1995).
36. M. Ohtsubo and J. Roberts, Science 259 1908 (1993); D. Quelle et al., Genes Dev. 7, 1559 (1993); D Resnitzky and S. Reed, Mol. Cell. Biol. 15, 3463 (1995).
37. M. Ohtsubo and J. Roberts, Science 259 1908 (1993); D. Quelle et al., Genes Dev. 7, 1559 (1993); D Resnitzky and S. Reed, Mol. Cell. Biol. 15, 3463 (1995).
38. J. Lees et al., EMBO J. 10, 4279 (1991); K. Buchkovich, L. Duffy, E. Harlow, Cell 58, 1097 (1989); R. Chen et al., ibid., p. 1193; J. Kato et al., Genes Dev. 7, 331 (1993); B. Lin et al., EMBO J. 10, 857 (1991).
39. J. Lees et al., EMBO J. 10, 4279 (1991); K. Buchkovich, L. Duffy, E. Harlow, Cell 58, 1097 (1989); R. Chen et al., ibid., p. 1193; J. Kato et al., Genes Dev. 7, 331 (1993); B. Lin et al., EMBO J. 10, 857 (1991).
40. J. Lees et al., EMBO J. 10, 4279 (1991); K. Buchkovich, L. Duffy, E. Harlow, Cell 58, 1097 (1989); R. Chen et al., ibid., p. 1193; J. Kato et al., Genes Dev. 7, 331 (1993); B. Lin et al., EMBO J. 10, 857 (1991).
41. J. Lees et al., EMBO J. 10, 4279 (1991); K. Buchkovich, L. Duffy, E. Harlow, Cell 58, 1097 (1989); R. Chen et al., ibid., p. 1193; J. Kato et al., Genes Dev. 7, 331 (1993); B. Lin et al., EMBO J. 10, 857 (1991).
42. J. Lees et al., EMBO J. 10, 4279 (1991); K. Buchkovich, L. Duffy, E. Harlow, Cell 58, 1097 (1989); R. Chen et al., ibid., p. 1193; J. Kato et al., Genes Dev. 7, 331 (1993); B. Lin et al., EMBO J. 10, 857 (1991).
43. R. Herrera, R. Weinberg, T. Jacks, unpublished data.
44. H. Matsushime, M. Roussel, R. Ashmun, C. Sherr, Cell 65, 701 (1991); A. Koff et al., Science 257, 1689 (1992).
45. H. Matsushime, M. Roussel, R. Ashmun, C. Sherr, Cell 65, 701 (1991); A. Koff et al., Science 257, 1689 (1992).
46. E. Firpo, A. Koff, M. Solomon, J. Roberts, Mol. Cell. Biol. 14, 4889 (1994).
47. B. Froehler, Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Humana, Totowa, NJ, 1993); B. Froehler, S. Wadwani, T. Terhost, S. Gerrard, Tetrahedron Lett. 33, 5307 (1992); B. Froehler, R. Jones, X. Cao, T. Terhost, ibid. 34, 1003 (1993).
48. B. Froehler, Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Humana, Totowa, NJ, 1993); B. Froehler, S. Wadwani, T. Terhost, S. Gerrard, Tetrahedron Lett. 33, 5307 (1992); B. Froehler, R. Jones, X. Cao, T. Terhost, ibid. 34, 1003 (1993).
49. B. Froehler, Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Humana, Totowa, NJ, 1993); B. Froehler, S. Wadwani, T. Terhost, S. Gerrard, Tetrahedron Lett. 33, 5307 (1992); B. Froehler, R. Jones, X. Cao, T. Terhost, ibid. 34, 1003 (1993).
50. J. Lewis et al., Proc. Natl. Acad. Sci. U.S.A. 93, 2400 (1996).
51. T. Fisher, T. Terhost, X. Cao, R. Wagner, Nucleic Acids Res. 21, 3857 (1993); J. Hanvey et al., Science 258, 1481 (1992); R. Wagner et al., ibid. 260, 1510 (1993); C. Moulds et al., Biochemistry 34, 5044 (1995).
52. T. Fisher, T. Terhost, X. Cao, R. Wagner, Nucleic Acids Res. 21, 3857 (1993); J. Hanvey et al., Science 258, 1481 (1992); R. Wagner et al., ibid. 260, 1510 (1993); C. Moulds et al., Biochemistry 34, 5044 (1995).
53. T. Fisher, T. Terhost, X. Cao, R. Wagner, Nucleic Acids Res. 21, 3857 (1993); J. Hanvey et al., Science 258, 1481 (1992); R. Wagner et al., ibid. 260, 1510 (1993); C. Moulds et al., Biochemistry 34, 5044 (1995).
54. T. Fisher, T. Terhost, X. Cao, R. Wagner, Nucleic Acids Res. 21, 3857 (1993); J. Hanvey et al., Science 258, 1481 (1992); R. Wagner et al., ibid. 260, 1510 (1993); C. Moulds et al., Biochemistry 34, 5044 (1995).
55. F. Girard, U. Strausfeld, A. Fernandez, N. Lamb, Cell 67, 1169 (1991).
56. Single-letter abbreviations for the amino acid residues are as follows: A. Ala; D, Asp; E, Glu; L, Leu; Q, Gln; and S, Ser.
57. 112. These amino acids had been converted to Thr and Ser, respectively. Electrophoretically, the tagged p27 wobble mutant migrates slightly more slowly than do endogenous murine p27 and exogenous wild-type p27.
58. We thank members of the Roberts lab for suggestions and comments throughout the course of this work; R. Herrera, R. Weinberg, and T. Jacks for communicating results before publication; and D. Grant for technical assistance. Supported by a postdoctoral fellowship from the American Cancer Society (S.C.), an NIH Cancer Biology Training grant (J.N.), and a Howard Hughes Medical Insti-tute (grant to G. Crabtree) and the NIH (J.R.).