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. 1991 Dec 1;115(5):1275–1282. doi: 10.1083/jcb.115.5.1275

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

PMCID: PMC2289235  PMID: 1955475

Abstract

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.

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Selected References

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  1. Abe K., Yoshida M., Usui T., Horinouchi S., Beppu T. Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases. Exp Cell Res. 1991 Jan;192(1):122–127. doi: 10.1016/0014-4827(91)90166-r. [DOI] [PubMed] [Google Scholar]
  2. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed] [Google Scholar]
  3. Arion D., Meijer L., Brizuela L., Beach D. cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF. Cell. 1988 Oct 21;55(2):371–378. doi: 10.1016/0092-8674(88)90060-8. [DOI] [PubMed] [Google Scholar]
  4. Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
  5. Blow J. J., Nurse P. A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell. 1990 Sep 7;62(5):855–862. doi: 10.1016/0092-8674(90)90261-c. [DOI] [PubMed] [Google Scholar]
  6. Brizuela L., Draetta G., Beach D. p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 1987 Nov;6(11):3507–3514. doi: 10.1002/j.1460-2075.1987.tb02676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Broek D., Bartlett R., Crawford K., Nurse P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature. 1991 Jan 31;349(6308):388–393. doi: 10.1038/349388a0. [DOI] [PubMed] [Google Scholar]
  8. Dasso M., Newport J. W. Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus. Cell. 1990 Jun 1;61(5):811–823. doi: 10.1016/0092-8674(90)90191-g. [DOI] [PubMed] [Google Scholar]
  9. Davis P. D., Hill C. H., Keech E., Lawton G., Nixon J. S., Sedgwick A. D., Wadsworth J., Westmacott D., Wilkinson S. E. Potent selective inhibitors of protein kinase C. FEBS Lett. 1989 Dec 18;259(1):61–63. doi: 10.1016/0014-5793(89)81494-2. [DOI] [PubMed] [Google Scholar]
  10. Draetta G., Beach D. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell. 1988 Jul 1;54(1):17–26. doi: 10.1016/0092-8674(88)90175-4. [DOI] [PubMed] [Google Scholar]
  11. Draetta G., Brizuela L., Potashkin J., Beach D. Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+. Cell. 1987 Jul 17;50(2):319–325. doi: 10.1016/0092-8674(87)90227-3. [DOI] [PubMed] [Google Scholar]
  12. Enoch T., Nurse P. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell. 1990 Feb 23;60(4):665–673. doi: 10.1016/0092-8674(90)90669-6. [DOI] [PubMed] [Google Scholar]
  13. Giordano A., Whyte P., Harlow E., Franza B. R., Jr, Beach D., Draetta G. A 60 kd cdc2-associated polypeptide complexes with the E1A proteins in adenovirus-infected cells. Cell. 1989 Sep 8;58(5):981–990. doi: 10.1016/0092-8674(89)90949-5. [DOI] [PubMed] [Google Scholar]
  14. Hartwell L. H. Cell division from a genetic perspective. J Cell Biol. 1978 Jun;77(3):627–637. doi: 10.1083/jcb.77.3.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartwell L. H., Culotti J., Pringle J. R., Reid B. J. Genetic control of the cell division cycle in yeast. Science. 1974 Jan 11;183(4120):46–51. doi: 10.1126/science.183.4120.46. [DOI] [PubMed] [Google Scholar]
  16. Hayles J., Beach D., Durkacz B., Nurse P. The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function. Mol Gen Genet. 1986 Feb;202(2):291–293. doi: 10.1007/BF00331653. [DOI] [PubMed] [Google Scholar]
  17. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  18. Hindley J., Phear G., Stein M., Beach D. Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe. Mol Cell Biol. 1987 Jan;7(1):504–511. doi: 10.1128/mcb.7.1.504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirano T., Funahashi S., Uemura T., Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis. EMBO J. 1986 Nov;5(11):2973–2979. doi: 10.1002/j.1460-2075.1986.tb04594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  21. Kase H., Iwahashi K., Matsuda Y. K-252a, a potent inhibitor of protein kinase C from microbial origin. J Antibiot (Tokyo) 1986 Aug;39(8):1059–1065. doi: 10.7164/antibiotics.39.1059. [DOI] [PubMed] [Google Scholar]
  22. Kase H., Iwahashi K., Nakanishi S., Matsuda Y., Yamada K., Takahashi M., Murakata C., Sato A., Kaneko M. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun. 1987 Jan 30;142(2):436–440. doi: 10.1016/0006-291x(87)90293-2. [DOI] [PubMed] [Google Scholar]
  23. Kimura G., Itagaki A., Summers J. Rat cell line 3y1 and its virogenic polyoma- and sv40- transformed derivatives. Int J Cancer. 1975 Apr 15;15(4):694–706. doi: 10.1002/ijc.2910150419. [DOI] [PubMed] [Google Scholar]
  24. Kung A. L., Sherwood S. W., Schimke R. T. Cell line-specific differences in the control of cell cycle progression in the absence of mitosis. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9553–9557. doi: 10.1073/pnas.87.24.9553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee M., Nurse P. Cell cycle control genes in fission yeast and mammalian cells. Trends Genet. 1988 Oct;4(10):287–290. doi: 10.1016/0168-9525(88)90171-0. [DOI] [PubMed] [Google Scholar]
  26. Loomis C. R., Bell R. M. Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C. J Biol Chem. 1988 Feb 5;263(4):1682–1692. [PubMed] [Google Scholar]
  27. Murray A. W. Cell biology. Remembrance of things past. Nature. 1991 Jan 31;349(6308):367–368. doi: 10.1038/349367a0. [DOI] [PubMed] [Google Scholar]
  28. Nishimoto T., Eilen E., Basilico C. Premature of chromosome condensation in a ts DNA- mutant of BHK cells. Cell. 1978 Oct;15(2):475–483. doi: 10.1016/0092-8674(78)90017-x. [DOI] [PubMed] [Google Scholar]
  29. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  30. Noguchi P. D., Browne W. C. The use of chicken erythrocyte nuclei as a biological standard for flow microfluorometry. J Histochem Cytochem. 1978 Sep;26(9):761–763. doi: 10.1177/26.9.361885. [DOI] [PubMed] [Google Scholar]
  31. Nurse P., Bissett Y. Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature. 1981 Aug 6;292(5823):558–560. doi: 10.1038/292558a0. [DOI] [PubMed] [Google Scholar]
  32. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  33. Osada H., Sonoda T., Tsunoda K., Isono K. A new biological role of sangivamycin; inhibition of protein kinases. J Antibiot (Tokyo) 1989 Jan;42(1):102–106. doi: 10.7164/antibiotics.42.102. [DOI] [PubMed] [Google Scholar]
  34. Osada H., Takahashi H., Tsunoda K., Kusakabe H., Isono K. A new inhibitor of protein kinase C, RK-286C (4'-demethylamino-4'-hydroxystaurosporine). I. Screening, taxonomy, fermentation and biological activity. J Antibiot (Tokyo) 1990 Feb;43(2):163–167. doi: 10.7164/antibiotics.43.163. [DOI] [PubMed] [Google Scholar]
  35. Osmani S. A., Engle D. B., Doonan J. H., Morris N. R. Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell. 1988 Jan 29;52(2):241–251. doi: 10.1016/0092-8674(88)90513-2. [DOI] [PubMed] [Google Scholar]
  36. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  37. Piggott J. R., Rai R., Carter B. L. A bifunctional gene product involved in two phases of the yeast cell cycle. Nature. 1982 Jul 22;298(5872):391–393. doi: 10.1038/298391a0. [DOI] [PubMed] [Google Scholar]
  38. Pondaven P., Meijer L., Beach D. Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components. Genes Dev. 1990 Jan;4(1):9–17. doi: 10.1101/gad.4.1.9. [DOI] [PubMed] [Google Scholar]
  39. Rao P. N., Johnson R. T. Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature. 1970 Jan 10;225(5228):159–164. doi: 10.1038/225159a0. [DOI] [PubMed] [Google Scholar]
  40. Schlegel R., Belinsky G. S., Harris M. O. Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine. Cell Growth Differ. 1990 Apr;1(4):171–178. [PubMed] [Google Scholar]
  41. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  42. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  43. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  44. Wittenberg C., Sugimoto K., Reed S. I. G1-specific cyclins of S. cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase. Cell. 1990 Jul 27;62(2):225–237. doi: 10.1016/0092-8674(90)90361-h. [DOI] [PubMed] [Google Scholar]
  45. Yamada K., Kimura G. Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts. J Cell Physiol. 1985 Feb;122(2):210–214. doi: 10.1002/jcp.1041220208. [DOI] [PubMed] [Google Scholar]
  46. Yoshida M., Beppu T. Reversible arrest of proliferation of rat 3Y1 fibroblasts in both the G1 and G2 phases by trichostatin A. Exp Cell Res. 1988 Jul;177(1):122–131. doi: 10.1016/0014-4827(88)90030-4. [DOI] [PubMed] [Google Scholar]
  47. Yoshida M., Nishikawa M., Nishi K., Abe K., Horinouchi S., Beppu T. Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells. Exp Cell Res. 1990 Mar;187(1):150–156. doi: 10.1016/0014-4827(90)90129-x. [DOI] [PubMed] [Google Scholar]

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