Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Sep;17(9):5640–5647. doi: 10.1128/mcb.17.9.5640

Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.

D Resnitzky 1
PMCID: PMC232412  PMID: 9271439

Abstract

Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams P. D., Kaelin W. G., Jr Transcriptional control by E2F. Semin Cancer Biol. 1995 Apr;6(2):99–108. doi: 10.1006/scbi.1995.0013. [DOI] [PubMed] [Google Scholar]
  2. Baldin V., Lukas J., Marcote M. J., Pagano M., Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993 May;7(5):812–821. doi: 10.1101/gad.7.5.812. [DOI] [PubMed] [Google Scholar]
  3. Beijersbergen R. L., Carlée L., Kerkhoven R. M., Bernards R. Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes. Genes Dev. 1995 Jun 1;9(11):1340–1353. doi: 10.1101/gad.9.11.1340. [DOI] [PubMed] [Google Scholar]
  4. Böhmer R. M., Scharf E., Assoian R. K. Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1. Mol Biol Cell. 1996 Jan;7(1):101–111. doi: 10.1091/mbc.7.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Draetta G. F. Mammalian G1 cyclins. Curr Opin Cell Biol. 1994 Dec;6(6):842–846. doi: 10.1016/0955-0674(94)90054-x. [DOI] [PubMed] [Google Scholar]
  6. Fang F., Orend G., Watanabe N., Hunter T., Ruoslahti E. Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science. 1996 Jan 26;271(5248):499–502. doi: 10.1126/science.271.5248.499. [DOI] [PubMed] [Google Scholar]
  7. Folkman J., Moscona A. Role of cell shape in growth control. Nature. 1978 Jun 1;273(5661):345–349. doi: 10.1038/273345a0. [DOI] [PubMed] [Google Scholar]
  8. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guadagno T. M., Assoian R. K. G1/S control of anchorage-independent growth in the fibroblast cell cycle. J Cell Biol. 1991 Dec;115(5):1419–1425. doi: 10.1083/jcb.115.5.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guadagno T. M., Ohtsubo M., Roberts J. M., Assoian R. K. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science. 1993 Dec 3;262(5139):1572–1575. doi: 10.1126/science.8248807. [DOI] [PubMed] [Google Scholar]
  11. Hatakeyama M., Brill J. A., Fink G. R., Weinberg R. A. Collaboration of G1 cyclins in the functional inactivation of the retinoblastoma protein. Genes Dev. 1994 Aug 1;8(15):1759–1771. doi: 10.1101/gad.8.15.1759. [DOI] [PubMed] [Google Scholar]
  12. Henglein B., Chenivesse X., Wang J., Eick D., Bréchot C. Structure and cell cycle-regulated transcription of the human cyclin A gene. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5490–5494. doi: 10.1073/pnas.91.12.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hinds P. W., Mittnacht S., Dulic V., Arnold A., Reed S. I., Weinberg R. A. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992 Sep 18;70(6):993–1006. doi: 10.1016/0092-8674(92)90249-c. [DOI] [PubMed] [Google Scholar]
  14. Hinds P. W. The retinoblastoma tumor suppressor protein. Curr Opin Genet Dev. 1995 Feb;5(1):79–83. doi: 10.1016/s0959-437x(95)90057-8. [DOI] [PubMed] [Google Scholar]
  15. Hunter T., Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994 Nov 18;79(4):573–582. doi: 10.1016/0092-8674(94)90543-6. [DOI] [PubMed] [Google Scholar]
  16. La Thangue N. B. DP and E2F proteins: components of a heterodimeric transcription factor implicated in cell cycle control. Curr Opin Cell Biol. 1994 Jun;6(3):443–450. doi: 10.1016/0955-0674(94)90038-8. [DOI] [PubMed] [Google Scholar]
  17. Lees E. Cyclin dependent kinase regulation. Curr Opin Cell Biol. 1995 Dec;7(6):773–780. doi: 10.1016/0955-0674(95)80060-3. [DOI] [PubMed] [Google Scholar]
  18. Lukas J., Bartkova J., Rohde M., Strauss M., Bartek J. Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity. Mol Cell Biol. 1995 May;15(5):2600–2611. doi: 10.1128/mcb.15.5.2600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MACPHERSON I., MONTAGNIER L. AGAR SUSPENSION CULTURE FOR THE SELECTIVE ASSAY OF CELLS TRANSFORMED BY POLYOMA VIRUS. Virology. 1964 Jun;23:291–294. doi: 10.1016/0042-6822(64)90301-0. [DOI] [PubMed] [Google Scholar]
  20. Matsuhisa T., Mori Y. An anchorage-dependent locus in the cell cycle for the growth of 3T3 cells. Exp Cell Res. 1981 Oct;135(2):393–398. doi: 10.1016/0014-4827(81)90176-2. [DOI] [PubMed] [Google Scholar]
  21. Michalovitz D., Halevy O., Oren M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell. 1990 Aug 24;62(4):671–680. doi: 10.1016/0092-8674(90)90113-s. [DOI] [PubMed] [Google Scholar]
  22. Morgan D. O. Principles of CDK regulation. Nature. 1995 Mar 9;374(6518):131–134. doi: 10.1038/374131a0. [DOI] [PubMed] [Google Scholar]
  23. Nigg E. A. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays. 1995 Jun;17(6):471–480. doi: 10.1002/bies.950170603. [DOI] [PubMed] [Google Scholar]
  24. Ohtsubo M., Roberts J. M. Cyclin-dependent regulation of G1 in mammalian fibroblasts. Science. 1993 Mar 26;259(5103):1908–1912. doi: 10.1126/science.8384376. [DOI] [PubMed] [Google Scholar]
  25. Ohtsubo M., Theodoras A. M., Schumacher J., Roberts J. M., Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. doi: 10.1128/mcb.15.5.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Otsuka H., Moskowitz M. Arrest of 3T3 cells in G1 phase in suspension culture. J Cell Physiol. 1975 Dec;87(2):213–219. doi: 10.1002/jcp.1040870209. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Pines J. Cyclins, CDKs and cancer. Semin Cancer Biol. 1995 Apr;6(2):63–72. doi: 10.1006/scbi.1995.0009. [DOI] [PubMed] [Google Scholar]
  29. Quelle D. E., Ashmun R. A., Shurtleff S. A., Kato J. Y., Bar-Sagi D., Roussel M. F., Sherr C. J. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 1993 Aug;7(8):1559–1571. doi: 10.1101/gad.7.8.1559. [DOI] [PubMed] [Google Scholar]
  30. Resnitzky D., Gossen M., Bujard H., Reed S. I. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994 Mar;14(3):1669–1679. doi: 10.1128/mcb.14.3.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Resnitzky D., Hengst L., Reed S. I. Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1. Mol Cell Biol. 1995 Aug;15(8):4347–4352. doi: 10.1128/mcb.15.8.4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Resnitzky D., Reed S. I. Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol Cell Biol. 1995 Jul;15(7):3463–3469. doi: 10.1128/mcb.15.7.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ruoslahti E., Obrink B. Common principles in cell adhesion. Exp Cell Res. 1996 Aug 25;227(1):1–11. doi: 10.1006/excr.1996.0243. [DOI] [PubMed] [Google Scholar]
  34. Schulze A., Zerfass-Thome K., Bergès J., Middendorp S., Jansen-Dürr P., Henglein B. Anchorage-dependent transcription of the cyclin A gene. Mol Cell Biol. 1996 Sep;16(9):4632–4638. doi: 10.1128/mcb.16.9.4632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwartz M. A., Ingber D. E. Integrating with integrins. Mol Biol Cell. 1994 Apr;5(4):389–393. doi: 10.1091/mbc.5.4.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schwartz M. A. Signaling by integrins: implications for tumorigenesis. Cancer Res. 1993 Apr 1;53(7):1503–1506. [PubMed] [Google Scholar]
  37. Sherr C. J. D-type cyclins. Trends Biochem Sci. 1995 May;20(5):187–190. doi: 10.1016/s0968-0004(00)89005-2. [DOI] [PubMed] [Google Scholar]
  38. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  39. Sherr C. J. The ins and outs of RB: coupling gene expression to the cell cycle clock. Trends Cell Biol. 1994 Jan;4(1):15–18. doi: 10.1016/0962-8924(94)90033-7. [DOI] [PubMed] [Google Scholar]
  40. Shin S. I., Freedman V. H., Risser R., Pollack R. Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4435–4439. doi: 10.1073/pnas.72.11.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Slack J. K., Higgins P. J. Cytoarchitecture and cell growth control. Cell Motil Cytoskeleton. 1996;33(2):83–87. doi: 10.1002/(SICI)1097-0169(1996)33:2<83::AID-CM1>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  42. Sofer-Levi Y., Resnitzky D. Apoptosis induced by ectopic expression of cyclin D1 but not cyclin E. Oncogene. 1996 Dec 5;13(11):2431–2437. [PubMed] [Google Scholar]
  43. Stoker M., O'Neill C., Berryman S., Waxman V. Anchorage and growth regulation in normal and virus-transformed cells. Int J Cancer. 1968 Sep 15;3(5):683–693. doi: 10.1002/ijc.2910030517. [DOI] [PubMed] [Google Scholar]
  44. Weinberg R. A. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323–330. doi: 10.1016/0092-8674(95)90385-2. [DOI] [PubMed] [Google Scholar]
  45. Whyte P. The retinoblastoma protein and its relatives. Semin Cancer Biol. 1995 Apr;6(2):83–90. doi: 10.1006/scbi.1995.0011. [DOI] [PubMed] [Google Scholar]
  46. Xiao Z. X., Ginsberg D., Ewen M., Livingston D. M. Regulation of the retinoblastoma protein-related protein p107 by G1 cyclin-associated kinases. Proc Natl Acad Sci U S A. 1996 May 14;93(10):4633–4637. doi: 10.1073/pnas.93.10.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xu G., Livingston D. M., Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1357–1361. doi: 10.1073/pnas.92.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zauberman A., Lupo A., Oren M. Identification of p53 target genes through immune selection of genomic DNA: the cyclin G gene contains two distinct p53 binding sites. Oncogene. 1995 Jun 15;10(12):2361–2366. [PubMed] [Google Scholar]
  49. Zhu X., Ohtsubo M., Böhmer R. M., Roberts J. M., Assoian R. K. Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein. J Cell Biol. 1996 Apr;133(2):391–403. doi: 10.1083/jcb.133.2.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES