Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Apr 30;93(9):4350–4354. doi: 10.1073/pnas.93.9.4350

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

S N Khleif 1, J DeGregori 1, C L Yee 1, G A Otterson 1, F J Kaye 1, J R Nevins 1, P M Howley 1
PMCID: PMC39540  PMID: 8633069

Abstract

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

Full text

PDF
4350

Images in this article

4351Fig. 1
on p.4351
4352Fig. 2
on p.4352
4352Fig. 3
on p.4352
4353Fig. 4
on p.4353
4353Fig. 5
on p.4353

Selected References

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

  1. 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]
  2. Bandara L. R., Adamczewski J. P., Hunt T., La Thangue N. B. Cyclin A and the retinoblastoma gene product complex with a common transcription factor. Nature. 1991 Jul 18;352(6332):249–251. doi: 10.1038/352249a0. [DOI] [PubMed] [Google Scholar]
  3. Banks-Schlegel S. P., Howley P. M. Differentiation of human epidermal cells transformed by SV40. J Cell Biol. 1983 Feb;96(2):330–337. doi: 10.1083/jcb.96.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cairns P., Mao L., Merlo A., Lee D. J., Schwab D., Eby Y., Tokino K., van der Riet P., Blaugrund J. E., Sidransky D. Rates of p16 (MTS1) mutations in primary tumors with 9p loss. Science. 1994 Jul 15;265(5170):415–417. doi: 10.1126/science.8023167. [DOI] [PubMed] [Google Scholar]
  5. Chan F. K., Zhang J., Cheng L., Shapiro D. N., Winoto A. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4. Mol Cell Biol. 1995 May;15(5):2682–2688. doi: 10.1128/mcb.15.5.2682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  7. Chellappan S., Kraus V. B., Kroger B., Munger K., Howley P. M., Phelps W. C., Nevins J. R. Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4549–4553. doi: 10.1073/pnas.89.10.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  9. Chittenden T., Livingston D. M., Kaelin W. G., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. doi: 10.1016/0092-8674(91)90559-h. [DOI] [PubMed] [Google Scholar]
  10. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  11. DeGregori J., Kowalik T., Nevins J. R. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol. 1995 Aug;15(8):4215–4224. doi: 10.1128/mcb.15.8.4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DeGregori J., Leone G., Ohtani K., Miron A., Nevins J. R. E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity. Genes Dev. 1995 Dec 1;9(23):2873–2887. doi: 10.1101/gad.9.23.2873. [DOI] [PubMed] [Google Scholar]
  13. Dowdy S. F., Hinds P. W., Louie K., Reed S. I., Arnold A., Weinberg R. A. Physical interaction of the retinoblastoma protein with human D cyclins. Cell. 1993 May 7;73(3):499–511. doi: 10.1016/0092-8674(93)90137-f. [DOI] [PubMed] [Google Scholar]
  14. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  15. Ewen M. E., Sluss H. K., Sherr C. J., Matsushime H., Kato J., Livingston D. M. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell. 1993 May 7;73(3):487–497. doi: 10.1016/0092-8674(93)90136-e. [DOI] [PubMed] [Google Scholar]
  16. Guan K. L., Jenkins C. W., Li Y., Nichols M. A., Wu X., O'Keefe C. L., Matera A. G., Xiong Y. Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function. Genes Dev. 1994 Dec 15;8(24):2939–2952. doi: 10.1101/gad.8.24.2939. [DOI] [PubMed] [Google Scholar]
  17. Hannon G. J., Beach D. p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. Nature. 1994 Sep 15;371(6494):257–261. doi: 10.1038/371257a0. [DOI] [PubMed] [Google Scholar]
  18. Harbour J. W., Lai S. L., Whang-Peng J., Gazdar A. F., Minna J. D., Kaye F. J. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science. 1988 Jul 15;241(4863):353–357. doi: 10.1126/science.2838909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Helin K., Ed H. The retinoblastoma protein as a transcriptional repressor. Trends Cell Biol. 1993 Feb;3(2):43–46. doi: 10.1016/0962-8924(93)90150-y. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hirai H., Roussel M. F., Kato J. Y., Ashmun R. A., Sherr C. J. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol Cell Biol. 1995 May;15(5):2672–2681. doi: 10.1128/mcb.15.5.2672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horowitz J. M., Park S. H., Bogenmann E., Cheng J. C., Yandell D. W., Kaye F. J., Minna J. D., Dryja T. P., Weinberg R. A. Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2775–2779. doi: 10.1073/pnas.87.7.2775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kamb A., Gruis N. A., Weaver-Feldhaus J., Liu Q., Harshman K., Tavtigian S. V., Stockert E., Day R. S., 3rd, Johnson B. E., Skolnick M. H. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994 Apr 15;264(5157):436–440. doi: 10.1126/science.8153634. [DOI] [PubMed] [Google Scholar]
  24. Kato J., Matsushime H., Hiebert S. W., Ewen M. E., Sherr C. J. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev. 1993 Mar;7(3):331–342. doi: 10.1101/gad.7.3.331. [DOI] [PubMed] [Google Scholar]
  25. Kaye F. J., Kratzke R. A., Gerster J. L., Horowitz J. M. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6922–6926. doi: 10.1073/pnas.87.17.6922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. La Thangue N. B. DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci. 1994 Mar;19(3):108–114. doi: 10.1016/0968-0004(94)90202-x. [DOI] [PubMed] [Google Scholar]
  27. Li Y., Nichols M. A., Shay J. W., Xiong Y. Transcriptional repression of the D-type cyclin-dependent kinase inhibitor p16 by the retinoblastoma susceptibility gene product pRb. Cancer Res. 1994 Dec 1;54(23):6078–6082. [PubMed] [Google Scholar]
  28. 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]
  29. Matsushime H., Ewen M. E., Strom D. K., Kato J. Y., Hanks S. K., Roussel M. F., Sherr C. J. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell. 1992 Oct 16;71(2):323–334. doi: 10.1016/0092-8674(92)90360-o. [DOI] [PubMed] [Google Scholar]
  30. Matsushime H., Quelle D. E., Shurtleff S. A., Shibuya M., Sherr C. J., Kato J. Y. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol. 1994 Mar;14(3):2066–2076. doi: 10.1128/mcb.14.3.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Medema R. H., Herrera R. E., Lam F., Weinberg R. A. Growth suppression by p16ink4 requires functional retinoblastoma protein. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6289–6293. doi: 10.1073/pnas.92.14.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mihara K., Cao X. R., Yen A., Chandler S., Driscoll B., Murphree A. L., T'Ang A., Fung Y. K. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science. 1989 Dec 8;246(4935):1300–1303. doi: 10.1126/science.2588006. [DOI] [PubMed] [Google Scholar]
  33. Münger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol. 1989 Oct;63(10):4417–4421. doi: 10.1128/jvi.63.10.4417-4421.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Münger K., Werness B. A., Dyson N., Phelps W. C., Harlow E., Howley P. M. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 1989 Dec 20;8(13):4099–4105. doi: 10.1002/j.1460-2075.1989.tb08594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424–429. doi: 10.1126/science.1411535. [DOI] [PubMed] [Google Scholar]
  36. Nobori T., Miura K., Wu D. J., Lois A., Takabayashi K., Carson D. A. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature. 1994 Apr 21;368(6473):753–756. doi: 10.1038/368753a0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Okamoto A., Demetrick D. J., Spillare E. A., Hagiwara K., Hussain S. P., Bennett W. P., Forrester K., Gerwin B., Serrano M., Beach D. H. Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11045–11049. doi: 10.1073/pnas.91.23.11045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Otterson G. A., Kratzke R. A., Coxon A., Kim Y. W., Kaye F. J. Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB. Oncogene. 1994 Nov;9(11):3375–3378. [PubMed] [Google Scholar]
  40. Parry D., Bates S., Mann D. J., Peters G. Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J. 1995 Feb 1;14(3):503–511. doi: 10.1002/j.1460-2075.1995.tb07026.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Qin X. Q., Chittenden T., Livingston D. M., Kaelin W. G., Jr Identification of a growth suppression domain within the retinoblastoma gene product. Genes Dev. 1992 Jun;6(6):953–964. doi: 10.1101/gad.6.6.953. [DOI] [PubMed] [Google Scholar]
  42. Quelle D. E., Ashmun R. A., Hannon G. J., Rehberger P. A., Trono D., Richter K. H., Walker C., Beach D., Sherr C. J., Serrano M. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene. 1995 Aug 17;11(4):635–645. [PubMed] [Google Scholar]
  43. 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]
  44. Reuter S., Delius H., Kahn T., Hofmann B., zur Hausen H., Schwarz E. Characterization of a novel human papillomavirus DNA in the cervical carcinoma cell line ME180. J Virol. 1991 Oct;65(10):5564–5568. doi: 10.1128/jvi.65.10.5564-5568.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Scheffner M., Münger K., Byrne J. C., Howley P. M. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5523–5527. doi: 10.1073/pnas.88.13.5523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990 Dec 21;63(6):1129–1136. doi: 10.1016/0092-8674(90)90409-8. [DOI] [PubMed] [Google Scholar]
  47. Schlegel R., Phelps W. C., Zhang Y. L., Barbosa M. Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J. 1988 Oct;7(10):3181–3187. doi: 10.1002/j.1460-2075.1988.tb03185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schwarz E., Freese U. K., Gissmann L., Mayer W., Roggenbuck B., Stremlau A., zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature. 1985 Mar 7;314(6006):111–114. doi: 10.1038/314111a0. [DOI] [PubMed] [Google Scholar]
  49. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993 Dec 16;366(6456):704–707. doi: 10.1038/366704a0. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Spruck C. H., 3rd, Gonzalez-Zulueta M., Shibata A., Simoneau A. R., Lin M. F., Gonzales F., Tsai Y. C., Jones P. A. p16 gene in uncultured tumours. Nature. 1994 Jul 21;370(6486):183–184. doi: 10.1038/370183a0. [DOI] [PubMed] [Google Scholar]
  52. Tam S. W., Shay J. W., Pagano M. Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4. Cancer Res. 1994 Nov 15;54(22):5816–5820. [PubMed] [Google Scholar]
  53. 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]
  54. Weintraub S. J., Chow K. N., Luo R. X., Zhang S. H., He S., Dean D. C. Mechanism of active transcriptional repression by the retinoblastoma protein. Nature. 1995 Jun 29;375(6534):812–815. doi: 10.1038/375812a0. [DOI] [PubMed] [Google Scholar]
  55. Weintraub S. J., Prater C. A., Dean D. C. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 1992 Jul 16;358(6383):259–261. doi: 10.1038/358259a0. [DOI] [PubMed] [Google Scholar]
  56. Werness B. A., Levine A. J., Howley P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76–79. doi: 10.1126/science.2157286. [DOI] [PubMed] [Google Scholar]
  57. Wölfel T., Hauer M., Schneider J., Serrano M., Wölfel C., Klehmann-Hieb E., De Plaen E., Hankeln T., Meyer zum Büschenfelde K. H., Beach D. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science. 1995 Sep 1;269(5228):1281–1284. doi: 10.1126/science.7652577. [DOI] [PubMed] [Google Scholar]
  58. Yee C., Krishnan-Hewlett I., Baker C. C., Schlegel R., Howley P. M. Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines. Am J Pathol. 1985 Jun;119(3):361–366. [PMC free article] [PubMed] [Google Scholar]
  59. Yokota J., Akiyama T., Fung Y. K., Benedict W. F., Namba Y., Hanaoka M., Wada M., Terasaki T., Shimosato Y., Sugimura T. Altered expression of the retinoblastoma (RB) gene in small-cell carcinoma of the lung. Oncogene. 1988 Oct;3(4):471–475. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES