Intestinal Cell Cycle Regulation: Interactions of Cyclin D1, Cdk4, and p21cip1

R. Daniel Beauchamp, Hong Miao Sheng, Jin Yi Shao, E Aubrey Thompson, Tien C. Ko

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Objective The p21cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (ECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with Cyclin D/Cdk4 protein complexes were associated with cell growth arrest. Methods Density arrest was achieved by prolonged culture of IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DMA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. Results The EC-6 [3H]-thymidine incorporation was decreased 7.5-fold from day 1 of confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex.

Original languageEnglish (US)
Pages (from-to)620-628
Number of pages9
JournalAnnals of Surgery
Volume223
Issue number5
StatePublished - 1996
Externally publishedYes

Fingerprint

Cyclin D1
Cell Cycle
Cyclin-Dependent Kinase 4
Thymidine
Contact Inhibition
Growth
Messenger RNA
Phosphotransferases
Western Blotting
Epithelial Cells
Cyclin D
Proteins
Cyclin-Dependent Kinases
Antibodies
Cell Cycle Checkpoints
Immunoblotting
Northern Blotting
Polyacrylamide Gel Electrophoresis
Cell Culture Techniques
Cell Count

ASJC Scopus subject areas

  • Surgery

Cite this

Beauchamp, R. D., Sheng, H. M., Shao, J. Y., Thompson, E. A., & Ko, T. C. (1996). Intestinal Cell Cycle Regulation: Interactions of Cyclin D1, Cdk4, and p21cip1. Annals of Surgery, 223(5), 620-628.

Intestinal Cell Cycle Regulation : Interactions of Cyclin D1, Cdk4, and p21cip1. / Beauchamp, R. Daniel; Sheng, Hong Miao; Shao, Jin Yi; Thompson, E Aubrey; Ko, Tien C.

In: Annals of Surgery, Vol. 223, No. 5, 1996, p. 620-628.

Research output: Contribution to journalArticle

Beauchamp, RD, Sheng, HM, Shao, JY, Thompson, EA & Ko, TC 1996, 'Intestinal Cell Cycle Regulation: Interactions of Cyclin D1, Cdk4, and p21cip1', Annals of Surgery, vol. 223, no. 5, pp. 620-628.
Beauchamp, R. Daniel ; Sheng, Hong Miao ; Shao, Jin Yi ; Thompson, E Aubrey ; Ko, Tien C. / Intestinal Cell Cycle Regulation : Interactions of Cyclin D1, Cdk4, and p21cip1. In: Annals of Surgery. 1996 ; Vol. 223, No. 5. pp. 620-628.
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abstract = "Objective The p21cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (ECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with Cyclin D/Cdk4 protein complexes were associated with cell growth arrest. Methods Density arrest was achieved by prolonged culture of IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DMA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. Results The EC-6 [3H]-thymidine incorporation was decreased 7.5-fold from day 1 of confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9{\%} and 68.7{\%}, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170{\%} of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex.",
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T2 - Interactions of Cyclin D1, Cdk4, and p21cip1

AU - Beauchamp, R. Daniel

AU - Sheng, Hong Miao

AU - Shao, Jin Yi

AU - Thompson, E Aubrey

AU - Ko, Tien C.

PY - 1996

Y1 - 1996

N2 - Objective The p21cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (ECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with Cyclin D/Cdk4 protein complexes were associated with cell growth arrest. Methods Density arrest was achieved by prolonged culture of IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DMA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. Results The EC-6 [3H]-thymidine incorporation was decreased 7.5-fold from day 1 of confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex.

AB - Objective The p21cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (ECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with Cyclin D/Cdk4 protein complexes were associated with cell growth arrest. Methods Density arrest was achieved by prolonged culture of IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DMA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. Results The EC-6 [3H]-thymidine incorporation was decreased 7.5-fold from day 1 of confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex.

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