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TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression.
Exp Cell Res. 2000 Aug 25; 259(1):149-57.EC

Abstract

Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors.

Authors+Show Affiliations

Department of Environmental Health Sciences, Case Western Reserve University, Cleveland, Ohio, 44106, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10942587

Citation

Rorke, E A., et al. "TGF-beta-mediated Cell Cycle Arrest of HPV16-immortalized Human Ectocervical Cells Correlates With Decreased E6/E7 mRNA and Increased P53 and p21(WAF-1) Expression." Experimental Cell Research, vol. 259, no. 1, 2000, pp. 149-57.
Rorke EA, Zhang D, Choo CK, et al. TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression. Exp Cell Res. 2000;259(1):149-57.
Rorke, E. A., Zhang, D., Choo, C. K., Eckert, R. L., & Jacobberger, J. W. (2000). TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression. Experimental Cell Research, 259(1), 149-57.
Rorke EA, et al. TGF-beta-mediated Cell Cycle Arrest of HPV16-immortalized Human Ectocervical Cells Correlates With Decreased E6/E7 mRNA and Increased P53 and p21(WAF-1) Expression. Exp Cell Res. 2000 Aug 25;259(1):149-57. PubMed PMID: 10942587.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF-1) expression. AU - Rorke,E A, AU - Zhang,D, AU - Choo,C K, AU - Eckert,R L, AU - Jacobberger,J W, PY - 2000/8/16/pubmed PY - 2000/9/23/medline PY - 2000/8/16/entrez SP - 149 EP - 57 JF - Experimental cell research JO - Exp Cell Res VL - 259 IS - 1 N2 - Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors. SN - 0014-4827 UR - https://www.unboundmedicine.com/medline/citation/10942587/TGF_beta_mediated_cell_cycle_arrest_of_HPV16_immortalized_human_ectocervical_cells_correlates_with_decreased_E6/E7_mRNA_and_increased_p53_and_p21_WAF_1__expression_ DB - PRIME DP - Unbound Medicine ER -