TGF-β-Mediated Cell Cycle Arrest of HPV16-Immortalized Human Ectocervical Cells Correlates with Decreased E6/E7 mRNA and Increased p53 and p21WAF-1 Expression

• Published in: Experimental cell research 2000-08, Vol.259 (1), p.149-157
• Main Authors: Rorke, Ellen A., Zhang, Desheng, Choo, C.K., Eckert, Richard L., Jacobberger, James W.
• Format: Article
• Language: English
• Subjects: carcinogenesis; cell cycle; cell proliferation; cell regulation; cervix; cyclin-dependent kinase; retinoblastoma protein; signaling; viral transformation
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.2000.4953

Transforming growth factor β (TGF-β) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-β1 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-β1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21WAF1, 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, p27Kip1, p16INK4a, or RNA levels of p15INK4b. 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 p21WAF1 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-β1 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, p21WAF1. 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.