TGF-beta-mediated cell cycle arrest of HPV16-immortalized human ectocervical cells correlates with decreased E6/E7 mRNA and increased p53 and p21(WAF1) expression
Ea. Rorke 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(WAF1) expression, EXP CELL RE, 259(1), 2000, pp. 149-157
Transforming growth factor beta (TGF-beta) suppresses proliferation and pot
entiates apoptosis of HPV16-immortalized human cervical epithelial cells (E
CE16-1). Exposure of ECE16-1 to TGF-beta 1 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 acti
ve, since TGF-beta 1 treatment increased the activity of a p53 transcriptio
nal response element in a luciferase reporter plasmid. Additionally, the pr
oteins of the p53-regulated genes, p21(waf1), mdm2, and fax, were increased
with similar time and dose responses. We did not observe consistent change
s in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kipl), p16
(INK4a) or RNA levels of p15(INK4b). activity Of CDK4 or 6, measured by pho
sphorylation of an Rb fragment, remained constant during the response perio
d; however, activity of CDK2 (phosphorylation of histone H1) decreased. Con
cordantly, 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; howev
er, significant changes in the E7 protein were not observed, while increase
d levels of Rb immunoprecipitated with anti-E7 antibodies were observed. Th
ese data are consistent with the following model. In ECE16-1 cells, there e
xists a fine balance between inhibitory levels of p53 and Rb and the antago
nists, E6 and E7. TG;TGF-beta 1 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 cycl
e inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex
that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphoryla
ted state, resulting in G1 arrest, presumably by binding E2F transcription
factors. (C) 2000 Academic Press.