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.
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.
Rorke
E A
EA
Department of Environmental Health Sciences, Case Western Reserve University, Cleveland, Ohio, 44106, USA.
Zhang
D
D
Choo
C K
CK
Eckert
R L
RL
Jacobberger
J W
JW
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002584:Cervix Uteri / Q000166:cytology; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology / Q000821:virology; D005260:Female; D016193:G1 Phase / Q000187:drug effects* / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics; D030361:Papillomavirus Infections / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D025521:Tumor Suppressor Proteins; D014412:Tumor Virus Infections / Q000235:genetics
C493956:CDKN1A protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019927:Cyclin E; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C118860:E6 protein, Human papillomavirus type 6; D008869:Microtubule-Associated Proteins; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C062495:oncogene protein E7, Human papillomavirus type 6; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495911:CDK6 protein, human; D051357:Cyclin-Dependent Kinase 2; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases