Defects in TGF-beta signaling overcome senescence of mouse keratinocytes expressing v-Ha-ras.
Previous studies have shown that TGFbeta1 expression is upregulated in mouse keratinocytes infected with a v-rasHa retrovirus, although the functional significance of this has not been clear. Here we show that v-rasHa retrovirus transduced primary mouse keratinocytes undergo hyperproliferation followed by a TGFbeta1 dependent G1 growth arrest and senescence. The growth arrest is accompanied by a 15-fold increase in total TGFbeta1 secreted and a fourfold increase in secreted active TGFbeta1. When cultured in the presence of a neutralizing antibody to TGFbeta1, the senescence response is suppressed. Levels of the TGFbeta1 target p15ink4b increase during senescence as does association of this kinase inhibitor with cyclinD/cdk4 complexes. However, p16ink4a, p53 and p19ARF expression also increase during senescence. Genetic analysis shows that TGFbeta1 null and dominant negative TbetaBRII expressing v-rasHa keratinocytes resist the G1 growth arrest and do not senescence. This resistance is associated with low expression of p15ink4b and p16ink4a, constitutive Rb phosphorylation and high levels of cdk4 and cdk2 kinase activity. In contrast, inactivation of TGFbetabeta1 secretion or response does not block the induction of p53 and p19ARF, but the level of p21waf1, a p53 target gene, is reduced in cyclin D/cdk4 and cyclin E/cdk2 complexes. Thus, although multiple senescence pathways are activated in response to a ras oncogene, inactivation of TGFbeta1 secretion or response is sufficient to block the senescence program. Since v-rasHa transduced TGFbeta1-/- keratinocytes form squamous cell carcinomas following skin grafting, these results suggest that in mouse keratinocytes, defects in TGFbeta1 signaling accelerate malignant progression by overcoming oncogene induced replicative senescence.
Tremain
R
R
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892, USA.
Marko
M
M
Kinnimulki
V
V
Ueno
H
H
Bottinger
E
E
Glick
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D016922:Cellular Senescence / Q000502:physiology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D056741:Cyclin D; D051358:Cyclin-Dependent Kinase 4; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D016193:G1 Phase; D005786:Gene Expression Regulation; D016158:Genes, p53; D011905:Genes, ras; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D015689:Oncogene Protein p21(ras) / Q000172:deficiency / Q000502:physiology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D025521:Tumor Suppressor Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C493968:Cdkn2b protein, mouse; D018797:Cell Cycle Proteins; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D056741:Cyclin D; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016213:Cyclins; D046911:Macromolecular Substances; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016212:Transforming Growth Factor beta; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D015689:Oncogene Protein p21(ras)