Abstract
Previous studies have shown that TGFβ1 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 TGFβ1 dependent G1 growth arrest and senescence. The growth arrest is accompanied by a 15-fold increase in total TGFβ1 secreted and a fourfold increase in secreted active TGFβ1. When cultured in the presence of a neutralizing antibody to TGFβ1, the senescence response is suppressed. Levels of the TGFβ1 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 TGFβ1 null and dominant negative TβRII 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 TGFβ1 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 TGFβ1 secretion or response is sufficient to block the senescence program. Since v-rasHa transduced TGFβ1−/− keratinocytes form squamous cell carcinomas following skin grafting, these results suggest that in mouse keratinocytes, defects in TGFβ1 signaling accelerate malignant progression by overcoming oncogene induced replicative senescence.
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Acknowledgements
The authors thank Chris Lam for technical assistance, Brett Cheney for maintenance of transgenic and knockout mice and Dr Luowei Li for Adβgal, and Dr Fernandez-Salas for advice on purification of recombinant adenoviruses. We also thank Dr Dana Ramujak and Dr Beverly Mock for advice on the immuncomplex kinase assays, and Dr Stuart Yuspa and members of the Yuspa laboratory for helpful discussions and critical reading of the manuscript.
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Tremain, R., Marko, M., Kinnimulki, V. et al. Defects in TGFβ signaling overcome senescence of mouse keratinocytes expressing v-rasHa. Oncogene 19, 1698–1709 (2000). https://doi.org/10.1038/sj.onc.1203471
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DOI: https://doi.org/10.1038/sj.onc.1203471
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