Arrest of G₁-S Progression by the p53-Inducible Gene PC3 Is Rb Dependent and Relies on the Inhibition of Cyclin D1 Transcription

Abstract

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G₁ arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G₁ arrest was Rb dependent. Furthermore, (i) the arrest of G₁-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G₁-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.

ACKNOWLEDGMENTS

We are grateful to J. A. DeCaprio for the gift of Rb^−/− 3T3 cells; to R. Weinberg for primary Rb^+/+ and Rb^−/− MEFs and for the gift of pRcCMV-cycA, pRcCMV-cycD1, pRcCMV-cycD3, and pRcCMV-cycE; to P. Sicinski and R. Weinberg for the gift of primary cyclin D1^+/+ and cyclin D1^−/− MEFs; to L. Zhu and E. Harlow for the gift of pCMV-CD20 and of CMVcdc2 constructs; to M. Ewen and D. M. Livingston for the gift of pRcCMV-CDK2 and pRcCMV-CDK4; to K. Poliak, D. Morgan, and C. Sherr for the gift of baculoviruses expressing cyclin A, cyclin B1, cdc2, CDK2, cyclin D1, and CDK4; to C. Sherr for the gift of the Flag-cyclin D1 construct; and to D. Beach and J. Massaguè for the gifts of pXp16 and pCMX-p27, respectively. We thank L. Baron for outstanding technical assistance. We are all very grateful to Francesca de Santa for her qualified help with the in vivo kinase experiments, given in a critical moment.

We gratefully acknowledge the support of F.T. by Donazione Bianchi and the help of A. Cesari who made it possible. This work was also carried out under a research contract with N.E.F.A.C., Pomezia, Italy, within the Neurobiological Systems National Research Plan of the Ministero dell' Università e della Ricerca Scientifica e Tecnologica.