Anchorage-Independent Growth of Pocket Protein-Deficient Murine Fibroblasts Requires Bypass of G₂ Arrest and Can Be Accomplished by Expression of TBX2

Abstract

Mouse embryonic fibroblasts (MEFs) deficient for pocket proteins (i.e., pRB/p107-, pRB/p130-, or pRB/p107/p130-deficient MEFs) have lost proper G₁ control and are refractory to Ras^V12-induced senescence. However, pocket protein-deficient MEFs expressing Ras^V12 were unable to exhibit anchorage-independent growth or to form tumors in nude mice. We show that depending on the level of pocket proteins, loss of adhesion induces G₁ and G₂ arrest, which could be alleviated by overexpression of the TBX2 oncogene. TBX2-induced transformation occurred only in the absence of pocket proteins and could be attributed to downregulation of the p53/p21^CIP1 pathway. Our results show that a balance between the pocket protein and p53 pathways determines the level of transformation of MEFs by regulating cyclin-dependent kinase activities. Since transformation of human fibroblasts also requires ablation of both pathways, our results imply that the mechanisms underlying transformation of human and mouse cells are not as different as previously claimed.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank George Q. Daley for the pEYK-MCF7 library, Daniel Peeper and Theo van Laar for help with recovery of cDNA vectors, Anja van der Wal for generation of MEFs, Tanja van Harn for kinase assays, Elly Delzenne-Goette and Sjaak Greven for nude mice injections and tumor analyses, Lauran Oomen and Lenny Brocks for help with digital microscopy, and Frank van Diepen and Anita Pfauth for help with FACS. We are grateful to Marieke Aarts, Jan-Hermen Dannenberg, Tanja van Harn, and Rob Wolthuis for helpful discussions and critically reading the manuscript.

This work was financially supported by the Dutch Cancer Society (NKI 2002-2634).