Abstract
The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21(Waf1/Cip1) in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15(INK4B)/p16(INK4A) that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-ras(V12)/c-myc-induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21(-/-); cdk4r24c MEFs expressing H-ras(V12) and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-ras(V12), and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-ras(V12)/c-myc-induced loss of contact inhibition. In addition, absence of pRb function renders H-ras(V12) + c-myc-transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein-deficient cells requires apoptotic suppression.
Highlights
Quiescent cells require exposure to mitogenic growth factors to enter the cell cycle and pass through the restriction point
We show for the first time the cooperativity between loss of INK4 family function and p21 in culture-induced senescence, pRb inactivation, and immortalization of mouse embryo fibroblasts (MEF)
We show that p21Waf1/Cip1 is robustly induced via a p53-dependent manner on explantation of MEFs into the standard tissue culture environment, but not when MEFs are grown in physiologic oxygen concentration
Summary
Quiescent cells require exposure to mitogenic growth factors to enter the cell cycle and pass through the restriction point. The key effectors promoting cell cycle entry and progression through the G1-S transition are the cyclin-dependent kinase (Cdk) 4/Cdk6cyclin D and Cdk2-cyclin E kinase complexes [1]. These kinase complexes phosphorylate pocket proteins pRb, p107, and p130, leading to release of the E2F family of transcriptional regulators and transcription of genes required for cell cycle progression and DNA synthesis. Cdk activity is negatively regulated by the INK4 (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) and CIP/ Kip (p21Waf1/Cip, p27Kip, and p57Kip2) family of inhibitors [2]. Induction of these proteins by stress, growth-inhibitory, or differentiation-inducing signals provides a means of halting cell cycle progression to allow for repair or promoting exit from the cell cycle
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