c-Rel Arrests the Proliferation of HeLa Cells and Affects Critical Regulators of the G₁/S-Phase Transition

Abstract

A tetracycline-regulated system was used to characterize the effects of c-Rel on cell proliferation. The expression of c-Rel in HeLa cells led to growth arrest at the G 1 /S-phase transition, which correlated with its nuclear localization and the induction of endogenous IB␣ expression. These changes were accompanied by a decrease in E2F DNA binding and the accumulation of the hypophosphorylated form of Rb. In vitro kinase assays showed a reduction in Cdk2 kinase activity that correlated with elevated levels of p21 WAF1 Cdk inhibitor and p53 tumor suppressor protein. While the steady-state levels of WAF1 transcripts were increased, pulsechase analysis revealed a sharp increase in p53 protein stability. Importantly, the deletion of the C-terminal transactivation domains of c-Rel abolished these effects. Together, these studies demonstrate that c-Rel can affect cell cycle control and suggest the involvement of the p21 WAF1 and p53 cell cycle regulators.c-Rel is a member of the Rel/NF-B family of transcription factors, which also includes the retroviral v-Rel oncoprotein, the cellular RelA, RelB, p105 (NF-B1), and p100 (NF-B2) precursor proteins, Xenopus X-Rel1, and the Drosophila Dorsal and Dif factors (reviewed in references 5 and 84). Rel proteins have homologous N-terminal domains that control their nuclear localization, dimer formation, and binding to DNA. Their distinct C-termini participate in transcriptional activation or in the control of Rel activity. These proteins also have similar modes of regulation. The prototypic NF-B factor is a heterodimer of p50 (NF-B1) and p65 (RelA) that is retained in an inactive cytoplasmic form by one of several IB inhibitors in unstimulated cells. In vivo activation of these complexes by a variety of stimuli results in the phosphorylation and rapid degradation of IB through the ubiquitin-proteasome pathway (reviewed in reference 82). The nuclear entry of active NF-B complexes is followed by their binding to B DNA sites and the activation of gene expression (reviewed in references 4 and 79).Several members of the Rel/NF-B family have been implicated in the control of cell proliferation and differentiation. For instance, the temporal expression of c-Rel correlates with lymphoid cell differentiation (14,15,18,40,55,63). However, their effects have been the object of conflicting observations. While the DNA-binding activity of NF-B factors is increased in response to proliferative signals (6, 67), the induction of these factors was also implicated in cell growth arrest and differentiation (26,35). Rel/NF-B factors have also been shown to regulate cell response to a variety of stimuli ranging from stress to infection (5,7,36,66,79) and were recently implicated in the promotion or the inhibition of apoptosis (1,8,9,11,37,52,56,65,83,85,87,91,94). The rearrangement and amplification of several rel and nf-b genes in hematopoietic and solid tumors are consistent with their participation in the control of cell growth and survival (reviewed in references 31 and 57).In this study, we use...

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