hMad4, c-Myc endogenous inhibitor, induces a replicative senescence-like state when overexpressed in human fibroblasts.

Mad family proteins have an antagonistic action on Myc-dependent cell proliferation and transformation. We isolated a human cDNA clone, human Mad4 (hMad4), encoding a polypeptide of 209 amino acid residues, exhibiting 90% identity with mouse Mad4. Northern blot analysis shows that hMad4 probe hybridizes to a 3.8 kb message; its expression is highest in quiescent human WI38 fibroblasts. Among tissues, hMad4 mRNA is most abundant in brain, lung, and muscle. Consistent with other members of the Mad family, hMad4 can repress the transactivation activity of Myc/Max heterodimers on an E-box chloramphenicol acteyl transferase (CAT) reporter plasmid; inhibition of both proliferation and clonogenic formation of hMad4-infected cells correlates with the in vitro reporter repression. Moreover, infection of young human fibroblasts induces a replicative senescence-like state. This phenotype was accompanied by s-beta-galactosidase and PAI-1 expression. These results suggest that hMad4 might be an important regulator of replicative senescence in human cells.

Marcotte R ，Qian JF ，Chen J ，Wang E ... -  《JOURNAL OF CELLULAR BIOCHEMISTRY》

c-Myc creates an activation loop by transcriptionally repressing its own functional inhibitor, hMad4, in young fibroblasts, a loop lost in replicatively senescent fibroblasts.

c-Myc transcriptional activity in cells is dampened by the Mad family of transcriptional repressors. The expression of one member, hMad4, is increased in growth-arrested states such as quiescence or replicative senescence; hMad4 mRNA levels in replicatively senescent fibroblasts are about twice those seen in their young contact-inhibited quiescent counterparts. Moreover, the repression of hMad4 transcription following serum stimulation observed in quiescent young fibroblasts is lost in senescent cells. This loss results in persistent expression of hMad4, which leads to an inability to switch from an hMad4/Max complex to a c-Myc/Max complex on selected c-Myc target genes following serum stimulation. We have located an initiator element (Inr), a candidate for Miz-1 binding, in the hMad4 promoter. In reporter assays, Miz-1 enhances reporter GFP expression; this enhancement is inhibited by co-expressing c-Myc. Thus hMad4, as does its murine counterpart, contains the Inr element through which Miz-1 activates its expression; but this action is inhibited in the presence of c-Myc. This inhibition may explain the down-regulation of hMad4, corresponding to the up-regulation of c-Myc, in young serum-starved quiescent fibroblasts upon serum stimulation. However, this reciprocal change does not occur in replicatively senescent fibroblasts upon serum stimulation; instead, hMad4 persists in the presence of high levels of c-Myc activation. Our results suggest that: (1) replicative senescence-specific factors may block c-Myc inhibition of Miz-1 activation of hMad4 expression; and (2) the continual presence of hMad4 protein may transcriptionally repress selected c-Myc target genes, whose functions are key to the signaling pathways leading to apoptosis inhibition and permanent exit of cell cycle traverse in normal human fibroblasts.

Marcotte R ，Chen JM ，Huard S ，Wang E ... -  《JOURNAL OF CELLULAR BIOCHEMISTRY》

Kinetics of myc-max-mad gene expression during hepatocyte proliferation in vivo: Differential regulation of mad family and stress-mediated induction of c-myc.

Mad proteins (Mad1, Mxi1, Mad3, Mad4, Mnt/Rox) are biochemical and biological antagonists of c-Myc oncoprotein. Mad-Max dimers repress the transcription of the same target genes activated by Myc-Max dimers. Despite the critical role of Max and Mad proteins as modulators of c-Myc functions, there are no comparative data on their regulation in vivo. We carried out a systematic analysis of c-myc, max, and mad family expression in a model of synchronized cell proliferation in vivo in adult tissues, that is, rat hepatocytes after partial hepatectomy. We confirmed the previously reported early peak of c-myc expression after hepatectomy but we show that it did not correlate with hepatocyte proliferation as it also occurred in sham-operated animals as a result of surgical stresses. A second peak of c-myc expression was observed later, at the time of the wave of DNA synthesis. No such expression was detected in sham-operated rat quiescent hepatocytes. max expression increased around 4-16 h after hepatectomy, before the peaks of c-myc and DNA synthesis. mxi1 and mad4 were slightly downregulated during liver regeneration. mnt/rox expression did not change. These expression patterns suggest a role of Myc-Max for efficient mitogenic response of hepatocytes. We also analyzed the effects of Myc and Max ectopic expression on the clonogenic growth of the rat hepatoma cells. Expression of c-Myc and Max increased clonogenic growth, whereas the reduction of c-Myc levels by an antisense vector decreased growth. The results suggest nonredundant roles for mad genes in hepatocyte proliferation and point to c-Myc as a putative target for anticancer therapy of liver cancer.

Mauleon I ，Lombard MN ，Muñoz-Alonso MJ ，Cañelles M ，Leon J ... -  《MOLECULAR CARCINOGENESIS》

Microarray analysis of E-box binding-related gene expression in young and replicatively senescent human fibroblasts.

Semov A ，Marcotte R ，Semova N ，Ye X ，Wang E ... -  《-》

Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock.

Munro J ，Barr NI ，Ireland H ，Morrison V ，Parkinson EK ... -  《EXPERIMENTAL CELL RESEARCH》