Biomedical subjects

Richard Marcotte

Publications and source records attributed to Richard Marcotte.

7 recordsLinked to original sources

PubMed2005

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.

Base Sequence↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

Alterations in TNF- and IL-related gene expression in space-flown WI38 human fibroblasts.

Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long-term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor- (TNF) or interleukin- (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS-93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor-associated proteins, NSMAF and PTPN13; three TNF-inducible genes, ABC50, PTX3, and SCYA13; TNF-alpha converting enzyme, IL-1 receptor antagonist, and IL-15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.

Cell Line↗

PubMed2002

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

An E-box (CACGTG) designer microarray was developed to monitor a group of genes whose expressions share a particular regulatory mode. Sensitivity and specificity of microarray hybridization, as well as variability of microarray data, were evaluated. This designer microarray was used to generate expression profiles of E-box binding-related genes in WI-38 fibroblast cultures at three different growth states: low-passage replicating, low-passage contact-inhibited quiescent, and replicatively senescent. Microarray gene screening reveals that quiescent and senescent cells, in comparison with replicating ones, are characterized by downregulation of Pam, a protein associated with c-Myc, and upregulation of Mad family genes, Max dimerization proteins. Moreover, quiescence and senescence can be distinguished by increased expression of Irlb, c-Myc transcription factor, and Miz-1, c-Myc-interacting Zn finger protein 1, only in the former state. Senescence is characterized by downregulation of Id4, inhibitor of DNA binding 4, and Mitf, microphthalmia-associated transcription factor, in comparison with young replicating and quiescent states. Differential expression of genes detected by microarray hybridization was independently confirmed by reverse transcription polymerase chain reaction technique. Alterations in the expression of E-box-binding transcription factors and c-Myc-binding proteins demonstrate the importance of these genes in establishing the contact-inhibited quiescent or senescent phenotypes.

Cell Division↗

PubMed2002

Replicative senescence revisited.

Forty years after its discovery, replicative senescence remains a rich source of information about cell-cycle regulation and the progression from a normal to a transformed phenotype. Effectors of this growth-arrested state are being discovered at a great pace. This review discusses the latest findings on the players responsible for establishing replicative senescence, as well as the associated telomere shortening.

Animals↗

PubMed2001

Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis.

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1alpha, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1alpha to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1alpha in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1alpha, whereas cells transfected with antisense eEF1A-1/EF-1alpha are protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1alpha and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.

Adenoviridae↗

PubMedDate not supplied

Senescent fibroblasts resist apoptosis by downregulating caspase-3.

In replicative senescence, cells undergo permanent exit from cell cycle traverse; this is traditionally thought to occur at the end of a culture's in vitro life span, after serial passaging. In general, the checkpoint for replicative senescence is found at the G(1)/S border, controlled by the modulation of a battery of proteins, typified by gaining inhibitors of cell cycle traverse, such as cyclin-dependent kinases or RB hyperphosphorylation, and losing pro-proliferation gene expressions such as c-fos, c-myc, and a cadre of proliferation-dependent kinases. Here, we present evidence that replicatively senescent fibroblasts are resistant to apoptotic death, associated with a lack of key enzyme activities, caspase-3 being the chief executioner. This observation, coupled with our earlier report that senescent fibroblasts maintain persistently high levels of pro-survival factor Bcl-2, suggests that the molecular signaling program present in fibroblasts at the end of their in vitro life span may not only cater to the state of permanent exit from cell cycle traverse, but also dictate an inability to commit cellular suicide. Future experiments will reveal whether replicatively senescent fibroblasts that can neither proliferate nor die contribute to organismic aging, and whether their accumulation over time in tissue becomes detrimental to the normal aging process.

Apoptosis↗