Role of Prion protein in premature senescence of human fibroblasts.

Prion protein (PrP) is essentially known for its capacity to induce neurodegenerative prion diseases in mammals caused by a conformational change in its normal cellular isoform (PrPC) into an infectious and disease-associated misfolded form, called scrapie isoform (PrPSc). Although its sequence is highly conserved, less information is available on its physiological role under normal conditions. However, increasing evidence supports a role for PrPC in the cellular response to oxidative stress. In the present study, a new link between PrP and senescence is highlighted. The role of PrP in premature senescence induced by copper was investigated. WI-38 human fibroblasts were incubated with copper sulfate (CuSO4) to trigger premature senescence. This induced an increase of PrP mRNA level, an increase of protein abundance of the normal form of PrP and a nuclear localization of the protein. Knockdown of PrP expression using specific small interfering RNA (siRNA) gave rise to appearance of several biomarkers of senescence as a senescent morphology, an increase of senescence associated ß-galactosidase activity and a decrease of the cellular proliferative potential. Overall these data suggest that PrP protects cells against premature senescence induced by copper.

Role of p38MAPK and oxidative stress in copper-induced senescence.

In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38(MAPK) regulated the appearance of premature senescence.

Copper(II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response.

The potential toxic effects of two types of copper(II) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu(2+) released in cell culture medium suggested that Cu(2+) cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.

Differential toxicity of copper (II) oxide nanoparticles of similar hydrodynamic diameter on human differentiated intestinal Caco-2 cell monolayers is correlated in part to copper release and shape.

The potential toxic effects of copper oxide (CuO) nanoparticles (NPs) were studied on differentiated Caco-2 cell monolayers, a classical in vitro model of human small intestine epithelium. Two types of CuO NPs, with different specific surface area, different sizes as raw material but the same hydrodynamic diameter in suspension, differentially disturbed the monolayer integrity, were cytotoxic and triggered an increase of the abundance of several transcripts coding for pro-inflammatory cytokines and chemokines. Specific surface area was not a major variable explaining the increased toxicity when intestinal epithelium is exposed to rod-shaped CuO NPs, compared with spherical CuO NPs. The results suggest that release of Cu(II) cations and shape of these CuO NPs are likely to be implicated in the toxicity of these CuO NPs.

p38(MAPK) in the senescence of human and murine fibroblasts.

Oncogenic and environmental stresses, such as reactive oxygen species, UV radiation etc, can induce premature cellular senescence without critical telomere shortening. The role of the Ras/Raf/ERK signal transduction cascade in this process has been previously established, but recent evidence also indicates a critical role of the p38 MAP kinases pathway. Oncogenic and environmental stresses impinge upon the p38(MAPK) pathway, suggesting a major role of this pathway in senescence induced by stresses. Prematurely senescent cells are most likely to appear in several age-relatedpathologies associated with a stressful environment and/or the release of pro-inflammatory cytokines.

Repeated exposures to UVB induce differentiation rather than senescence of human keratinocytes lacking p16(INK-4A).

Skin cancers and extrinsic aging are delayed consequences of cumulative UV radiation insults. Exposure of human keratinocytes to UVB has been previously shown to trigger premature senescence. In order to explore the involvement of the cyclin-dependent kinase inhibitor p16(INK-4a) in UVB-induced premature senescence, we developed an original model of repeated sublethal exposures of human keratinocytes deficient in p16(INK-4a). We did not observe any significant increase of senescence-associated beta-galactosidase activity positive cells following UVB exposure in this cell line in contrast to primary keratinocytes, suggesting a role for p16(INK-4a) in UVB-induced senescence. However, we detected sustained DNA damage, prolonged cell cycle arrest, and induction of markers of epidermal differentiation like involucrin and filaggrin as consequences of the repeated exposures. Keratinocytes exposed to the same dose of UVB in a single exposure died. Furthermore, the abundance of the keratins 6, 16 and 17 was increased in keratinocytes exposed repeatedly to UVB suggesting an alternative differentiation. This model allows the induction of a state of differentiation observed in vivo with differentiation uncoupled from premature senescence.

Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts.

In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.

Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts.

Repeated exposures to sublethal concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of WI-38 human diploid fibroblasts. We found 16 replicative senescence-related genes with similar alterations in expression level in replicative senescence and two models of stress-induced premature senescence. Among these genes was IGFBP-3. Using a siRNA approach, we showed that IGFBP-3 regulates the appearance of several biomarkers of senescence after repeated exposures of WI-38 fibroblasts to tert-butylhydroperoxide and ethanol.

Heme oxygenase-1 and interleukin-11 are overexpressed in stress-induced premature senescence of human WI-38 fibroblasts induced by tert-butylhydroperoxide and ethanol.

Acute repeated exposures to subcytotoxic concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of human diploid fibroblasts. In the present work we found an increased mRNA and protein level of interleukin-11 and heme oxygenase-1 in premature senescence of WI-38 human diploid foetal lung fibroblasts induced by both tert-butylhydroperoxide and ethanol. We tested whether interleukin-11 and heme oxygenase-1 could protect against tert-butylhydroperoxide- or ethanol-induced premature senescence when stable overexpression was established using a retroviral vector-based transduction. No protective effect was found against the decrease of the proliferative potential, the increase of the proportion of senescence-associated ss-galactosidase positive cells and the increase of the mRNA levels of six senescence-associated genes.