A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence.

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 microM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 microM H2O2 stress or five repeated 30 microM t-BHP stresses along the same PD, respectively a 322 +/- 55 and 380 +/- 129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

The recombination-activating gene 1 of Pleurodeles waltl (urodele amphibian) is transcribed in lymphoid tissues and in the central nervous system.

The recombination-activating gene 1 (RAG1) product is required for the somatic rearrangement of immunoglobulin and T-cell receptor genes. We cloned and sequenced the large continuous open reading frame coding for the salamander Pleurodeles waltl RAG1 protein. Semi-quantitative RT-PCR experiments were performed to quantify the expression of RAG1 in different tissues. The strongest signal was observed in the thymus of juvenile animals, confirming the primary lymphoid nature of that organ. Weaker expression was observed in the spleen, brain, and eyes of adults. Signals in these tissues represented 5.5%, 4.6%, and 2.0%, respectively, of the signal detected in the thymus. Expression in brain was confirmed by in situ hybridization. Similarly, low amounts of RAG1 transcripts were previously detected in the mouse brain. Moreover, the transcription of RAG1 begins as early as the neurula stages of development. These data suggest that the RAG1 protein could play a role in the central nervous system of vertebrates.

Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts.

Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H(2)O(2), with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-beta1 (TGF-beta1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated beta-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-beta1 or its receptor (TGF-beta RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated beta-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-beta1 in stressful conditions, leading to SIPS and overexpression of these four genes.

Cell cycle regulation in H(2)O(2)-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virus E6 and E7 proteins.

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.

Consideration of heteroduplexes and homoduplexes for the quantification by competitive PCR of human mitochondrial DNA deletions with ageing of tissues and cells.

The purpose of this work was first to construct two internal standards for human mitochondrial DNA mt DNA corresponding respectively to the fragment resulting from the 4,977 bp common deletion (H2del) and a fragment which was never reported to be deleted (H1). Secondly, we wished to consider the possible effect of annealing between the target and corresponding internal standard which forms heteroduplexes. These experiments show that the correction of the number of copies found by competitive PCR by considering the percentage of heteroduplexes allows a more accurate quantification of the number of target copies present in mt DNA samples. The design of internal standards specific to the fragment resulting from other deletions could also help a more accurate quantification of the frequency of other mt DNA deletions as well, and reconsideration of the role of mt DNA deletions in ageing.

Stress-induced premature senescence as alternative toxicological method for testing the long-term effects of molecules under development in the industry.

No alternative in vitro method exists for detecting the potential long-term genotoxic effects of molecules at subcytotoxic concentrations, in terms of days and weeks after exposure(s) to the molecule tested. A theoretical model of cellular senescence led to the concept that subcytotoxic stresses under any molecules at subcytotoxic doses, such as molecules under development in the pharmaceutical, cosmetics and food industry, might lead human fibroblasts into a state closely related to in vitro senescence. This concept was then experimentally confirmed in vitro: many biomarkers of replicative senescence of human fibroblasts were found 72 h after their exposure to various kinds of stressors used at non-cytotoxic concentrations. This phenomenon has been termed stress-induced premature senescence (SIPS). Moreover, proteomics studies have revealed that, besides their effects on the appearance of the biomarkers of senescence, sublethal stresses under a variety of stressors also lead to long-term specific changes in the expression level of proteins which are stress-specific. These changes have been coined the molecular scars of stress. The proteins corresponding to these molecular scars may be identified using the latest developments in mass spectrometry. This model of stress-induced premature senescence may be applied to the toxicological sciences when testing for the potential irreversible long-term effects of molecules on the cell fate.