Mojgan Djavaheri-Mergny

Oxidized low density lipoprotein induces activation of the transcription factor NFκB in fibroblasts, endothelial and smooth muscle cells

The effect of cupric ions or endothelial cell‐oxidized LDL on the transcription factor NFκB activation was investigated by electrophoretic mobility shift assay. Oxidized LDL induced NFkB activation in fibroblasts, endothelial and smooth muscle cells. The extent of NFκB activation was proportional to the degree of LDL oxidation, as assessed by the lipid peroxidation product and the conjugated diene level. A similar activation was observed with the lipid extract of coopper‐oxidized LDL, which indicates that lipid peroxidation products are involved in the activation mechanism. Furthermore, α‐tocopherol, a lipophilic free radical scavenger, partially inhibited the stimulatory effect of Cu2+‐oxidized LDL. Since NFκB is considered as an oxidative stress‐responsive transcription factor, our study supports the evidence that the stress induced by oxidized LDL causes NFκB activation in different cell types, and that this effect can be ascribed to the lipid peroxidation products.

Ultraviolet‐A induces activation of AP‐1 in cultured human keratinocytes

UV‐A irradiation induces a time‐dependent activation of AP‐1 in NCTC 2544 human keratinocytes. 4 h after irradiation, a 2–3‐fold increase in AP‐1 activity is observed in human keratinocytes and fibroblasts. Activation is still detectable 24 h later. The UV‐A induced AP‐1 binding complex is shown to contain c‐Fos and c‐Jun proteins. Lipophilic vitamin E impedes UV‐A induced lipid peroxidation but does not prevent AP‐1 activation which is inhibited by N‐acetylcysteine, a hydrophilic antioxidant. This finding suggests that UV‐A‐dependent AP‐1 activation is sensitive to the cellular redox state but is not related to membrane lipid peroxidation.

Copper and cell-oxidized low-density lipoprotein induces activator protein 1 in fibroblasts, endothelial and smooth muscle cells

The effect of cupric ion‐ or endothelial cell‐oxidized low‐density lipoproteins (LDL) on transcription factor AP1 activation was investigated by electrophoretic mobility shift assay. Both oxidized LDL induced AP1 activation in fibroblasts, endothelial and smooth muscle cells. This phenomenon was also observed in the presence of cycloheximide. α‐Tocopherol, a lipophilic free radical scavenger, and N‐acetylcysteine, an hydrophilic antioxidant, partially inhibited the stimulatory effect of Cu2+‐oxidized LDL. LDL modified by the mixture of the oxygen radicals OH· and O2·−, which generated lipid peroxidation products, also initiated AP1 activation, whereas LDL modified by OH· alone, which did not lead to marked LDL lipid peroxidation, was ineffective. Thus, lipid peroxidation products seem at least partially involved in the activation mechanism. Since AP1 activity is essential for the regulation of genes involved in cell growth and differentiation, our study suggests that the oxidative stress induced by oxidized LDL might be related to the fibroproliferative response observed in the atherosclerotic plaque.

Unraveling the relationship between structure and stabilization of triarylpyridines as G-quadruplex binding ligands

A series of novel 2,4,6-triarylpyridines have been synthesized and their interactions with intramolecular G-quadruplexes have been measured by Förster Resonance Energy Transfer (FRET) melting and Fluorescent Intercalator Displacement (FID) assays. A few of these compounds exhibit stabilization of G4-DNA that is comparable to other benchmark G4-DNA ligands with fair to excellent G4-DNA vs. duplex selectivity and significant cytotoxicity towards HeLa cells. The nature of the 4-aryl substituents along with side chain length governs the G4-DNA stabilization ability of the compounds. In addition, we demonstrate that there is a strong correlation between the ability of the compounds to stabilize the same G4-DNA sequence in K(+) and Na(+) conditions and a strong correlation between the ability of the compounds to stabilize different G4-DNA sequences in K(+) or Na(+) buffer.

UV-A-induced decrease in nuclear factor-kappaB activity in human keratinocytes.

Previous reports have demonstrated an increase in nuclear factor-kappaB (NF-kappaB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320-400 nm) on NF-kappaB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-kappaB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-kappaB inducers. Moreover, UV-A radiation induces a decrease in NF-kappaB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IkappaBalpha (IkappaB is the NF-kappaB inhibitor), which is closely associated with NF-kappaB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-kappaB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-kappaB activity in mammalian cells, probably through degradation of NF-kappaB protein subunits. These findings suggest that UV-A could modulate the NF-kappaB-dependent gene expression.

EARLY ALTERATIONS OF ACTIN IN CULTURED HUMAN KERATINOCYTES AND FIBROBLASTS EXPOSED TO LONG‐WAVELENGTH RADIATIONS. POSSIBLE INVOLVEMENT IN THE UVA‐INDUCED PERTURBATIONS OF ENDOCYTOTIC PROCESSES

Abstract Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320‐400 nm) radiations markedly decreased the actin reactivity with fluorescein‐labeled phalloidin. This indicates a change in the degree of poly merization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA‐induced inhibition of specific and nonspecific endocytotic processes.

ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL’s hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1 expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.

EXPOSURE TO LONG WAVELENGTH ULTRAVIOLET RADIATION DECREASES PROCESSING OF LOW DENSITY LIPOPROTEIN BY CULTURED HUMAN FIBROBLASTS

Exposure of MRC5 human fibroblasts to UVA radiation (365 nm) resulted in a dose‐dependent decrease in low density lipoprotein (LDL) uptake and degradation by cells. Following a 25 J/cm2 irradiation dose, about 45% and 70% reduction in 125I‐LDL uptake and degradation were observed, respectively. Under the same conditions, the 14C‐sucrose uptake was also decreased to about the same extent as LDL uptake. Cell pretreatment with the antioxidants vitamin E and vitamin C did not prevent the UVA‐induced fall in LDL degradation. These results point to the possible effects of UVA radiation on receptor‐mediated and nonspecific uptake of exogenous molecules. With special regard to the alterations in receptor‐mediated processing of exogenous ligands, such a phenomenon could be of importance in UVA‐induced skin degenerative processes.

Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP‐ribosylating and apoptotic effects

To assess Pseudomonas exotoxinu2003A (ETA) compartmentalization, processing and cytotoxicity inu2003vivo, we have studied the fate of internalized ETA with the use of the inu2003vivo rodent liver model following toxin administration, cell‐free hepatic endosomes, and pure inu2003vitro protease assays. ETA taken up into rat liver inu2003vivo was rapidly associated with plasma membranes (5–30u2003min), internalized within endosomes (15–60u2003min), and later translocated into the cytosolic compartment (30–90u2003min). Coincident with endocytosis of intact ETA, inu2003vivo association of the catalytic ETA‐A subunit and low molecular mass ETA‐A fragments was observed in the endosomal apparatus. After an inu2003vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA‐degrading activity was attributed to the luminal species of endosomal acidic cathepsinsu2003B and D, with the major cleavages generated inu2003vitro occurring mainly within domainu2003III of ETA‐A. Cell‐free endosomes preloaded inu2003vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA‐A inu2003vitro in a pH‐dependent and ATP‐dependent manner. Rat hepatic cells underwent inu2003vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochromeu2003c, caspase‐9 and caspase‐3 activation, and DNA fragmentation. In an inu2003vitro assay, intact ETA induced ADP‐ribosylation of EF‐2 and mitochondrial release of cytochromeu2003c, with the former effect being efficiently increased by a cathepsinu2003B/cathepsinu2003D pretreatment. The data show a novel processing pathway for internalized ETA, involving cathepsinsu2003B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction.

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.