Lipodystrophy-linked LMNA p.R482W mutation induces clinical early atherosclerosis and in vitro endothelial dysfunction.

OBJECTIVE: Some mutations in LMNA, encoding A-type lamins, are responsible for Dunnigan-type-familial partial lipodystrophy (FPLD2), with altered fat distribution and metabolism. The high prevalence of early and severe cardiovascular outcomes in these patients suggests that, in addition to metabolic risk factors, FPLD2-associated LMNA mutations could have a direct role on the vascular wall cells. APPROACH AND RESULTS: We analyzed the cardiovascular phenotype of 19 FPLD2 patients aged >30 years with LMNA p.R482 heterozygous substitutions, and the effects of p.R482W-prelamin-A overexpression in human coronary artery endothelial cells. In 68% of FPLD2 patients, early atherosclerosis was attested by clinical cardiovascular events, occurring before the age of 45 in most cases. In transduced endothelial cells, exogenous wild-type-prelamin-A was correctly processed and localized, whereas p.R482W-prelamin-A accumulated abnormally at the nuclear envelope. Patients' fibroblasts also showed a predominant nuclear envelope distribution with a decreased rate of prelamin-A maturation. Only p.R482W-prelamin-A induced endothelial dysfunction, with decreased production of NO, increased endothelial adhesion of peripheral blood mononuclear cells, and cellular senescence. p.R482W-prelamin-A also induced oxidative stress, DNA damages, and inflammation. These alterations were prevented by treatment of endothelial cells with pravastatin, which inhibits prelamin-A farnesylation, or with antioxidants. In addition, pravastatin allowed the correct relocalization of p.R482W-prelamin-A within the endothelial cell nucleus. These data suggest that farnesylated p.R482W-prelamin-A accumulation at the nuclear envelope is a toxic event, leading to cellular oxidative stress and endothelial dysfunction. CONCLUSIONS: LMNA p.R482 mutations, responsible for FPLD2, exert a direct proatherogenic effect in endothelial cells, which could contribute to patients' early atherosclerosis.

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation.

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.

Ad-IFN gamma induces antiproliferative and antitumoral responses in malignant mesothelioma.

PURPOSE: The aim of the work was to investigate the effects of adenovirus(Ad)-mediated IFN gamma gene transfer on human mesothelioma (HM) cell proliferation in vitro and growth in nude mice. EXPERIMENTAL DESIGN: We constructed an E1E3-deleted replication-defective recombinant Ad carrying the human IFN gamma gene (Ad-IFN gamma) and tested its activity in vitro on HM cell lines established in our laboratory and in a nude mice model. RESULTS: In vitro, infection of HM cells with Ad-IFN gamma led to a prolonged production of an active cytokine in the 10 HM cell lines tested and also led to an antiproliferative effect on the HM cells previously demonstrated as responsive to exogenous recombinant human IFN gamma. In nude mice, s.c. inoculation of HM cells from one responsive HM cell line previously infected with Ad-IFN gamma resulted in a delay in tumor development, and injection of Ad-IFN gamma in preestablished tumors restrained tumor development. CONCLUSIONS: These results indicate for the first time that HM cells are efficiently transduced by Ad-IFN gamma and produce an active cytokine for several days. IFN gamma produced by gene transfer is shown to have both an antiproliferative effect in vitro and an antitumoral effect in vivo in a nude mice model.

Glycogen synthase kinase-3 inhibitors augment TRAIL-induced apoptotic death in human hepatoma cells.

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.