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Adenovirus-mediated Overexpression (adenovirus-mediated + overexpression)
Selected AbstractsScavenger receptor class B, type I is expressed in porcine brain capillary endothelial cells and contributes to selective uptake of HDL-associated vitamin EJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Daniel Goti It is clearly established that an efficient supply to the brain of ,-tocopherol (,TocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of ,TocH into cells constituting the blood,brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of ,TocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated ,TocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of ,TocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated ,TocH uptake. In accordance with the proposed function of SR-BI, selective HDL,CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C],TocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3 -associated ,TocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated ,TocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient. [source] Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cellsMOLECULAR CARCINOGENESIS, Issue 3 2005Dong Xiao Abstract The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in apoptosis induction by phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived cancer chemopreventive agent, with DU145 and LNCaP human prostate cancer cells as a model. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c- jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. Exposure of DU145 and LNCaP cells to growth suppressive concentrations of PEITC resulted in activation of ERK1/2 and JNKs, but not p38 MAPK, in both cell lines. In DU145 cells, the apoptosis induction by PEITC was statistically significantly attenuated by pharmacological inhibition of JNKs with SP600125. Adenovirus-mediated overexpression of Flag-tagged JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), an inhibitor of JNK, also inhibited PEITC-induced apoptosis in DU145 cells. On the other hand, inhibition of ERK1/2 activation with MEK1 inhibitor PD98059 failed to offer protection against PEITC-induced apoptosis in DU145 cells. In LNCaP cells, the PEITC-induced cell death was not affected by either pretreatment with PD98059 or SP600125 or overexpression of JBD of JIP-1. These results indicate that involvement of MAPKs in apoptosis induction by PEITC in human prostate cancer cells is cell line-specific. © 2005 Wiley-Liss, Inc. [source] Overexpression of hepatocyte nuclear factor-3, induces apoptosis through the upregulation and accumulation of cytoplasmic p53 in prostate cancer cells,THE PROSTATE, Issue 4 2010Hyun Joo Lee Abstract BACKGROUND Hepatocyte nuclear factor-3, (HNF-3,) has been known to act as a repressor in the pathogenesis of many cancers. Herein, we investigated the effect of HNF-3, overexpression in prostate cancer cells. METHODS HNF-3, was overexpressed in prostate cancer cells using an adenovirus recombinant expressing wild-type HNF-3,. The apoptosis of prostate cancer cells was determined by TUNEL, FACS, and caspase activity analyses. RESULTS Adenovirus-mediated overexpression of HNF-3, caused cell death in prostate cancer cells as assessed by changes in cellular and nuclear morphology, TUNEL analysis, and caspase activations. Furthermore, FACS analysis showed an increased sub-G1 phase of cell cycle as well as the G2/M phase with a corresponding decrease in S phases. HNF-3, overexpression caused the upregulation of p53 protein and its accumulation, together with HNF-3,, in the cytoplasm. It also causes Bax protein to localize to the mitochondria-enriched fraction. These findings suggest that multiple apoptotic pathways seem to be involved in the HNF-3,-induced cell death: pathways involving the accumulation of p53 protein in the cytoplasm and subsequent cytochrome c release, and other pathways involving death receptor signaling and caspase-8 activation. CONCLUSIONS The results of the current study suggest a novel function of HNF-3, as a killer of malignant prostate cancer cells, which reveals HNF-3, as a promising therapeutic molecule for prostate cancers. Prostate 70: 353,361, 2010. © 2009 Wiley-Liss, Inc. [source] Endothelin 1 contributes to the effect of transforming growth factor ,1 on wound repair and skin fibrosisARTHRITIS & RHEUMATISM, Issue 3 2010David Lagares Objective To characterize the pathways induced by transforming growth factor ,1 (TGF,1) that lead to the expression of endothelin 1 (ET-1) in human dermal fibroblasts, and to study the effects of TGF,1 and ET-1 on the acquisition of a profibrotic phenotype and assess the contribution of the TGF,1/ET-1 axis to skin wound healing and fibrosis in vivo. Methods The mechanism of induction of ET-1 expression by TGF,1 and its effect on the expression of ,-smooth muscle actin and type I collagen were studied in human dermal fibroblasts, in experiments involving the TGF, receptor inhibitor GW788388 and the ET receptor antagonist bosentan, by real-time reverse transcription,polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, immunofluorescence, Western blotting, and promoter/reporter transient transfection analyses. Experiments assessing dermal wound healing in mice were performed with adenovirus-driven overexpression of active TGF,1 and ET-1, with or without treatment with bosentan. The contributions of TGF,1 and ET-1 to the fibrotic response were also assessed in a mouse model of bleomycin-induced skin fibrosis, by histologic, immunohistochemical, RT-PCR, and protein analyses. Results TGF,1 induced ET-1 expression in human dermal fibroblasts through Smad- and activator protein 1/JNK,dependent signaling. The ability of TGF,1 to induce the expression of profibrotic genes was dependent on ET-1. Adenovirus-mediated overexpression of TGF,1 and ET-1 in mouse skin was associated with accelerated wound closure, increased fibrogenesis, and excessive scarring. Treatment with bosentan prevented the effects of TGF,1. In the bleomycin-induced fibrosis model, treatment with GW788388 and bosentan prevented the fibrotic response. Conclusion Our results strongly support the notion that the TGF,1/ET-1 axis has a role in wound repair and skin fibrosis. ET-1 receptor antagonists, such as bosentan, may represent a useful therapeutic tool in the treatment of excessive scarring and fibrosis-related diseases. [source] Clearance of coagulation factor VIII in very low-density lipoprotein receptor knockout miceBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Niels Bovenschen Summary Low-density lipoprotein receptor-related protein (LRP) contributes to factor VIII (FVIII) catabolism in vivo. Besides LRP, FVIII also interacts with very low-density lipoprotein receptor (VLDLR) in vitro. We investigated the physiological role of VLDLR in FVIII catabolism, using knockout mouse models for VLDLR and LRP, alone and in combination. VLDLR,/, mice displayed normal plasma FVIII, whereas VLDLR,/, LRP, double-knockout mice had slightly increased FVIII compared with LRP-deficient mice. Remarkably, VLDLR deficiency slightly accelerated FVIII clearance. Adenovirus-mediated overexpression of VLDLR did not lower plasma FVIII in LRP-deficient mice. We conclude that VLDLR does not act in concert with LRP in FVIII clearance in vivo. [source] | ||||||||||