Neurovascular alterations of muscularis propria in the human anterior vaginal wall in pelvic organ prolapse.

In the pathophysiology and progression of pelvic organ prolapse (POP), it has been demonstrated that there is a reorganisation of the muscularis propria of the anterior vaginal wall due to a phenotypic smooth muscle cell to myofibroblast switch. An abnormal deposition of collagen type III seems to be influenced by the involvement of advanced glycation end-products. The aim of the present study was to evaluate the hypothesis that this connective tissue remodelling could also be associated with neurovascular alterations of the muscularis in women with POP compared with control patients. We examined 30 women with POP and 10 control patients treated for uterine fibromatosis. Immunohistochemical analysis, using glial fibrillary acidic protein, S-100 protein, receptor tyrosine kinase, neurofilament and α-smooth muscle actin antibodies, was performed. S-100, receptor tyrosine kinase and neurofilament were also evaluated using Western blot analysis. We observed a decrease in all neurovascular-tested markers in nerve bundles, ganglia and interstitial cells of Cajal from POP samples as compared with controls. Even if the processes responsible for these morphological alterations are still not known, it is conceivable that collagen III deposition in the anterior vaginal wall affects not only the architecture of the muscle layer but could also modify the intramuscular neurovascularisation and account for an alteration of the neuromuscular plasticity of the layer.

Correction to: The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma.

The original article [1] contained an error whereby Fig. 4 displayed incorrect magnification scales.

c-Myc Sustains Transformed Phenotype and Promotes Radioresistance of Embryonal Rhabdomyosarcoma Cell Lines.

We have previously reported that the MEK/ERK pathway sustains in vitro and in vivo transformed phenotype and radioresistance of embryonal rhabdomyosarcoma (ERMS) cell lines. Furthermore, we found that aberrant MEK/ERK signaling activation promotes c-Myc oncoprotein accumulation. In this study, the role of c-Myc in sustaining the ERMS transformed and radioresistant phenotype is characterized. RD and TE671 cell lines conditionally expressing MadMyc chimera protein, c-Myc-dominant negative and shRNA directed to c-Myc were used. Targeting c-Myc counteracted in vitro ERMS adherence and in suspension, growth motility and the expression of pro-angiogenic factors. c-Myc depletion decreased MMP-9, MMP-2, u-PA gelatinolytic activity, neural cell adhesion molecule sialylation status, HIF-1α, VEGF and increased TSP-1 protein expression levels. Rapid but not sustained targeting c-Myc radiosensitized ERMS cells by radiation-induced apoptosis, DNA damage and impairing the expression of DNA repair proteins RAD51 and DNA-PKcs, thereby silencing affected ERMS radioresistance. c-Myc sustains ERMS transformed phenotype and radioresistance by protecting cancer cells from radiation-induced apoptosis and DNA damage, while promoting radiation-induced DNA repair. This data suggest that c-Myc targeting can be tested as a promising treatment in cancer therapy.

Vitamin D protects endothelial cells from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis.

PURPOSE: Radiotherapy toxicity is related to oxidative stress-mediated endothelial dysfunction. Here, we investigated on radioprotective properties of Vitamin D (Vit.D) on human endothelial cells (HUVEC). METHODS: HUVEC, pre-treated with Vit.D, were exposed to ionizing radiation (IR): ROS production, cellular viability, apoptosis, senescence and western blot for protein detection were performed. The role of MAPKs pathway was investigated by using U0126 (10 µM) MEKs/ERKs-, SB203580 (2.5 µM) p38-inhibitor or by over/expressing MKK6 p38-upstream activator. RESULTS: Vit.D reduced IR-induced ROS production protecting proliferating and quiescent HUVEC from cellular apoptosis or senescence, respectively, by regulating MAPKs pathways. In proliferating HUVEC, Vit.D prevented IR-induced apoptosis by activating ERKs while in quiescent HUVEC counteracted IR-induced senescence by inhibiting the p38-IR-induced activation. MEKs&ERKs inhibition in proliferating or MKK6/mediated p38 activation in quiescent HUVEC, respectively, reverted anti-apoptotic or anti-senescent Vit.D properties. SirT1 protein expression levels were up-regulated by Vit.D. ERKs inhibition blocked Vit.D-induced SirT1 protein up-regulation in proliferating cells. In quiescent HUVEC cells, p38 inhibition counteracted the IR-induced SirT1 protein down-regulation, while MKK6 transfection abrogated the Vit.D positive effects on SirT1 protein levels after irradiation. SirT1 inhibition by sirtinol blocked the Vit.D radioprotective effects. CONCLUSION: Vit.D protects HUVEC from IR induced/oxidative stress by positively regulating the MAPKs/SirT1 axis.

The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.

Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.

beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.

Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.

The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels.

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.

Screening for anti-human T-lymphotropic virus antibody in blood donors and polytransfused patients in Apulia (Italy).

A stringent procedure for the diagnosis of human T-lymphotropic virus (HTLV) infection was applied to 1,732 volunteer blood donors, 401 patients with various hematological disorders and 78 individuals at high risk for HIV infection. It consisted of a viral lysate-based screening assay (Abbott Laboratories, North Chicago, Ill., USA), and two confirmatory assays (Western blot and radioimmunoprecipitation assay). A confirmed positive sample had to react with at least two different HTLV gene products. Evidence of HTLV infection was not found in either blood donors or patients with hematological disorders. In fact, HTLV infection was only observed in 10 intravenous drug abusers or their sexual partners. Contrary to previous reports that claimed HTLV seroprevalences of between 0.3 and 8% in blood donors from Apulia (Italy), our data suggest that infection with this virus is principally restricted to intravenous drug abusers.