Spectrum of ALMS1 variants and evaluation of genotype-phenotype correlations in Alström syndrome

Alstr?m syndrome is a monogenic recessive disorder featuring an array of clinical manifestations, with systemic fibrosis and multiple organ involvement, including retinal degeneration, hearing loss, childhood obesity, diabetes mellitus, dilated cardiomyopathy (DCM), urological dysfunction, and pulmonary, hepatic, and renal failure. We evaluated a large cohort of patients with Alstr?m syndrome for mutations in the ALMS1 gene. In total, 79 disease-causing variants were identified, of which 55 are novel mutations. The variants are primarily clustered in exons 8, 10, and 16, although we also identified novel mutations in exons 12 and 18. Most alleles were identified only once (45/79), but several were found recurrently. Founder effects are likely in families of English and Turkish descent. We also identified 66 SNPs and assessed the functional significance of these variants based on the conserved identity of the protein and the severity of the resulting amino acid substitution. A genotype-phenotype association study examining 18 phenotypic parameters in a subset of 58 patients found suggestive associations between disease-causing variants in exon 16 and the onset of retinal degeneration before the age of 1 year (P = 0.02), the occurrence of urological dysfunction (P = 0.02), of DCM (P = 0.03), and of diabetes (P = 0.03). A significant association was found between alterations in exon 8 and absent, mild, or delayed renal disease (P = 0.0007). This data may have implications for the understanding of the molecular mechanisms of ALMS1 and provides the basis for further investigation of how alternative splicing of ALMS1 contributes to the severity of the disease.     

The ‘6K1’ protein of a strain of Soybean mosaic virus localizes to the cell periphery

Summary The ‘6K1’ protein of the Pinellia isolate of Soybean mosaic virus was cloned into a prokaryotic expression vector and a polyclonal antiserum raised to the expressed fusion protein. In immunogold labeling of thin sections of infected leaves of Pinellia ternata, specific labeling occurred at the cell periphery. This might suggest that the potyvirus ‘6K1’ protein plays some role in viral cell-to-cell movement but the lack of transmembrane domains suggests that it does not conform to currently-recognized patterns of viral movement proteins.     

Regulation of Alström syndrome gene expression during adipogenesis and its relationship with fat cell insulin sensitivity

Alstr?m syndrome (ALMS) is an autosomal recessive genetic disease with characteristic phenotypical features including multi-organ fibrosis, insulin resistance, obesity and type 2 diabetes. ALMS1, a ubiquitously expressed gene mutated in ALMS patients, gives rise to a protein of unknown function localized to basal bodies of ciliated cells and centrosomes. Together with Bardet-Biedl syndrome, ALMS is a member of genetic ciliopathies, but the link between cilia/centrosome deficits and metabolic abnormalities remains to be determined. In this study for the first time we quantified Alms1 expression in a cellular model of adipogenesis during the differentiation of 3T3-L1 cells. An early decrease in Alms1 mRNA was observed during preadipocyte to adipocyte conversion. However, acute treatment of preadipocytes with the adipogenic factors did not result in significant change of Alms1 expression. In addition, to study the possible relationship between Alms1 and the degree of fat cell insulin sensitivity, as assessed with an insulin-dependent 2-[1-3H]-deoxyglucose uptake assay, we induced either a reduction or an increase in 3T3-L1 adipocytes insulin sensitivity by a chronic treatment with insulin or rosiglitazone respectively. In all these conditions Alms1 expression remained unchanged. In conclusion, our results show that Alms1 is expressed at higher level in preadipocytes suggesting a role of the gene in the early phase of adipogenesis. Moreover, changes in fat cell insulin sensitivity do not imply any effect on Alms1 expression.     

Integrin α3β1 regulates tumor cell responses to stromal cells and can function to suppress prostate cancer metastatic colonization

Integrin α3β1 promotes tumor cell adhesion, migration, and invasion on laminin isoforms, and several clinical studies have indicated a correlation between increased tumoral α3β1 integrin expression and tumor progression, metastasis, and poor patient outcomes. However, several other clinical and experimental studies have suggested that α3β1 can possess anti-metastatic activity in certain settings. To help define the range of α3β1 functions in tumor cells in vivo, we used RNAi to silence the α3 integrin subunit in an aggressive, in vivo-passaged subline of PC-3 prostate carcinoma cells. Loss of α3 integrin impaired adhesion and proliferation on the α3β1 integrin ligand, laminin-332 in vitro. Despite these deficits in vitro, the α3-silenced cells were significantly more aggressive in a lung colonization model in vivo, with a substantially increased rate of tumor growth that significantly reduced survival. In contrast, silencing the related α6 integrin subunit delayed metastatic growth in vivo. The increased colonization of α3-silenced tumor cells in vivo was recapitulated in 3D collagen co-cultures with lung fibroblasts or pre-osteoblast-like cells, where α3-silenced cells showed dramatically enhanced growth. The increased response of α3-silenced tumor cells to stromal cells in co-culture could be reproduced by fibroblast conditioned medium, which contains one or more heparin-binding factors that selectively favor the growth of α3-silenced cells. Our new data suggest a scenario in which α3β1 regulates tumor–host interactions within the metastatic tumor microenvironment to limit growth, providing some of the first direct evidence that specific loss of α3 function in tumor cells can have pro-metastatic consequences in vivo.     

5,7-Dihydroxy-3,4,6-trimethoxyflavone inhibits intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 via the Akt and nuclear factor-κB-dependent pathway, leading to suppression of adhesion of monocytes and eosinophils to bronchial epithelial cells

5,7-Dihydroxy-3',4',6'-trimethoxyflavone (eupatilin), the active pharmacological ingredient from Artemisia asiatica Nakai (Asteraceae), is reported to have a variety of anti-inflammatory properties in intestinal epithelial cells. However, little information is known about the molecular mechanism of eupatilin-induced attenuation of bronchial epithelial inflammation. This study investigates the role of eupatilin in the adhesion of inflammatory cells such as monocytes and eosinophils to bronchial epithelial cells. Stimulation of a human bronchial epithelial cell line (BEAS-2B) with tumour necrosis factor-α (TNF-α) increased the expression of surface adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), in which eupatilin significantly inhibited the expression of those adhesion molecules in a dose-dependent manner. Eupatilin suppressed the TNF-α-induced activation of IκBα and nuclear factor-κB (NF-κB) signals in BEAS-2B cells. The IκB kinase (IKK) activation was also significantly reduced in eupatilin-pre-treated BEAS-2B and primary normal human bronchial epithelial (NHBE) cells. However, eupatilin did not influence AP-1 activity in TNF-α-stimulated cells. Suppression of NF-κB signalling induced by eupatilin resulted in the inhibition of the expression of adhesion molecules and the adhesion of monocytes and eosinophils to BEAS-2B cells. Furthermore, eupatilin suppressed the phosphorylation of Akt in TNF-α-stimulated BEAS-2B and NHBE cells, leading to down-regulation of NF-κB activation and adhesion molecule expression and finally to suppression of the inflammatory cell adhesion to epithelial cells. These results suggest that eupatilin can inhibit the adhesion of inflammatory cells to bronchial epithelial cells via a signalling pathway, including activation of Akt and NF-κB, as well as expression of adhesion molecules.     

Guanylate-binding protein-1 is expressed at tight junctions of intestinal epithelial cells in response to interferon-γ and regulates barrier function through effects on apoptosis

Guanylate-binding protein-1 (GBP-1) is an interferon inducible large GTPase involved in endothelial cell proliferation and invasion. In this report, expression and function of GBP-1 were investigated in vitro in intestinal epithelia after exposure to interferon-γ and in human colonic mucosa from individuals with inflammatory bowel disease (IBD). Interestingly, in contrast to other epithelia, GBP-1 distributed to the plasma membrane in intestinal epithelial cells where it colocalized with the tight junction protein coxsackie- and adenovirus receptor. In addition, expression of GBP-1 was upregulated in colonic epithelia of individuals with IBD. Downregulation of GBP-1 by siRNA resulted in enhanced permeability that correlated with increased apoptosis. Indeed, inhibition of caspase activity prevented the inhibition of barrier formation induced by the loss of GBP-1. These data suggest that GBP-1 is a novel marker of intestinal mucosal inflammation that may protect against epithelial apoptosis induced by inflammatory cytokines and subsequent loss of barrier function.     

C-kit mutations determine dasatinib mechanism of action in HMC-1 neoplastic mast cells: dasatinib differently regulates PKCδ translocation in HMC-1560 and HMC-1560,816 cell lines

Abstract

Purpose: The second generation of tyrosine kinase inhibitors is a group of compounds that inhibit c-kit receptor activity and therefore widely used in the treatment of mastocytosis. In this research, the relationship between the mechanism of action of tyrosine kinase inhibitors and protein kinase C is investigated in HMC-1⁵⁶⁰ or HMC-1^560,816 cell lines.

Results: From all the tyrosine kinase inhibitors tested, nilotinib is the compound that has the highest cytotoxic effect against HMC-1⁵⁶⁰ mast cell line, while midostaurin is the most potent in HMC-1^560,816. Moreover, an increase on histamine release is observed after protein kinase C activation either in HMC-1⁵⁶⁰ or HMC-1^560,816 cells. Furthermore, dasatinib increases histamine release in both mast cell lines, which could be related with the secondary reactions previously described in dasatinib-treated patients. Dasatinib also induces Ca²⁺-dependent protein kinase C isoforms translocation from the cytosol to the membrane, whereas protein kinase Cδ is translocated from the cytosol to the nucleus in the HMC-1^560,816 cell line, but not in HMC-1⁵⁶⁰ cells.

Conclusion: Results obtained demonstrate that dasatinib induces an important cytotoxic effect in both HMC-1 cell lines and differently regulates protein kinase Cδ in HMC-1⁵⁶⁰ and HMC-1^560,816 cells. Finally, our results confirm that PKCδ is an essential target for dasatinib.     

Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways

Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.     

Recombinant AexU effector protein of Aeromonas veronii bv. sobria disrupts the actin cytoskeleton by downregulation of Rac1 and induces direct cytotoxicity to β4-integrin expressing cell lines

AexU is a type three secretion system (TTSS) effector of Aeromonas hydrophila which has an in vitro ADP-ribosyltransferase (ART) and GTPase-activating protein (GAP) activities on Rac1, RhoA and Cdc42. Here we show that, AexU of Aeromonas veronii bv. sobria AeG1 strain disrupts actin cytoskeleton of HeLa cells during AeG1 infection, aexU transfection or direct application of AexU protein. Such cellular disruption was rescued by either inactivation of AexU-GAP activity by substitution of arginine residue 143 to alanine or expression of a constitutively active (CA) Rac1 but not CA RhoA or CA Cdc42. On the other hand, AexU was found co-localized with β4-integrin probably through its Arg-Gly-Asp (RGD) integrin binding motif (319–321) residues. Interestingly, direct application of GST-AexU-HA fusion protein caused significant cytotoxic effect on β4-integrin expressing HT-29 cells. In contrast, β4-integrin blockade with a specific antibody reduced such cytotoxicity. Consequently, AexU cytotoxic effect was exaggerated with a greater expression of β4-integrin in Caco-2 and HeLa cells, while it was incompetent on β4-integrin non-expressing CHO cells. As far as we know, this is a novel TTSS effector which specifically inactivates Rac1 to disrupt actin cytoskeleton and has an alternative cytotoxic pathway through β4-integrin mediation.     

Oxidative stress and TGF-β1 induction by metformin in MCF-7 and MDA-MB-231 human breast cancer cells are accompanied with the downregulation of genes related to cell proliferation,invasion and metastasis

High doses of metformin induces oxidative stress (OS) and transforming growth factor β1 (TGF-β1) in breast cancer cells, which was associated with increased cancer stem cell population, local invasion, liver metastasis and treatment resistance. Considering the impact of TGF- β1 and OS in breast cancer and the interrelation between these two pathways, the objective of this work was to investigate the effects of consecutive metformin treatments, at a non-cytotoxic dosage, in TGF- β1 targets in MCF-7 and MDA-MB-231 cells. Cells were exposed to 6 μM of metformin for seven consecutive passages. Samples were collected to immunocytochemistry (evaluation of p53, Nf-кB, NRF2 and TGF-β1), biochemical (determination of lipoperoxidation, total thiols and nitric oxide/peroxynitrite levels) and molecular biology analyzes (microarray and Real-time quantitative array PCR). Microarray analysis confirmed alterations in genes related to OS and TGF-β1. Treatment interfered in several TGF-β1 target-genes. Metformin upregulated genes involved in OS generation and apoptosis, and downregulated genes associated with metastasis and epithelial mesenchymal transition in MCF-7 cells. In MDA-MB-231 cells, metformin downregulated genes involved with cell invasion, viability and proliferation. The results shows that even a non-cytotoxic dosage of metformin can promote a less aggressive profile of gene expression in breast cancer cells.     

Significance of cell proliferation markers (Minichromosome maintenance protein 7, topoisomerase IIα and Ki‐67) in cavital fluid cytology: Can we differentiate reactive mesothelial cells from malignant cells?

The aim of this study was to evaluate whether immunocytochemical expressions of proliferation markers, such as minichromosome maintenance protein 7 (MCM 7), topoisomerase IIα (topo IIα), and Ki‐67, in reactive mesothelial cells and malignant cells obtained from cavital fluids could be useful for their differential diagnosis. Samples diagnosed as reactive mesothelial cells (14 cases) or malignant tumors (28 cases) in cavital fluids were examined. Immunocytochemical staining of MCM 7, topo IIα, and Ki‐67 was performed with the universal immunoperoxidase polymer method. In reactive mesothelial cells, MCM 7 was stained in a fine granular pattern and its distribution was uniform in the nuclei. Topo IIα and Ki‐67 were stained in a coarse granular pattern and the distributions were the same as MCM 7. In contrast, in malignant cells, MCM 7 was stained in an irregular and fine granular pattern, and topo IIα and Ki‐67 were stained in a uniform and coarse granular pattern. Labeling indices of MCM 7 (cut‐off value; 30%, sensitivity; 100%, and specificity; 100%), topo IIα (cut‐off value; 15%, sensitivity; 89.3%, and specificity; 92.9%) and Ki‐67 (cut‐off value; 30%, sensitivity; 64.3%, and specificity; 92.9%) of malignant cells were significantly higher than those of reactive mesothelial cells. MCM 7, topo IIα, and Ki‐67 are different types of cell proliferation markers. MCM 7 and topo IIα, in particular, could be reliable tools for differential diagnosis between reactive mesothelial cells and malignant cells. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.