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Beaslas O.,Minerva Foundation Institute for Medical Research | Vihervaara T.,Minerva Foundation Institute for Medical Research | Li J.,Jinan University | Laurila P.-P.,Institute for Molecular Medicine Finland | And 4 more authors.
Experimental Cell Research | Year: 2012

ORP8 is an oxysterol/cholesterol binding protein anchored to the endoplasmic reticulum and the nuclear envelope, and is abundantly expressed in the macrophage. We created and characterized mouse RAW264.7 macrophages with ORP8 stably silenced using shRNA lentiviruses. A microarray transcriptome and gene ontology pathway analysis revealed significant alterations in several nuclear pathways and ones associated with centrosome and microtubule organization. ORP8 knockdown resulted in increased expression and altered subcellular distribution of an interaction partner of ORP8, nucleoporin NUP62, with an intranuclear localization aspect and association with cytoplasmic vesicular structures and lamellipodial edges of the cells. Moreover, ORP8 silenced cells displayed enhanced migration, and a more pronounced microtubule cytoskeleton than controls expressing a non-targeting shRNA. ORP8 was shown to compete with Exo70 for interaction with NUP62, and NUP62 knockdown abolished the migration enhancement of ORP8-silenced cells, suggesting that the endogenous ORP8 suppresses migration via binding to NUP62. As a conclusion, the present study reveals new, unexpected aspects of ORP8 function in macrophages not directly involving lipid metabolism, but rather associated with nuclear functions, microtubule organization, and migration capacity. © 2012 Elsevier Inc. Source

Bockelman C.,University of Helsinki | Hagstrom J.,University of Helsinki | Makinen L.K.,University of Helsinki | Keski-Santti H.,University of Helsinki | And 5 more authors.
British Journal of Cancer | Year: 2011

Background:No reliable prognostic markers exist for squamous cell carcinoma of the tongue, and its prognosis can even in early stages be unpredictable and survival poor despite treatment. A potential marker is oncoprotein cancerous inhibitor of PP2A (CIP2A), which acts as a prognostic marker in gastric and non-small cell lung cancers.Methods:We collected specimens of 73 stage T1N0M0 and T2N0M0 oral squamous cell carcinomas of the tongue, as well as samples from normal oral mucosa, dysplastic lesions, and invasive carcinomas (n39). All samples were stained for CIP2A by immunohistochemistry. Survival curves were constructed according to the Kaplan-Meier method. The Cox proportional hazard model served for univariate and multivariate survival analysis.Results:High CIP2A immunoreactivity predicted poor survival in tongue cancer patients (P0.027, logrank test). In multivariate survival analysis, CIP2A was an independent prognostic factor (HR 2.02, 95% confidence interval 1.07-3.82, P0.030). Cytoplasmic CIP2A expression was higher in severe dysplasia than in mild dysplasia.Conclusion:Our results suggest that high CIP2A expression characterises aggressive disease. Acting as a prognostic marker it might be of help when choosing patients for adjuvant treatment in tongue cancer patients. © 2011 Cancer Research UK All rights reserved. Source

Khan N.A.,University of Helsinki | Auranen M.,University of Helsinki | Paetau I.,University of Helsinki | Pirinen E.,Ecole Polytechnique Federale de Lausanne | And 8 more authors.
EMBO Molecular Medicine | Year: 2014

Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD+/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD+ precursor, was previously shown to boost NAD+ levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD+ levels are a promising treatment strategy for mitochondrial myopathy. © 2014 The Authors. Published under the terms of the CC BY license. Source

Orpana A.K.,University of Helsinki | Ho T.H.,Minerva Foundation Institute for Medical Research | Stenman J.,Minerva Foundation Institute for Medical Research | Stenman J.,Institute for Molecular Medicine Finland | Stenman J.,Karolinska Institutet
Analytical Chemistry | Year: 2012

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele. © 2012 American Chemical Society. Source

Bockelman C.,University of Helsinki | Koskensalo S.,University of Helsinki | Hagstrom J.,University of Helsinki | Lundin M.,Institute for Molecular Medicine Finland | And 2 more authors.
Cancer Biology and Therapy | Year: 2012

Background: To improve the prognostic evaluation of colorectal cancer requires new molecular markers. Cancerous inhibitor of protein phosphatase 2A (CIP2A) serves as an oncoprotein by targeting PP 2A-mediated inhibition of c-Myc. A prognostic role for CIP2A has been demonstrated in gastric, lung and tongue cancers. Results: CIP2A was overexpressed in 661 (87.9%) specimens. CIP2A overexpression was associated with tumor differentiation grade (p = 0.014), p53 immunopositivity (p = 0.042), EGFR immunopositivity (p = 0.007) and c-Myc nuclear immunopositivity (p = 0.018). In survival analysis, CIP2A failed to show any prognostic significance (p = 0.270, log-rank test). Methods: 863 consecutive colorectal cancer patients treated at Helsinki University Central Hospital in 1983-2001 were collected with 752 scored successfully for CIP2A immunohistochemical expression from tumor tissue microarrays. Associations with clinicopathologic variables and molecular markers were explored by the chi-square test, and the Kaplan-Meier method served for survival analysis. Conclusions: Overexpression of CIP2A in colorectal cancer patients may be an important step in colorectal carcinogenesis. Based on our findings, CIP2A shows no association with patient prognosis in colorectal cancer, but is associated with nuclear c-Myc. © 2012 Landes Bioscience. Source

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