Search

// Gabrielis Kundrotas 1,2 , Evelina Gasperskaja 1 , Grazina Slapsyte 1 , Zivile Gudleviciene 2 , Jan Krasko 3 , Ausra Stumbryte 2 and Regina Liudkeviciene 2 1 Department of Botany and Genetics, Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania 2 Biobank, National Cancer Institute, Vilnius, Lithuania 3 Laboratory of Immunology, National Cancer Institute, Vilnius, Lithuania Correspondence to: Gabrielis Kundrotas, email: // Keywords : human mesenchymal stem cells, genomic stability, long-term expansion, PCR arrays, senescence markers, Gerotarget Received : August 04, 2015 Accepted : February 05, 2016 Published : February 17, 2016 Abstract Human bone marrow mesenchymal stem cells (hBM-MSCs) hold promise for treating incurable diseases and repairing of damaged tissues. However, hBM-MSCs face the disadvantages of painful invasive isolation and limited cell numbers. In this study we assessed characteristics of MSCs isolated from residual human bone marrow transplantation material and expanded to clinically relevant numbers at passages 3-4 and 6-7. Results indicated that early passage hBM-MSCs are genomically stable and retain identity and high proliferation capacity. Despite the chromosomal stability, the cells became senescent at late passages, paralleling the slower proliferation, altered morphology and immunophenotype. By qRT-PCR array profiling, we revealed 13 genes and 33 miRNAs significantly differentially expressed in late passage cells, among which 8 genes and 30 miRNAs emerged as potential novel biomarkers of hBM-MSC aging. Functional analysis of genes with altered expression showed strong association with biological processes causing cellular senescence. Altogether, this study revives hBM as convenient source for cellular therapy. Potential novel markers provide new details for better understanding the hBM-MSC senescence mechanisms, contributing to basic science, facilitating the development of cellular therapy quality control, and providing new clues for human disease processes since senescence phenotype of the hematological patient hBM-MSCs only very recently has been revealed.

// Suchitra Natarajan 1 , Farhana Begum 1 , Jeonga Gim 1 , Landon Wark 1 , Dana Henderson 1 , James R. Davie 2, 3 , Sabine Hombach-Klonisch 1, 4, * , Thomas Klonisch 1, 5, 6, * 1 Department of Human Anatomy and Cell Science, College of Medicine, University of Manitoba, Winnipeg, Canada 2 Children’s Hospital Research Institute of Manitoba, Winnipeg, Canada 3 Department of Biochemistry and Medical Genetics, College of Medicine, University of Manitoba, Winnipeg, Canada 4 Department of Obstetrics, Gynecology and Reproductive Medicine, College of Medicine, University of Manitoba, Winnipeg, Canada 5 Department of Surgery, College of Medicine, University of Manitoba, Winnipeg, Canada 6 Department of Medical Microbiology and Infectious Diseases, College of Medicine, University of Manitoba, Winnipeg, Canada * These authors have contributed equally to this work Correspondence to: Thomas Klonisch, e-mail: thomas.klonisch@umanitoba.ca Keywords: telomeres, HMGA2, TRF2, genomic stability, shelterin Received: April 12, 2015 Accepted: December 07, 2015 Published: January 18, 2016 ABSTRACT The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) plays important roles in the repair and protection of genomic DNA in embryonic stem cells and cancer cells. Here we show that HMGA2 localizes to mammalian telomeres and enhances telomere stability in cancer cells. We present a novel interaction of HMGA2 with the key shelterin protein TRF2. We found that the linker (L1) region of HMGA2 contributes to this interaction but the ATI-L1-ATII molecular region of HMGA2 is required for strong interaction with TRF2. This interaction was independent of HMGA2 DNA-binding and did not require the TRF2 interacting partner RAP1 but involved the homodimerization and hinge regions of TRF2. HMGA2 retained TRF2 at telomeres and reduced telomere-dysfunction despite induced telomere stress. Silencing of HMGA2 resulted in (i) reduced binding of TRF2 to telomere DNA as observed by ChIP, (ii) increased telomere instability and (iii) the formation of telomere dysfunction-induced foci (TIF). This resulted in increased telomere aggregation, anaphase bridges and micronuclei. HMGA2 prevented ATM-dependent pTRF2 T188 phosphorylation and attenuated signaling via the telomere specific ATM-CHK2-CDC25C DNA damage signaling axis. In summary, our data demonstrate a unique and novel role of HMGA2 in telomere protection and promoting telomere stability in cancer cells. This identifies HMGA2 as a new therapeutic target for the destabilization of telomeres in HMGA2 + cancer cells.

BRCA1 is an important player in the DNA damage response signaling, and its deficiency results in genomic instability. A complete loss or significantly reduced BRCA1 protein expression is often found in sporadic breast cancer cases despite the absence of genetic or epigenetic aberrations, suggesting the existence of other regulatory mechanisms controlling BRCA1 protein expression. Herein, we demonstrate that Fyn-related kinase (Frk)/Rak plays an important role in maintaining genomic stability, possibly in part through positively regulating BRCA1 protein stability and function via tyrosine phosphorylation on BRCA1 Tyr1552. In addition, Rak deficiency confers cellular sensitivity to DNA damaging agents and poly(ADP-ribose) polymerase (PARP) inhibitors. Overall, our findings highlight a critical role of Rak in the maintenance of genomic stability, at least in part, through protecting BRCA1 and provide novel treatment strategies for patients with breast tumors lacking Rak.

The RNA helicase A (RHA) is involved in several steps of RNA metabolism, such as RNA processing, cellular transit of viral molecules, ribosome assembly, regulation of transcription and translation of specific mRNAs. RHA is a multifunctional protein whose roles depend on the specific interaction with different molecular partners, which have been extensively characterized in physiological situations. More recently, the functional implication of RHA in human cancer has emerged. Interestingly, RHA was shown to cooperate with both tumor suppressors and oncoproteins in different tumours, indicating that its specific role in cancer is strongly influenced by the cellular context. For instance, silencing of RHA and/or disruption of its interaction with the oncoprotein EWS-FLI1 rendered Ewing sarcoma cells more sensitive to genotoxic stresses and affected tumor growth and maintenance, suggesting possible therapeutic implications. Herein, we review the recent advances in the cellular functions of RHA and discuss its implication in oncogenesis, providing a perspective for future studies and potential translational opportunities in human cancer.

Chromosome ends in mammalian cells are protected by a specialized 6 subunit complex called shelterin. Mutations in one component of this complex, POT1, have been associated with a variety of cancers including solid and lymphoid tumors [1]. Interestingly, the vast majority of the POT1 mutations cluster within its DNAbinding domains, the so-called OB-fold domains. We have recently uncovered the molecular mechanism by which these mutations contribute to cancer progression [2]. Surprisingly we found that not all the cancer-associated POT1 mutations alter the ability of POT1 to bind telomeric DNA. Instead, our study revealed that POT1 mutations compromise telomere replication, leading to telomere dysfunction. Indeed, the expression of these mutations in various cellular systems resulted in frequent stalling of the replication machinery at telomeric repeats, triggering the activation of the ATR kinase, and ultimately leading to increased genome instability [2]. We determined that this is caused by alterations in the recruitment of the CST (CTC1-TEN1-STN1) complex at telomeres. CST is an RPA like complex that acts as a pol-α primase accessory factor [3] that enables replication fork restart at telomeric DNA. To address the physiological implication of our findings we generated a mouse model in which POT1 was inactivated in the common progenitor lymphoid (CLPs) cells. We chose this compartment because of the predominance of POT1 mutations in cancer of lymphoid origin. Analysis of these mice revealed that, when combined with p53 inactivation, POT1 inhibition resulted in highly aggressive thymic lymphomas. Tumor cells derived from these mice showed increased levels of telomere dysfunction with many end-to-end chromosomal fusions and signs of replication defects at telomeres [2]. The existence of telomere dysfunction in POT1depleted tumors created a paradox. How do POT1 mutated cells survive despite the high level of telomere dysfunction and the resultant DNA damage signaling? To get insight into this problem, we interrogated the cancer genomes for clues. Specifically, we compared by NGS the transcription profile of tumors derived from POT1-proficient and POT1-deficient tumors. This analysis revealed that the ATR pathway is significantly downregulated in tumors with POT1 mutations. This observation was confirmed in tissue culture based experiments as well as in vivo. The impairment of the ATR-pathway in POT1-mutated tumors at first seems counterintuitive, given that increased ATR signaling has been suggested to allow tumor cells to cope with oncogene-induced replication stress [4]. However, it is possible that the attenuation of the ATR signaling pathway enables tumor cells with replication defects to evade checkpoint activation and cell cycle arrest. In agreement with this, reduced ATR signaling in mice is sufficient to increase the incidence of tumors [5]. In addition, somatic mutations affecting the ATRsignaling pathway have been found in human tumors with microsatellite instability [6]. Several questions remain to be addressed to understand the link between replication stress at telomeres and cancer development. First of all, we still do not understand how telomere replication defects and fragility leads to chromosome fusions. Additionally, from a mechanistic point of view, how POT1 mutations impact telomere replication remains unknown. Our data implicate the CST complex in this process. However, additional biochemical and structural analysis are required to elucidate how mutations in POT1 compromise CST function. Next, it will be of interest to define whether other sources of replication stress, particularly other genetic alterations known to affect telomere replication, promote a similar telomere dysfunction-induced genomic instability and increased tumor formation. Finally, and most Editorial

// Ulrike Fischer 1 , Christina Backes 1, 3 , Abdulrahman Raslan 2 , Andreas Keller 3 , Carola Meier 2 , Eckart Meese 1 1 Department of Human Genetics, Saarland University, 66421 Homburg/Saar, Germany 2 Department of Anatomy and Cell Biology, Saarland University, 66421 Homburg/Saar, Germany 3 Clinical Bioinformatics, Saarland University, 66123 Saarbrucken, Germany Correspondence to: Ulrike Fischer, e-mail: ulrike.fischer@uks.eu Keywords: neurosphere, genomic stability, in vivo , array-CGH Received: November 04, 2014 Accepted: January 28, 2015 Published: February 19, 2015 ABSTRACT In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells