Regulation Of DNA Repair Pathways Research Articles

Understanding the Human RECQ5 Helicase-Connecting the Dots from DNA to Clinics.

RECQ5, a member of the conserved RECQ helicase family, is the sole human RECQ homolog that has not been linked to a hereditary developmental syndrome. Nonetheless, dysregulation of RECQ5 has emerged as a significant clinical concern, being linked to cancer predisposition, cardiovascular disease, and inflammation. In cells, RECQ5 assumes a crucial role in the regulation of DNA repair pathways, particularly in the repair of DNA double-strand breaks and inter-strand DNA crosslinks. Moreover, RECQ5 exhibits a capacity to modulate gene expression by interacting with transcription machineries and their co-regulatory proteins, thus safeguarding against transcription-induced DNA damage. This review aims to provide an overview of the multifaceted functions of RECQ5 and its implications in maintaining genomic stability. We will discuss the potential effects of clinical variants of RECQ5 on its cellular functions and their underlying mechanisms in the pathogenesis of cancer and cardiovascular disease. We will review the impact of RECQ5 variants in the field of pharmacogenomics, specifically their influence on drug responses, which may pave the way for novel therapeutic interventions targeting RECQ5 in human diseases.

Targeting the Interplay between HDACs and DNA Damage Repair for Myeloma Therapy.

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells, and accounts for 10% of all hematologic malignancies and 1% of all cancers. MM is characterized by genomic instability which results from DNA damage with certain genomic rearrangements being prognostic factors for the disease and patients’ clinical response. Following genotoxic stress, the evolutionary conserved DNA damage response (DDR) is activated and, in turn, coordinates DNA repair with cell-cycle events. However, the process of carcinogenesis cannot be attributed only to the genetic alterations, but also involves epigenetic processes. Regulation of expression and activity of key players in DNA repair and checkpoint proteins are essential and mediated partly by posttranslational modifications (PTM), such as acetylation. Crosstalk between different PTMs is important for regulation of DNA repair pathways. Acetylation, which is mediated by acetyltransferases (HAT) and histone deacetylases (HDAC), not only affects gene expression through its modulation of histone tails but also has recently been implicated in regulating non-histone proteins. Currently, several HDAC inhibitors (HDACi) have been developed both in pre-clinical and clinical studies, with some of them exhibiting significant anti-MM activities. Due to reversibility of epigenetic changes during the evolutionary process of myeloma genesis, the potency of epigenetic therapies seems to be of great importance. The aim of the present paper is the summary of all data on the role of HDACi in DDR, the interference with each DNA repair mechanism and the therapeutic implications of HDACi in MM.

Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair.

The proper function of DNA repair is indispensable for eukaryotic cells since accumulation of DNA damages leads to genome instability and is a major cause of oncogenesis. Ubiquitylation and deubiquitylation play a pivotal role in the precise regulation of DNA repair pathways by coordinating the recruitment and removal of repair proteins at the damaged site. Here, we summarize the most important post-translational modifications (PTMs) involved in DNA double-strand break repair. Although we highlight the most relevant PTMs, we focus principally on ubiquitylation-related processes since these are the most robust regulatory pathways among those of DNA repair.

FLT3-ITD cooperates with Rac1 to modulate the sensitivity of leukemic cells to chemotherapeutic agents via regulation of DNA repair pathways.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm, and patients with an internal tandem duplication (ITD) mutation of the FMS-like tyrosine kinase-3 (FLT3) receptor gene have a poor prognosis. FLT3-ITD interacts with DOCK2, a G effector protein that activates Rac1/2. Previously, we showed that knockdown of DOCK2 leads to decreased survival of FLT3-ITD leukemic cells. We further investigated the mechanisms by which Rac1/DOCK2 activity affects cell survival and chemotherapeutic response in FLT3-ITD leukemic cells. Exogenous expression of FLT3-ITD led to increased Rac1 activity, reactive oxygen species, phosphorylated STAT5, DNA damage response factors and cytarabine resistance. Conversely, DOCK2 knockdown resulted in a decrease in these factors. Consistent with the reduction in DNA damage response factors, FLT3-ITD cells with DOCK2 knockdown exhibited significantly increased sensitivity to DNA damage response inhibitors. Moreover, in a mouse model of FLT3-ITD AML, animals treated with the CHK1 inhibitor MK8776 + cytarabine survived longer than those treated with cytarabine alone. These findings suggest that FLT3-ITD and Rac1 activity cooperatively modulate DNA repair activity, the addition of DNA damage response inhibitors to conventional chemotherapy may be useful in the treatment of FLT3-ITD AML, and inhibition of the Rac signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3-ITD AML.

Identification of Novel Cyclin A2 Binding Site and Nanomolar Inhibitors

It has been shown that dysfunction in tight regulation of DNA repair pathways can induce various syndromes, increase the risk of cancer, and trigger neurological defect. However, there are cases where inhibition of DNA repair process is necessary. For example, after treating cancer cells with radiotherapy, it is essential to inhibit DNA repair function of the radiation-induced cancer cells for successful apoptosis. Therefore, the key is to target a protein that is responsible for controlling the rapid and proper resolution of DNA strand breaks. The recent discovery of CCNA2 (cyclin A2), the main mammalian S-phase cyclin, as a regulator of neuronal genomic stability and DNA repair positioned cyclin A2 as a potential therapeutic target for DNA repair dysfunction and as a radiation sensitizer. Thus, identifying small molecule regulators of cyclin A2 activity carries significant potential to regulate diverse cellular processes in both ageing/neurodegeneration and in cancer. No functional modulators of cyclin A2 are known to date. Here, we identified a potential allosteric cyclin A2 ligand binding pocket based on high-resolution structural data. Molecular dynamics simulations were used to generate diverse binding pocket conformations for application of the relaxed complex scheme. We then used structure-based virtual screening to find potential cyclin A2 inhibitors. Based on a consensus score of docked poses from Glide and AutoDock Vina, we identified about 60 promising hit compounds, where all PAINS scaffolds were removed from consideration. A biochemical luminescence assay of cyclin A2 function was used for experimental verification and identified three nanomolar inhibitors of cyclin A2. These novel cyclin A2 inhibitors are the first reported cyclin A2 inhibitors that do not target the cyclin A2 - CDK 2 protein-protein interface.

A Mammalian Genetic Complementation Assay for Assessing Cellular Resistance to Genotoxic Compounds.

A complementation assay was developed to determine whether alleles of DNA repair genes are necessary for repairing specific types of damage. The assay was established by measuring the resistance capacity of Rad51d-deficient mouse embryonic fibroblasts (MEFs) transfected with mammalian expression constructs. Here, we describe the methods used to assess colony survival following the treatment of transfected cells with genotoxic compounds. This approach provides a time-efficient and stringent strategy to screen genetic alleles for identifying regions or specific amino acid residues critical for function or regulation of DNA repair pathways.

Exposure to Engineered Nanomaterials: Impact on DNA Repair Pathways

Some engineered nanomaterials (ENMs) may have the potential to cause damage to the genetic material in living systems. The mechanistic machinery functioning at the cellular/molecular level, in the form of DNA repair processes, has evolved to help circumvent DNA damage caused by exposure to a variety of foreign substances. Recent studies have contributed to our understanding of the various DNA damage repair pathways involved in the processing of DNA damage. However, the vast array of ENMs may present a relatively new challenge to the integrity of the human genome; therefore, the potential hazard posed by some ENMs necessitates the evaluation and understanding of ENM-induced DNA damage repair pathways. This review focuses on recent studies highlighting the differential regulation of DNA repair pathways, in response to a variety of ENMs, and discusses the various factors that dictate aberrant repair processes, including intracellular signalling, spatial interactions and ENM-specific responses.

Nickel induces transcriptional down-regulation of DNA repair pathways in tumorigenic and non-tumorigenic lung cells.

The heavy metal nickel is a known carcinogen, and occupational exposure to nickel compounds has been implicated in human lung and nasal cancers. Unlike many other environmental carcinogens, however, nickel does not directly induce DNA mutagenesis, and the mechanism of nickel-related carcinogenesis remains incompletely understood. Cellular nickel exposure leads to signaling pathway activation, transcriptional changes and epigenetic remodeling, processes also impacted by hypoxia, which itself promotes tumor growth without causing direct DNA damage. One of the mechanisms by which hypoxia contributes to tumor growth is the generation of genomic instability via down-regulation of high-fidelity DNA repair pathways. Here, we find that nickel exposure similarly leads to down-regulation of DNA repair proteins involved in homology-dependent DNA double-strand break repair (HDR) and mismatch repair (MMR) in tumorigenic and non-tumorigenic human lung cells. Functionally, nickel induces a defect in HDR capacity, as determined by plasmid-based host cell reactivation assays, persistence of ionizing radiation-induced DNA double-strand breaks and cellular hypersensitivity to ionizing radiation. Mechanistically, we find that nickel, in contrast to the metalloid arsenic, acutely induces transcriptional repression of HDR and MMR genes as part of a global transcriptional pattern similar to that seen with hypoxia. Finally, we find that exposure to low-dose nickel reduces the activity of the MLH1 promoter, but only arsenic leads to long-term MLH1 promoter silencing. Together, our data elucidate novel mechanisms of heavy metal carcinogenesis and contribute to our understanding of the influence of the microenvironment on the regulation of DNA repair pathways.

FLT3/ITD Cooperates with Rac1/DOCK2 to Modulate the Sensitivity of Leukemic Cells to Chemotherapeutic Agents Via Regulation of DNA Repair Pathways

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis. FLT3/ITD activates Rac1 GTPase, and both FLT3/ITD and Rac1 play key roles in the signaling network that activates signal transducer and activator of transcription 5 (STAT5) and promotes leukemogenesis.Utilizing a co-immunoprecipitation/mass spectrometry analysis, we found that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity and leads to decreased survival of leukemic cells with elevated FLT3 activity, both alone and in combination with cytarabine (Ara-C) treatment in both in vitro studies and a mouse xenograft model.We further investigated the mechanisms by which Rac1/DOCK2 activity affects cell survival and chemotherapeutic response in FLT3/ITD leukemic cells. Exogenous expression of FLT3/ITD in TF-1 cells resulted in elevated Rac1 activity, an increase in the level of reactive oxygen species, and increased activation of STAT5. Western and qPCR studies in these cells demonstrated a concomitant statistically significant increase in DNA damage response (DDR) factors, including Chk1, Rad51, Wee1, and PIM-1. Furthermore, expression of FLT3/ITD led to increased expression of AP-1 and its downstream targets, the mismatch repair (MMR) factors MSH2, MLH1 and MSH6. These FLT3/ITD-expressing TF-1 cells exhibited markedly increased resistance to Ara-C treatment, and the increase in IC50 positively correlates with the expression level of FLT3/ITD. These effects on DDR factors appear to be dependent on FLT3/ITD activity, as they were not observed in TF-1 cells expressing even relatively high levels of wild-type (WT) FLT3. These findings suggest that enhanced DDR may provide a mechanism of resistance to Ara-C treatment in FLT3/ITD cells.We then utilized DOCK2 KD in patient-derived leukemia cell lines that express FLT3/ITD (MV4;11 and Molm 14) to evaluate whether decreased Rac1 activity would restore sensitivity to Ara-C. DOCK2 KD via shRNA resulted in significantly reduced expression of Meis1 and c-Myb, which are known regulators of FLT3 expression. Accordingly, the expression level and activity of FLT3 in cells with DOCK2 KD are markedly decreased. The DOCK2 KD cells showed reduced STAT5 activity and decreased expression of Chk1, Rad51, Wee1 and PIM-1, as well as lower expression levels of AP1, MSH2, MSH6 and MLH1. Consistent with the reduction in activity of DDR factors, DOCK2 KD cells exhibited significantly increased sensitivity to Chk1 inhibitor MK8776, Wee1 inhibitor MK1775 and Rad51 inhibitor B02. Moreover, synergistic effects between these DDR inhibitors and Ara-C, as indicated by increased apoptosis and reduction in cell proliferation, were observed at markedly lower concentrations in FLT3/ITD cells with DOCK2 KD. In contrast, DOCK2 KD in a leukemia cell line expressing WT FLT3 (REH) did not lead to down-regulation of the activity of FLT3 or DDR factors, and no enhancement in sensitivity toward DDR inhibitors was observed.These findings suggest that FLT3/ITD and Rac1 activity cooperatively modulate DDR and MMR activity in leukemic cells. The increased DNA damage repair activity of FLT3/ITD leukemia cells may play a role in the relatively poor response of FLT3/ITD AML to standard AML chemotherapeutic regimens. Therefore, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML, and pharmacologic inhibition of the Rac signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-029: Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic

Abstract Environmental exposure to certain heavy metals, such as nickel and arsenic, has been implicated in a variety of human cancers, including lung, skin, digestive track, and bladder cancers. Importantly, the mechanism underlying the carcinogenicity of nickel and arsenic remains poorly understood as they do not induce direct DNA mutagenesis. However, they do lead to global changes in chromatin structure and transcription, many similar to the effects of hypoxia. Since hypoxia is known to regulate many different DNA repair pathways, we investigated whether nickel and arsenic may similarly affect cellular DNA repair. We discovered that nickel and arsenic can lead to alterations in DNA repair gene expression, stable gene silencing, and decreased DNA repair capacity. First, we measured protein and mRNA levels of different DNA repair genes after NiCl2 or NaAsO2 treatment. We found that both metals induced down-regulation of BRCA1, FANCD2, and MLH1 over 24 to 48 hours at both the protein and mRNA levels. These results were observed in several different cells lines (HeLa, MCF7, BEAS-2B) with one notable exception that high dose arsenic induced up-regulation of BRCA1, FANCD2, and MLH1 in lung cancer-derived cell lines (A549, HCC827, NCI-H460). Next, to study the impact of long-term heavy metal exposure on DNA repair gene expression, we utilized an MLH1 promoter reporter construct that allows selection of cells harboring a silenced MLH1 promoter with ganciclovir. RKO cells stably expressing this construct were grown in the presence of 100 μM NiCl2, 0.5 μM NaAsO2, 1% oxygen, or control conditions. After 3 weeks, we observed that arsenic treatment, like hypoxia, led to a significant increase in promoter silencing compared to control cells, peaking at about 3.7-fold after 4 weeks. Nickel did not increase silencing, which may indicate a different mechanism of gene regulation. Finally, we used a luciferase assay to measure the effect of nickel and arsenic on the two primary DNA double-strand break (DSB) repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ). BEAS-2B cells pretreated with 250 μM NiCl2 or 5 μM NaAsO2 were transfected with a digested, inactive luciferase plasmid and allowed to conduct DSB repair to reactivate luciferase expression. We found that nickel and arsenic led to a 40-50% reduction in cellular HR capacity with no significant effect on NHEJ. To further pursue these results, we are performing chromatin immunoprecipitation studies to identify transcriptional or epigenetic factors mediating nickel and arsenic-induced down-regulation of DNA repair genes. In addition, we are using chromosomal-based assays to further characterize the impact of nickel and arsenic on DNA repair capacity. In conclusion, we have found that nickel and arsenic negatively regulate cellular DNA repair pathways, identifying a novel way in which heavy metals may contribute to carcinogenesis. Citation Format: Susan E. Scanlon, Christine D. Scanlon, Denise C. Hegan, Parker Sulkowski, Peter M. Glazer. Negative transcriptional and epigenetic regulation of DNA repair pathways by the heavy metals nickel and arsenic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-029.

Abstract 80: Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response

Abstract Background Hypoxia occurs within the majority of solid tumors. It has multiple impacts on tumor biology including angiogenesis, suppression of immune reactivity, enhanced receptor tyrosine kinase signaling, down regulation of DNA repair pathways, promotion of pro-survival phenotypes and increased proclivity for invasion and metastasis. Levels of hypoxia vary between and within tumors, correlate with patient outcomes, and can lead to differences in response to therapy. Despite considerable advances, our understanding of the regulatory processes that control these diverse and heterogeneous changes in cancer phenotype remain incomplete. A better understanding of how they are mediated will inform strategies for personalized medicine and promise to substantially advance our understanding of tumor heterogeneity. Methods Until recently, techniques for RNA-expression profiling have lacked the resolution necessary to detect genome wide changes in splicing. We exploited the increased precision offered by deep sequencing to investigate genome-wide remodelling of transcript architectures in both cell line and tumor samples. Using novel computational strategies we generated sample-specific gene models from RNA sequencing data generated over a timecourse of HCT116 colorectal carcinoma cells following a switch to 1% oxygenation and used these data to identify widespread changes in splicing. We applied similar strategies to raw RNA-sequencing data derived from TCGA. Results We detected the expression of thousands of novel isoforms and a systematic, pathway-dependent switch to the expression of noncoding transcripts at multiple genes, including many constituents of the Fanconi Anemia, nucleotide excision- and double strand break repair pathways. Of particular note were HDAC6 and 53BP1: critical regulators of DNA damage response. Both loci exhibited a hypoxia-dependent expression of retained-intron transcripts and a concomitant decline in protein levels, thus providing a novel and unanticipated mechanism by which hypoxia can drive genetic instability. Predictions made using our cell line model were recapitulated in a large cohort of 458 colorectal carcinomas derived from TCGA. Validation of the cell line data revealed, for the first time, a strong signature of isoform switching in which a widespread transition to noncoding expression is associated with poor patient outcome. These data were derived from independent patient biopsies sequenced by others, thus demonstrating the robustness of our findings. Conclusions These data therefore have clear, substantial, and immediate implications both for our understanding of the basic aetiology of solid tumors and for the development of RNA-based signatures and biomarkers in diagnostics and personalized medicine. Citation Format: Crispin Miller, Danish Memon, Keren Dawson, Christopher Smowton, Wei Xing, Caroline Dive. Programmatic re-splicing of the hypoxia transcriptome regulates the DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 80.

Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis.

The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage repair (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate, however, that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family. Targeting of these pathways, especially through PARP inhibition, is now being exploited therapeutically to effect significant clinical responses in subsets of individuals, particularly in patients with ovarian cancer or prostate cancer, including cancers with a marked metastatic burden. Targeting DNA repair-deficient tumors with drugs that take advantage of the fundamental differences between normal repair-proficient cells and repair-deficient tumors offers new avenues for treating advanced disease in the future. Clin Cancer Res; 22(13); 3132-7. ©2016 AACR.

Multifaceted control of DNA repair pathways by the hypoxic tumor microenvironment.

Hypoxia, as a pervasive feature in the microenvironment of solid tumors, plays a significant role in cancer progression, metastasis, and ultimately clinical outcome. One key cellular consequence of hypoxic stress is the regulation of DNA repair pathways, which contributes to the genomic instability and mutator phenotype observed in human cancers. Tumor hypoxia can vary in severity and duration, ranging from acute fluctuating hypoxia arising from temporary blockages in the immature microvasculature, to chronic moderate hypoxia due to sparse vasculature, to complete anoxia at distances more than 150 μM from the nearest blood vessel. Paralleling the intra-tumor heterogeneity of hypoxia, the effects of hypoxia on DNA repair occur through diverse mechanisms. Acutely, hypoxia activates DNA damage signaling pathways, primarily via post-translational modifications. On a longer timescale, hypoxia leads to transcriptional and/or translational downregulation of most DNA repair pathways including DNA double-strand break repair, mismatch repair, and nucleotide excision repair. Furthermore, extended hypoxia can lead to long-term persistent silencing of certain DNA repair genes, including BRCA1 and MLH1, revealing a mechanism by which tumor suppressor genes can be inactivated. The discoveries of the hypoxic modulation of DNA repair pathways have highlighted many potential ways to target susceptibilities of hypoxic cancer cells. In this review, we will discuss the multifaceted hypoxic control of DNA repair at the transcriptional, post-transcriptional, and epigenetic levels, and we will offer perspective on the future of its clinical implications.

Abstract 3408: FOXM1 regulates doxorubicin resistance through the non homologous end joining pathway and is associated with recurrence of bladder cancer

Abstract Non-muscle invasive bladder cancer (NMIBC) remains a very troublesome cancer to care because of high recurrence rates. In the previous study, we characterized a gene expression dataset from 102 NMIBC patients of the Korean cohort to identify a signature associated with disease recurrence. In recurrent bladder cancers, the expression of FOXM1 (Forkhead Box M1) was increased compared to primary cancers. Although there is a relationship between DNA repair pathway and FOXM1, it remains unclear whether FOXM1 is associated with DNA repair mediated cancer recurrence. To investigate this association, we used five different human bladder cancer cell lines to examine the expression level of FOXM1. Two cancer cell lines (5637 and EJ cells) showed the increased level of FOXM1 than other three cancer cell lines (UC5, LD611 and UC9 cells). We examined the chemoresistance with chemotherapy reagent (Doxorubicin) and checked the possible regulation of DNA repair pathway by FOXM1 in these cell lines. The cell lines which elevated FOXM1 expression showed high expression levels of Non-homologous end joining (NHEJ) pathway genes (Ku70, Ku80, LIG4 and XRCC4). To examine the direct interaction between the FOXM1 and NHEJ pathway genes, the binding activity was checked by a promoter assay and chromatin immunoprecipitation. We revealed that the expression levels of NHEJ pathway genes are elevated on activation of exogenous FOXM1. Therefore, we suggest a novel activity of FOXM1 has a role in cancer recurrence through the NHEJ pathway in bladder cancer cells. Citation Format: Yun-Gil Roh, Se-Ra Lee, Won-Tae Kim, Seon-Kyu Kim, In-Sun Chu, Sun-Hee Leem. FOXM1 regulates doxorubicin resistance through the non homologous end joining pathway and is associated with recurrence of bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3408. doi:10.1158/1538-7445.AM2014-3408

Abstract 3971: PPM1D/WIP1, a potential target in neuroblastoma

Abstract Background: Gain of 17q is the most powerful genetic predictor of adverse clinical outcome in neuroblastoma (NB). 17q+ correlated with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastoma tumors. Specifically, gain of WIP1 at 17q23 is frequently detected in NB. WIP1 is a serine/threonine phosphatase encoded by the gene PPM1D, member of the PP2C family known to be regulators of DNA repair pathways. Methods: A formazan-based spectrophotometric method was used to measure cell viability in several NB cell lines treated with different WIP1 inhibitors. Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for WIP1 expression using immunoblotting, immunohistochemistry and qPCR. Stable WIP1 knockdown NB cells were generated by shRNA transfections. Clonogenicity of WIP1 knockdown were examined by clonogenic assay. In vivo, WIP1 knocked- and non-silenced cells were injected in nude NMRI mice followed by daily evaluation of tumor formation. Results: Detailed CGH analysis revealed that genomic gain of WIP1 is evident with at least one extra copy in all tumor samples showing 17q gain. Results from our NB cell lines demonstrate 17q aberrations in all samples. These findings were corroborated by expression pattern of WIP1 mRNA and different immunostaining techniques. In a wide range of NB cell lines we found that all WIP1 inhibitors had good cytotoxic effect at higher concentrations (50µM) while some showed cytotoxic effects at lower concentrations. Transfection experiments with shRNA against WIP1 showed decreased cell viability, proliferation and colony formation in NB cells. Tumor xenograft development after WIP1 downregulation was significantly delayed with time to tumor development (0.10 mL) more than doubled compared to animals injected with cells transfected with the scrambled construct (15 days median, vs. 33 days). Conclusions: Our data suggest that WIP1 expression have significant oncogenic function in neuroblastoma development. Our results showed that WIP1 is present in at least one extra genomic copy in the majority of primary NB. WIP1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. In addition, WIP1 inhibition could be a potential target in treating high-risk neuroblastoma but more studies investigating the effects of WIP1 inhibition in neuroblastoma are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Malin Wickström, Diana Treis, Hjalmar Ståhlberg Nordegren, Lotta Elfman, Ninib Baryawno, Susanne Fransson, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/WIP1, a potential target in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2014-3971

Proteomic Changes in Rat Spermatogenesis in Response to In Vivo Androgen Manipulation; Impact on Meiotic Cells

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70–2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.

XPA-Mediated Regulation of Global Nucleotide Excision Repair by ATR Is p53-Dependent and Occurs Primarily in S-Phase

Cell cycle checkpoints play an important role in regulation of DNA repair pathways. However, how the regulation occurs throughout the cell cycle remains largely unknown. Here we demonstrate that nucleotide excision repair (NER) is regulated by the ATR/p53 checkpoint via modulation of XPA nuclear import and that this regulation occurs in a cell cycle-dependent manner. We show that depletion of p53 abrogated the UV-induced nuclear translocation of XPA, while silencing of Chk1 or MAPKAP Kinase-2 (MK2) had no effect. Inhibition of p53 transcriptional activities and silencing of p53-Ser15 phosphorylation also reduced the damage-induced XPA nuclear import. Furthermore, in G1-phase cells the majority of XPA remained in the cytoplasm even after UV treatment. By contrast, while most of the XPA in S-phase cells was initially located in the cytoplasm before DNA damage, UV irradiation stimulated bulk import of XPA into the nucleus. Interestingly, the majority of XPA molecules always were located in the nucleus in G2-phase cells no matter whether the DNA was damaged or not. Consistently, the UV-induced Ser15 phosphorylation of p53 occurred mainly in S-phase cells, and removal of cyclobutane pyrimidine dimers (CPDs) was much more efficient in S-phase cells than in G1-phase cells. Our results suggest that upon DNA damage in S phase, NER could be regulated by the ATR/p53-dependent checkpoint via modulation of the XPA nuclear import process. In contrast, the nuclear import of XPA in G1 or G2 phase appears to be largely independent of DNA damage and p53.

Aging Results in Differential Regulation of DNA Repair Pathways in Pachytene Spermatocytes in the Brown Norway Rat1

The present trend of increasing paternal age is accompanied by concerns for the development of complex multigene diseases (e.g., autism and schizophrenia) in progeny. Recent studies have established strong correlations between male age, increased oxidative stress, decreased sperm quality, and structural aberrations of chromatin and DNA in spermatozoa. We tested the hypothesis that increasing age would result in altered gene expression relating to oxidative stress and DNA damage/repair in germ cells. To test this hypothesis, pachytene spermatocytes and round spermatids were isolated from Brown Norway (BN) rats at 4 (young) and 18 (aged) mo of age. Microarray analysis was used to compare gene expression between the groups. The probe sets with significantly altered expression were linked to DNA damage/repair and oxidative stress in pachytene spermatocytes but not in round spermatids. Further analysis of pachytene spermatocytes demonstrated that genes involved in the base excision repair (BER) and nucleotide excision repair (NER) pathways were specifically altered. Quantitative RT-PCR confirmed that NER genes were upregulated (>1.5-fold), whereas BER genes were downregulated (>1.5-fold). At the protein level the members of the BER pathway were also altered by up to 2.3-fold; levels of NER proteins remained unchanged. Furthermore, there was an increase in 8-oxo-2'-deoxyguanosine (8-oxodG) immunoreactivity in testes from aged males and in the number of spermatozoa positive for 8-oxodG. In conclusion, aging is associated with differential regulation of DNA repair pathways with a decrease in the BER pathway leading to deficient repair of 8-oxo-dG lesions in germ cells and spermatozoa.

Stepping up melanocytes to the challenge of UV exposure

Exposure to solar ultraviolet radiation (UV) is the main etiological factor for skin cancer, including melanoma. Cutaneous pigmentation, particularly eumelanin, afforded by melanocytes is the main photoprotective mechanism, as it prevents UV-induced DNA damage in the epidermis. Therefore, maintaining genomic stability of melanocytes is crucial for prevention of melanoma, as well as keratinocyte-derived basal and squamous cell carcinoma. A critical independent factor for preventing melanoma is DNA repair capacity. The response of melanocytes to UV is mediated mainly by a network of paracrine factors that not only activate melanogenesis, but also DNA repair, anti-oxidant, and survival pathways that are pivotal for maintenance of genomic stability and prevention of malignant transformation or apoptosis. However, little is known about the stress response of melanocytes to UV and the regulation of DNA repair pathways in melanocytes. Unraveling these mechanisms might lead to strategies to prevent melanoma, as well as non-melanoma skin cancer.

DNA repair during organogenesis

DNA damage caused by genotoxic agents can impact on virtually any cellular process due to its ability to affect gene expression and subsequent gene products. The importance of repairing damaged DNA is evidenced by the variety of DNA repair pathways that have evolved in all living organisms, and the human syndromes caused by a lack of this repair ability. This review focuses on the expression and activity of DNA repair pathways during mammalian organogenesis, and the role of these pathways in ensuring the stability of the conceptal genome. DNA repair capacity may play a role also in the response of the conceptus to genotoxic agents that may induce malformations; the consequences of exposure to a genotoxic agent during organogenesis depend on the extent of the damage and on the ability of the embryo to respond by repairing DNA or arresting cell division. The four main repair pathways (nucleotide excision repair, base excision repair, mismatch repair, and recombination repair) are expressed to various degrees during organogenesis, as are members of the genotoxic stress-activated cell cycle checkpoint pathways. Developmental-stage-specific alterations in transcript levels, protein levels, as well as activity, indicate that the regulation of DNA repair pathways during development is complex. The importance of DNA repair pathways in endogenous damage control is illustrated by the sensitivity of development to their disruption if some of these genes are mutated. Furthermore, the conceptus has a limited capacity to alter DNA repair responses following exposure to genotoxic agents.