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Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

In mammalian cells, extracellular vesicles (EVs) derived from the endosomal system carry many different kinds of bioactive molecule to deliver to recipient cells in a paracrine or endocrine manner. EVs can mediate local and systemic intercellular communications, including reeducating stromal cells, remodeling the architecture of the tumor microenvironment, modulating cancer metabolism and metastases, or even conferring drug resistance. Because the molecular and functional characteristics of prostate cancer (PCa) evolve over time, the bioactive molecule profiles/signatures of tumor-derived EVs (TDEs) reflect the real-time status of cancer cells. TDEs appear to be valuable diagnostic and prognostic biomarkers as well as potential therapeutic vehicles, suggesting their essential role in precision medicine of disease management. We summarized critical aspects of TDEs in PCa and discussed their potential clinical applications.

This review focuses on DNA–dependent protein kinase (DNA–PK), which is the key regulator of canonical non–homologous end–joining (NHEJ), the predominant mechanism of DNA double–strand break (DSB) repair in mammals. DNA–PK consists of the DNA–binding Ku70/80 heterodimer and the catalytic subunit DNA–PKcs. They assemble at DNA ends, forming the active DNA–PK complex, which initiates NHEJ–mediated DSB repair. Paradoxically, both Ku and DNA–PKcs are associated with telomeres, and they play crucial roles in protecting the telomere against fusions. Herein, we discuss possible mechanisms and contributions of Ku and DNA–PKcs in telomere regulation.

Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activation of multiple LPA G protein-coupled receptors. We previously reported that LPA suppresses oxidative stress in premature aging Hutchinson-Gilford progeria syndrome (HGPS) patient fibroblasts via its type 3 receptor (LPA3). Mitochondria have been suggested to be the primary origin of oxidative stress via the overproduction of reactive oxygen species (ROS). Mitochondria are responsible for producing ATP through oxidative phosphorylation (OXPHOS) and have a calcium buffering capacity for the cell. Defects in mitochondria will lead to declined antioxidant capacity and cell apoptosis. Therefore, we aim to demonstrate the regulatory role of LPA3 in mitochondrial homeostasis. siRNA-mediated depletion of LPA3 leads to the depolarization of mitochondrial potential (ΔΨm) and cellular ROS accumulation. In addition, the depletion of LPA3 enhances cisplatin-induced cytochrome C releasing. This indicates that LPA3 is essential to suppress the mitochondrial apoptosis pathway. LPA3 is also shown to improve mitochondrial ADP-ATP exchange by enhancing the protein level of ANT2. On the other hand, LPA3 regulates calcium uptake from the ER to mitochondria via the IP3R1-VDAC1 channel. Moreover, activation of LPA3 by selective agonist OMPT rescues mitochondrial homeostasis of H2O2-induced oxidative stress cells and HGPS patient fibroblasts by improving mitochondrial ΔΨm and OXPHOS. In summary, our findings imply that LPA3 acts as the gatekeeper for mitochondrial healthiness to maintain cell youth. Furthermore, LPA3 can be a promising therapeutic target to prevent mitochondrial oxidative stress in aging and HGPS.

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.

Purpose: Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. Experimental Design: Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. Results: We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. Conclusions: In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.

Therapeutic drug synergism intervened in cancer treatments has been demonstrated to be more effective than using a single effector. However, it remains inherently challenging, with a limited cell count from tumor samples, to achieve potent personalized drug cocktails. To address the issue above, we herein present a nanodroplet cell processing platform. The platform incorporates an automatic nanodroplet dispenser with cell array ParaStamp chips, which were fabricated by a new wax stamping approach derived from laser direct writing. Such approach enables not only the on-demand de-wetting with hydrophobic wax films on substrates but also the mask-less fabrication of non-planar microstructures (i.e. no photolithography process). The ParaStamp chip was pre-occupied with anti-cancer drugs and their associate mixtures, enabling for the spatially addressable screening of optimal drug combinations simultaneously. Each droplet with a critical volume of 200 nl containing with 100 cells was utilized. Results revealed that the optimal combination reduces approximate 28-folds of conducted doses compared with single drugs. Tumor inhibition with the optimally selected drug combination was further confirmed by using PC-3 tumor-bearing mouse models. Together, the nanodroplet cell processing platform could therefore offer new opportunities to power the personalized cancer medicine at early-stage drug screening and discovery.

Purpose Harnessing the immune-stimulatory effects of radiation by combining it with immunotherapy is a promising new treatment strategy. However, more studies characterizing immunotherapy and radiation dose scheduling for the optimal therapeutic effect is essential for designing clinical trials. Methods and Materials A new ablative radiation dosing scheme, personalized ultrafractionated stereotactic adaptive radiation therapy (PULSAR), was tested in combination with α-PD-L1 therapy in immune-activated and resistant syngeneic immunocompetent mouse models of cancer. Specifically, tumor growth curves comparing immunotherapy and radiation therapy dose sequencing were evaluated in immunologically cold and hot tumor models. The response relative to cytotoxic killer T cells was evaluated using an α-CD8 depleting antibody, and immunologic memory was tested by tumor rechallenge of cured mice. Results We report that both radiation and immunotherapy sequencing, as well as radiation therapy fraction spacing, affect the combination treatment response. Better tumor control was achieved by giving α-PD-L1 therapy during or after radiation, and spacing fractions 10 days apart (PULSAR) achieved better tumor control than traditional daily fractions. We showed that CD8+ depleting antibody abrogated tumor control in the PULSAR combination treatment, and certain treatment schedules induced immunologic memory. Conclusions These results illustrate that radiation therapy dosing and scheduling affect tumor control, in combination with checkpoint blockade therapies. PULSAR-style radiation dosing is more complementary in combination with single-agent immunotherapy than traditional daily fractions in this preclinical model. Preclinical investigation could prove helpful in designing clinical trials investigating combination therapy.

Combining targeted therapeutic agents is an attractive cancer treatment strategy associated with high efficacy and low toxicity. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is an essential factor in DNA damage repair. Studies from us and others have revealed that DNA-PKcs also plays an important role in normal mitosis progression. Histone deacetylase (HDACs) inhibitors commonly lead to mitotic aberration and have been approved for treating various cancers in the clinic. We showed that DNA-PKcs depletion or kinase activity inhibition increases cancer cells’ sensitivity to HDACs inhibitors in vitro and in vivo. DNA-PKcs deficiency significantly enhances HDACs inhibitors (HDACi)-induced mitotic arrest and is followed by apoptotic cell death. Mechanistically, we found that DNA-PKcs binds to HDAC6 and facilitates its acetylase activity. HDACi is more likely to impair HDAC6-induced deacetylation of HSP90 and abrogate HSP90’s chaperone function on Aurora A, a critical mitotic kinase that regulates centrosome separation and mitotic spindle assembly in DNA-PKcs-deficient cells. Our current work indicates crosstalk between DNA-PKcs and HDACs signaling pathways, and highlights that the combined targeting of DNA-PKcs and HDACs can be used in cancer therapy. Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit, HDACs, Histone deacetylases, DSBs, DNA double-strand breaks, ATM, ataxia telangiectasia mutated, ATR, ATM-Rad3-related

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signaling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1 associated ring domain 1 (BARD1) complex, in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this event is essential for productive transcription. Our in vitro biochemical analyses showed TOP2B and BARD1 interaction and colocalization in the EGR1 transcription start site (TSS) and that the BRCA1-BARD1 complex ubiquitinates TOP2B, which appears to stabilize TOP2B protein in the cell and binding to DNA. Intriguingly, BRCA1 phosphorylation at S1524 controls this interaction. In addition, genomic analyses indicated colocalization between TOP2B and BRCA1 in a large number of protein-coding genes. Together, these findings reveal the novel function of the BRCA1-BARD1 complex in gene expression and in the regulation of TOP2B during Pol II transcription.Significance StatementMaintaining genomic integrity against cellular and extracellular genotoxic elements is essential for normal cell growth and function. Recent studies indicated that stimulus-induced transcription provokes topoisomerase IIβ-mediated DNA strand break and DNA damage response signaling, requiring DNA repair to be coupled with transcription. Here, we present a novel role for the BRCA1-BARD1 complex in regulating the transcription of serum-inducible genes and the stability of topoisomerase IIβ. The mechanism involving topoisomerase IIβ ubiquitination by the BRCA1-BARD1 complex and the phosphorylation of BRCA1 S1524 upon transcriptional activation appears to function as a switch to the reaction. Our findings provide the first evidence of functional interaction between the BRCA1-BARD1 complex and topoisomerase IIβ in transcription in humans.

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA(2) is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA(3) displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA(2) and LPA(3) determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA(2) agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA(3) agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA(2) predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA(3) increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA(3) signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA(2) and LPA(3) at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

Astronauts on interplanetary missions - such as to Mars - will be exposed to space radiation, a spectrum of highly-charged, fast-moving particles that includes 56Fe and 28Si. Earth-based preclinical studies show space radiation decreases rodent performance in low- and some high-level cognitive tasks. Given astronaut use of touchscreen platforms during training and space flight and given the ability of rodent touchscreen tasks to assess functional integrity of brain circuits and multiple cognitive domains in a non-aversive way, here we exposed 6-month-old C57BL/6J male mice to whole-body space radiation and subsequently assessed them on a touchscreen battery. Relative to Sham treatment, 56Fe irradiation did not overtly change performance on tasks of visual discrimination, reversal learning, rule-based, or object-spatial paired associates learning, suggesting preserved functional integrity of supporting brain circuits. Surprisingly, 56Fe irradiation improved performance on a dentate gyrus-reliant pattern separation task; irradiated mice learned faster and were more accurate than controls. Improved pattern separation performance did not appear to be touchscreen-, radiation particle-, or neurogenesis-dependent, as 56Fe and 28Si irradiation led to faster context discrimination in a non-touchscreen task and 56Fe decreased new dentate gyrus neurons relative to Sham. These data urge revisitation of the broadly-held view that space radiation is detrimental to cognition.

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N’ terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

Background/Aims: Hyperglycemia has been shown to increase the incidence and metastasis in various types of cancers. However, the correlation between hyperglycemia and lymphatic metastasis in prostate cancer (PCa) remains unclear. Our previous study demonstrated that lysophosphatidic acid (LPA) enhances vascular endothelial growth factor-C (VEGF-C) expression, a lymphangiogenic factor, through activating it receptors LPA1/3 in prostate cancer (PCa) cells. Moreover, hyperglycemia up-regulates autotaxin (ATX) expression, a LPA-generating enzyme. Therefore, we propose that high glucose promotes VEGF-C expression through LPA signaling in PCa cells. Methods: Pharmacological inhibitors and siRNAs were utilized to investigate the molecular mechanism of high glucose-induced VEGF-C expression. Real-time PCR and Western blot were used to determine the mRNA and protein expressions, respectively. Cellular bioenergetics analysis was performed to determine the glycolysis levels. Results: We demonstrated that the expressions of VEGF-C, ATX, and calreticulin were increased upon high glucose treatments in PC-3 cells. Moreover, high glucose-induced VEGF-C expression was mediated through the LPA1/3, PLC, Akt, ROS and LEDGF-dependent pathways. Additionally, high glucose enhanced the aerobic glycolysis via LPA1/3. Conclusion: These results indicated that hyperglycemia leads to LPA synthesis, and subsequent promoting pathological consequence of PCa. These novel findings could potentially provide new strategies for PCa treatments.

LILRB4, also known as ILT3, is an immune inhibitory transmembrane protein found on monocytes and antigen presenting cells, including dendritic cells (DCs). LILRB4 upregulation in DCs induces a tolerogenic phenotype that facilitates the generation of antigen-specific T regulatory cells. LILRB4 is also expressed on acute myeloid leukemia (AML) with monocytic differentiation, in which it promotes T cell suppression and tumor infiltration. IO-202 is a first-in-class LILRB4 antagonist antibody that is being evaluated in a Phase I trial (NCT04372433) for the treatment of AML and chronic myelomonocytic leukemia (CMML). RNA-seq data from TCGA database indicates that LILRB4 expression is upregulated in many solid tumor types. Therefore, the therapeutic potential of IO-202 in solid tumors was investigated in this study. Using computational biology approaches, we found that high LILRB4 expression in solid tumors from TCGA database is associated with macrophage infiltration in the tumor microenvironment. Flow cytometric analysis of cancer patients' blood and tumor samples confirmed LILRB4 expression on tumor-associated macrophages (TAMs) and on monocytic myeloid-derived suppressor cells (M-MDSCs) and DCs in tumor and periphery. These data raised the hypothesis that LILRB4 functions as a myeloid checkpoint and contributes to tumor immune evasion, not only in AML, but also in many solid tumor types. LILRB4 is not present in rodents. As such, the functional activity and anti-tumor efficacy of IO-202 were evaluated using human primary immune cells and immunocompetent LILRB4 transgenic mice, respectively. In healthy donor monocyte-derived DCs, IO-202 treatment promoted DC maturation, activation and an antigen presenting phenotype, as well as enhanced their ability to activate allogeneic T cells. In the radiation therapy (RT)-resistant Lewis lung carcinoma (LLC) syngeneic model, IO-202 in combination with RT resulted in tumor growth inhibition, which was associated with changes in the relative abundance of tumor-infiltrating immune cells, evidenced by a reduction in M-MDSCs and an increase in T cells, along with increased T cell activation. In conclusion, our data suggest that LILRB4 functions as a myeloid checkpoint in multiple cancer types and that IO-202, the first-in-class LILRB4 antagonist antibody, enhances dendritic cell function and T cell activation in vitro and anti-tumor immunity in a solid tumor model in vivo. These data demonstrate the therapeutic potential of IO-202 as a myeloid checkpoint inhibitor for solid tumors. Citation Format: Maria José Costa, Tao Huang, Jing-Tyan Ma, Caroline Bonnans, Azita Tabrizi, Xiaoling C. Liao, Paul Woodard, Benjamin P. Chen, Ching-Cheng Hsu, Mi Deng, Cheng Cheng Zhang, An Song. IO-202, a first-in-class LILRB4 antagonist antibody, activates dendritic cells and inhibits solid tumor growth in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1629.

Repetitive DNA is prone to replication fork stalling, which can lead to genome instability. Here, we find that replication fork stalling at telomeres leads to the formation of t‐circle‐tails, a new extrachromosomal structure that consists of circular telomeric DNA with a single‐stranded tail. Structurally, the t‐circle‐tail resembles cyclized leading or lagging replication intermediates that are excised from the genome by topoisomerase II‐mediated cleavage. We also show that the DNA damage repair machinery NHEJ is required for the formation of t‐circle‐tails and for the resolution of stalled replication forks, suggesting that NHEJ, which is normally constitutively suppressed at telomeres, is activated in the context of replication stress. Inhibition of NHEJ or knockout of DNA‐PKcs impairs telomere replication, leading to multiple‐telomere sites (MTS) and telomere shortening. Collectively, our results support a “looping‐out” mechanism, in which the stalled replication fork is cut out and cyclized to form t‐circle‐tails, and broken DNA is religated. The telomere loss induced by replication stress may serve as a new factor that drives replicative senescence and cell aging.

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G protein-coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish.

Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.

Astronauts on interplanetary space missions - such as to Mars - will be exposed to space radiation, a spectrum of highly-charged, fast-moving particles that includes 56Fe and 28Si. Earth-based preclinical studies with mature, “astronaut-aged” rodents show space radiation decreases performance in low- and some high-level cognitive tasks. Given the prevalence of touchscreens in astronaut training and in-mission assessment, and the ability of rodent touchscreen tasks to assess the functional integrity of brain circuits and multiple cognitive domains in a non-aversive way, it is surprising the effect of space radiation on rodent touchscreen performance is unknown. To fill this knowledge gap, 6-month-old C57BL/6J male mice were exposed to whole-body space radiation and assessed on a touchscreen battery starting 1-month later. Relative to Sham, 56Fe irradiation did not overtly change performance on tasks of visual discrimination, reversal learning, rule-based, or object-spatial paired associates learning, suggesting preserved functional integrity of supporting brain circuits. Surprisingly, 56Fe irradiation led to better performance on a dentate gyrus-reliant task of pattern separation ability. Irradiated mice discriminated similar visual cues in ∼40% fewer days and ∼40% more accurately than control mice. Improved pattern separation was not touchscreen-, radiation-particle, or neurogenesis-dependent, as both 56Fe and 28Si irradiation led to faster context discrimination (e.g. Sham Block 5 vs. 56Fe Block 2) in a non-touchscreen task and 56Fe led to fewer new dentate gyrus neurons relative to Sham. These data urge revisitation of the broadly-held view that space radiation is detrimental to cognition.SIGNIFICANCE STATEMENTAstronauts on an interplanetary mission - such as to Mars - will be unavoidably exposed to galactic cosmic radiation, a spectrum of highly-charged, fast-moving particles. Rodent studies suggest space radiation is detrimental to cognition. However, here we show this is not universally true. Mature mice that received whole body exposure to Mars-relevant space radiation perform similarly to control mice on high-level cognitive tasks, reflecting the functional integrity of key neural circuits. Even more surprisingly, irradiated mice perform better than controls in both appetitive and aversive tests of pattern separation, a mission-critical task reliant on dentate gyrus integrity. Notably, improved pattern separation was not touchscreen-, radiation-particle-, or neurogenesis-dependent. Our work urges revisitation of the generally-accepted conclusion that space radiation is detrimental to cognition.

The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately, no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries. We observed that the vanillin derivative VND3207 improved the survival of lethally irradiated mice by promoting intestinal regeneration and increasing the number of surviving crypts. Pre-treatment with VND3207 significantly increased the number of Lgr5+ intestinal stem cells (ISCs) and their daughter cells, the transient Ki67+ proliferating cells. Mechanistically, VND3207 decreased oxidative DNA damage and lipid peroxidation and maintained endogenous antioxidant status by increasing the level of superoxide dismutase and total antioxidant capacity. In addition, VND3207 maintained appropriate levels of activated p53 that triggered cell cycle arrest but were not sufficient to induce NOXA-mediated apoptosis, thus ensuring DNA damage repair in the irradiated small intestinal crypt cells. Furthermore, VND3207 treatment restores the intestinal bacterial flora structures altered by TBI exposure. In conclusion, VND3207 promoted intestinal repair following radiation injury by reducing reactive oxygen species-induced DNA damage and modulating appropriate levels of activated p53 in intestinal epithelial cells.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor‐like lipid mediator that regulates various physiological functions via activating multiple LPA G protein‐coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin‐transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1‐Oleoyl‐2‐O‐methyl‐rac‐glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish., In normal cells, activation of LPA3 stabilizes Nrf2 and enhances antioxidants to prevent accumulation of reactive oxygen species (ROS) and cell senescence. In Hutchinson–Gilford progeria syndrome (HGPS) cells, LPA3 is shown to be downregulated through high internalization and subsequent lysosomal degradation. The decline of LPA3 contributes to ROS accumulation and cell senescence of HGPS cells.

RNA polymerase II (Pol II) dependent transcription in stimulus-inducible genes requires topoisomerase IIβ-mediated DNA strand break and the activation of DNA damage response signaling. Here we report a novel function of DNA repair proteins, involving a breast cancer 1 (BRCA1)-BRCA1 associated RING domain 1 (BARD1) complex, in transcriptional activation of stimulus-inducible genes in humans. We have found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this phosphorylation event is essential for productive transcription. The major TOPIIβ binding site in the EGR1 TSS was mapped in the promoter between −132 and −14. We showed that BARD1 interacts with and could ubiquitinate TOPIIβ, and that BRCA1 regulates this interaction. Together, our findings indicate that the BRCA1-BARD1 complex controls TOPIIβ stability and gene association during Pol II transcription.

Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability.

Summary RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.

High-charge and -energy (HZE) particles comprise space radiation and they pose a challenge to astronauts on deep space missions. While exposure to most HZE particles decreases neurogenesis in the hippocampus&mdash, a brain structure important in memory&mdash, prior work suggests that 12C does not. However, much about 12C&rsquo, s influence on neurogenesis remains unknown, including the time course of its impact on neurogenesis. To address this knowledge gap, male mice (9&ndash, 11 weeks of age) were exposed to whole-body 12C irradiation 100 cGy (IRR, 1000 MeV/n, 8 kEV/&micro, m) or Sham treatment. To birthdate dividing cells, mice received BrdU i.p. 22 h post-irradiation and brains were harvested 2 h (Short-Term) or three months (Long-Term) later for stereological analysis indices of dentate gyrus neurogenesis. For the Short-Term time point, IRR mice had fewer Ki67, BrdU, and doublecortin (DCX) immunoreactive (+) cells versus Sham mice, indicating decreased proliferation (Ki67, BrdU) and immature neurons (DCX). For the Long-Term time point, IRR and Sham mice had similar Ki67+ and DCX+ cell numbers, suggesting restoration of proliferation and immature neurons 3 months post-12C irradiation. IRR mice had fewer surviving BrdU+ cells versus Sham mice, suggesting decreased cell survival, but there was no difference in BrdU+ cell survival rate when compared within treatment and across time point. These data underscore the ability of neurogenesis in the mouse brain to recover from the detrimental effect of 12C exposure.

High-charge and -energy (HZE) particles comprise space radiation and they pose a challenge to astronauts on deep space missions. While exposure to most HZE particles decreases neurogenesis in the hippocampus—a brain structure important in memory—prior work suggests that 12C does not. However, much about 12C’s influence on neurogenesis remains unknown, including the time course of its impact on neurogenesis. To address this knowledge gap, male mice (9–11 weeks of age) were exposed to whole-body 12C irradiation 100 cGy (IRR; 1000 MeV/n; 8 kEV/µm) or Sham treatment. To birthdate dividing cells, mice received BrdU i.p. 22 h post-irradiation and brains were harvested 2 h (Short-Term) or three months (Long-Term) later for stereological analysis indices of dentate gyrus neurogenesis. For the Short-Term time point, IRR mice had fewer Ki67, BrdU, and doublecortin (DCX) immunoreactive (+) cells versus Sham mice, indicating decreased proliferation (Ki67, BrdU) and immature neurons (DCX). For the Long-Term time point, IRR and Sham mice had similar Ki67+ and DCX+ cell numbers, suggesting restoration of proliferation and immature neurons 3 months post-12C irradiation. IRR mice had fewer surviving BrdU+ cells versus Sham mice, suggesting decreased cell survival, but there was no difference in BrdU+ cell survival rate when compared within treatment and across time point. These data underscore the ability of neurogenesis in the mouse brain to recover from the detrimental effect of 12C exposure.

Non-planar microstructure-based tissue culture devices have emerged as powerful tools to mimic in vivo physiological microenvironments in a wide range of medical applications. Here we report a spontaneous aqueous molding approach - inspired by Stenocara gracilipes beetles - to rapidly fabricate non-planar microstructure devices for facilitating tissue-based bioassays. The device fabrication is determined from the self-assembled liquid morphology, which is induced by condensation or guided by surface tension. Through experiments and modeling, we reveal that the molding mainly comprises two typical circular and striped domains, highlighting versatile applications for bioengineering. In addition, the molding characteristic is dependent on the geometry of the patterned wetting surfaces, the working volume of the liquid, and the interaction between the liquid and the substrate. The theoretical model, based on the geometry of the patterned liquid, is highly consistent with experimental data. We also demonstrate that our approach can facilitate the culturing of tumor spheroids incorporated with biomimic nano-cilia, rapid high-throughput drug screening, tumor spheroid migration assay, and in vitro modeling of blood vessels. Remarkably, the delivery of multiple concentrations of drugs and their associate mixtures (a total of 25 test spots in one device) can be carried out simultaneously within seconds. Taken together, these insights may offer new opportunities to tailor non-planar microstructures, and our proposed methodology can be applicable for the emerging needs in tumor cell biology and tissue engineering.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

Pit mud play a key role in the brewing process of Chinese Strong-flavour liquor. In this study, the organic acids and metal ions was determinated by Ion Chromatograph and Inductively Coupled Plasma Mass Spectrometry, respectively. The diversities and structures in pit mud were analyzed by using high-throughput sequencing based on Illumina Miseq platform. The results showed that the lactic acid in calcified pit mud was about 10 times higher than quality pit mud, and the calcium ions was about 20 times higher than quality pit mud. It could found a strange phenomenon, the calcified pit mud had more pyruvic acid and quality pit mud basically does not had this kind of material. The data analysis shown that the pit mud were detected prokaryotes 908 strains and the eukaryotes 226 strains. It was found that the Lactobacillus and Prevotella in calcified pit mud (the total of both increase 11 times) had a higher percentage. It can be guess that the lactic acid and metal ions would form lactic acid salt compounds and those compounds could affect the growth and reproduction of microbes in pit mud. This study provided a good comparison of physical and chemical properties between the calcified pit mud and the quality pit mud, which was significant for the maintenance of pit mud and the quality of liquor. Because of the first study for pit mud by using high-throughput sequencing, it could provide theoretical basis for improving microbes in pit mud.

Purpose: Variations in the radiosensitivity of tumor cells within and between tumors impact tumor response to radiation, including the dose required to achieve permanent local tumor control. The increased expression of DNA-PKcs, a key component of a major DNA damage repair pathway in tumors treated by radiation, suggests that DNA-PKcs– dependent repair is likely a cause of tumor cell radioresistance. This study evaluates the relative biological effect of spread-out Bragg-peak protons in DNA-PKcs–deficient cells and the same cells transfected with a functional DNA-PKcs gene. Materials and Methods: A cloned radiation-sensitive DNA-PKcs–deficient tumor line and its DNA-PKcs–transfected resistant counterpart were used in this study. Thepresence of functional DNA-PKcs was evaluated by DNA-PKcs autophosphorylation.Cells to be proton irradiated or x-irradiated were obtained from the same single cell suspension and dilution series to maximize precision. Cells were concurrently exposed to 6-MV x-rays or mid 137-MeV spread-out Bragg peak protons and cultured for colony formation. Results: The surviving fraction data were well fit by the linear-quadratic model for each of 8 survival curves. The results suggest that the relative biological effectiveness of mid spread-out Bragg peak protons is approximately 6% higher in DNA-PKcs–mediated resistant tumor cells than in their DNA-PKcs–deficient and radiation-sensitive counterpart.Conclusion: DNA-PKcs–dependent repair of radiation damage is less capable ofrepairing mid spread-out Bragg peak proton lesions than photon-induced lesions, suggesting protons may be more efficient at sterilizing DNA-PKcs–expressing cells that are enriched in tumors treated by conventional fractionated dose x-irradiation.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at the Thr2609 cluster is essential for its complete function in DNA repair and tissue stem cell homeostasis. This phenomenon is demonstrated by congenital bone marrow failure occurring in DNA-PKcs(3A/3A) mutant mice, which require bone marrow transplantation (BMT) to prevent early mortality. Surprisingly, an increased incidence of spontaneous tumors, especially skin cancer, was observed in adult BMT-rescued DNA-PKcs(3A/3A) mice. Upon further investigation, we found that spontaneous γH2AX foci occurred in DNA-PKcs(3A/3A) skin biopsies and primary keratinocytes and that these foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation. Consistently, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs(3A/3A) cells as compared with wild-type and DNA-PKcs-knockout cells. In addition, a previously identified DNA-PKcs Thr2609Pro mutation, found in breast cancer, also induces a similar impairment of telomere leading-end maturation. Taken together, our current analyses indicate that the functional DNA-PKcs T2609 cluster is required to facilitate telomere leading strand maturation and prevention of genomic instability and cancer development.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

// Lan Yu 1, * , Zeng-Fu Shang 1, 2, 4, * , Feng-Ming Hsu 1, 3 , Zhang Zhang 1 , Vasu Tumati 1 , Yu-Fen Lin 1 , Benjamin P.C. Chen 1 , Debabrata Saha 1 1 Department of Radiation Oncology, Simmons Comprehensive Cancer Center at UT Southwestern Medical Center, Dallas, TX, USA 2 School of Radiation Medicine and Protection, Medical College of Soochow University, Suzhou Industrial Park, China 3 Department of Urology and Department of Oncology, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan 4 Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Suzhou Industrial Park, China * These authors have contributed equally to this work Correspondence to: Debabrata Saha, e-mail: debabrata.saha@utsouthwestern.edu Keywords: Radiosensitizer, Mitotic Catastrophe, Apoptosis, Autophagy, Senescence Received: July 18, 2014 Accepted: December 20, 2014 Published: February 28, 2015 ABSTRACT The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown that the DNA-PKcs inhibitor NU7441 is a highly potent radiosensitizer, however, the mechanism of NU7441's anti-proliferation effect has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been investigated. We found that NU7441 significantly enhances the effect of IR in all cell lines. The notable findings in response to this combined treatment are (i) prolonged delay in IR-induced DNA DSB repair, (ii) induced robust G2/M checkpoint, (iii) increased aberrant mitosis followed by mitotic catastrophe specifically in H1299, (iv) dramatically induced autophagy in A549 and (v) IR-induced senescence specifically in H460. H1299 cells show greater G2 checkpoint adaptation after combined treatment, which can be attributed to higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. In conclusion, choice of cell death pathway is dependent on the mutation status and other genetic factors of the cells treated.

Astronauts traveling to Mars will be exposed to chronic low doses of galactic cosmic space radiation, which contains highly charged, high-energy (HZE) particles. 56Fe-HZE-particle exposure decreases hippocampal dentate gyrus (DG) neurogenesis and disrupts hippocampal function in young adult rodents, raising the possibility of impaired astronaut cognition and risk of mission failure. However, far less is known about how exposure to other HZE particles, such as 28Si, influences hippocampal neurogenesis and function. To compare the influence of 28Si exposure on indices of neurogenesis and hippocampal function with previous studies on 56Fe exposure, 9-week-old C57BL/6J and Nestin-GFP mice (NGFP; made and maintained for 10 or more generations on a C57BL/6J background) received whole-body 28Si-particle-radiation exposure (0, 0.2 and 1 Gy, 300 MeV/n, LET 67 KeV/µ, dose rate 1 Gy/min). For neurogenesis assessment, the NGFP mice were injected with the mitotic marker BrdU at 22 h postirradiation and brains were examined for indices of hippocampal proliferation and neurogenesis, including Ki67+, BrdU+, BrdU+NeuN+ and DCX+ cell numbers at short- and long-term time points (24 h and 3 months postirradiation, respectively). In the short-term group, stereology revealed fewer Ki67+, BrdU+ and DCX+ cells in 1-Gy-irradiated group relative to nonirradiated control mice, fewer Ki67+ and DCX+ cells in 0.2 Gy group relative to control group and fewer BrdU+ and DCX+ cells in 1 Gy group relative to 0.2 Gy group. In contrast to the clearly observed radiation-induced, dose-dependent reductions in the short-term group across all markers, only a few neurogenesis indices were changed in the long-term irradiated groups. Notably, there were fewer surviving BrdU+ cells in the 1 Gy group relative to 0- and 0.2-Gy-irradiated mice in the long-term group. When the short- and long-term groups were analyzed by sex, exposure to radiation had a similar effect on neurogenesis indices in male and female mice, although only male mice showed fewer surviving BrdU+ cells in the long-term group. Fluorescent immunolabeling and confocal phenotypic analysis revealed that most surviving BrdU+ cells in the long-term group expressed the neuronal marker NeuN, definitively confirming that exposure to 1 Gy 28Si radiation decreased the number of surviving adult-generated neurons in male mice relative to both 0- and 0.2-Gy-irradiated mice. For hippocampal function assessment, 9-week-old male C57BL/6J mice received whole-body 28Si-particle exposure and were then assessed long-term for performance on contextual and cued fear conditioning. In the context test the animals that received 0.2 Gy froze less relative to control animals, suggesting decreased hippocampal-dependent function. However, in the cued fear conditioning test, animals that received 1 Gy froze more during the pretone portion of the test, relative to controls and 0.2-Gy-irradiated mice, suggesting enhanced anxiety. Compared to previously reported studies, these data suggest that 28Si-radiation exposure damages neurogenesis, but to a lesser extent than 56Fe radiation and that low-dose 28Si exposure induces abnormalities in hippocampal function, disrupting fear memory but also inducing anxiety-like behavior. Furthermore, exposure to 28Si radiation decreased new neuron survival in long-term male groups but not females suggests that sex may be an important factor when performing brain health risk assessment for astronauts traveling in space.

We present a microarray chip integrated with novel round bottom μ-well arrays and biomimetic nano-cilia, which can, for the first time, not only recapitulate in vivo-like tumor microenvironment but also be achieved in situ for hallmarks of tumor-based bioassays.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5–8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

The combination of live cell imaging and laser micro-irradiation is an important technique to investigate the recruitment and kinetics of DNA repair molecules to DNA damage sites. In this chapter, we describe the detailed methods to study the dynamics of fluorescently tagged ATM protein kinase at laser-induced DNA double strand break (DSB) sites. The same protocol can be applied to analyze the recruitment and kinetics of other potential or known DNA repair proteins to DSB sites.

Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 μm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.

The ataxia telangiectasia mutated and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) axis is the major signaling pathway activated in response to replication stress and is essential for the intra-S checkpoint. ATR phosphorylates and activates a number of molecules to coordinate cell cycle progression. Chk1 is the major effector downstream from ATR and plays a critical role in intra-S checkpoint on replication stress. Activation of Chk1 kinase also requires its association with Claspin, an adaptor protein essential for Chk1 protein stability, recruitment and ATR-dependent Chk1 phosphorylation. We have previously reported that, on replication stress, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated by ATR at the stalled replication forks and is required for cellular resistance to replication stresses although the impact of DNA-PKcs onto the ATR signaling pathway remains elusive. Here we report that ATR-dependent Chk1 phosphorylation and Chk1 signaling are compromised in the absence of DNA-PKcs. Our investigation reveals that DNA-PKcs is required to maintain Chk1–Claspin complex stability and transcriptional regulation of Claspin expression. The impaired Chk1 activity results in a defective intra-S checkpoint response in DNA-PKcs–deficient cells. Taken together, these results suggest that DNA-PKcs, in addition to its direct role in DNA damage repair, facilitates ATR-Chk1 signaling pathway in response to replication stress.