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To investigate the population dynamics andRodents were captured using the mouse trap method in pastures from Batang Township and Longbao Township of Yushu City, Qinghai Province on May, August and October, 2018. The body weight and snout-vent length of all captured rodents were measured, and the species was identified according to the rodent morphology. Genomic DNA was extracted from rodent liver specimens and lesion specimens, and the mitochondrialA total of 285 small rodents were captured, including 143The small rodents around the human settlement in Yushu City of Qinghai Province mainly include

Lignification of cellulose limits the effective utilisation of fibre in plant cell wall. Lignocellulose-degrading bacteria secrete enzymes that decompose lignin and have the potential to improve fibre digestibility. Therefore, this study aimed to investigate the effect of whole-plant corn silage inoculated with lignocellulose-degrading bacteria on the growth performance, rumen fermentation, and rumen microbiome in sheep. Twelve 2-month-old male hybrid sheep (Dorper ♂ × small-tailed Han ♀) were randomly assigned into two dietary groups (n = 6): (1) untreated whole-plant corn silage (WPCS) and (2) WPCS inoculated with bacterial inoculant (WPCSB). Whole-plant corn silage inoculated with bacterial inoculant had higher in situ NDF digestibility than WPCS. Sheep in the WPCSB group had significantly higher average daily gain, DM intake, and feed conversion rate than those in the WPCS group (P 0.05). Furthermore, higher volatile fatty acid concentrations were detected in WPCSB rumen samples, leading to lower ruminal pH (P 0.05). The WPCSB group showed higher abundance of Bacteroidetes and lower abundance of Firmicutes in the rumen microbiome than the WPCS group (P 0.05). Multiple differential genera were identified, with Prevotella being the most dominant genus and more abundant in WPCSB samples. Moreover, the enriched functional attributes, including those associated with glycolysis/gluconeogenesis and citrate cycle, were more actively expressed in the WPCSB samples than in the WPCS samples. Additionally, certain glucoside hydrolases that hydrolyse the side chains of hemicelluloses and pectins were also actively expressed in the WPCSB microbiome. These findings suggested that WPCSB increased NDF digestibility in three ways: (1) by increasing the relative abundance of the most abundant genera, (2) by recruiting more functional features involved in glycolysis/gluconeogenesis and citrate cycle pathways, and (3) by increasing the relative abundance and/or expression activity of the glucoside hydrolases involved in hemicellulose and pectin metabolism. Our findings provide novel insights into the microbial mechanisms underlying improvement in the growth performance of sheep/ruminants. However, the biological mechanisms cannot be fully elucidated using only metagenomics tools; therefore, a combined multi-omics approach will be used in subsequent studies.

Coronaviruses are a common class of respiratory viruses that can cause human infections. 2019 novel coronavirus(2019-nCoV), a new coronavirus that has recently caused a pandemic, has affected millions of people and put tremendous pressure on the health systems of almost every country in the world. Coronaviruses are known to spread from person to person through droplets or contact. The 2019-nCoV has also been found in the conjunctival secretions and tears of some clinically diagnosed patients. To assess whether the eye is one of the transmission routes of the virus, we review literature, and summarize the anatomy of the eye-nose pathway, the expression of the virus receptor in the eye, the preclinical animal studies, and the clinical data. We analyze the possibility of eyes as a means of transmission and propose some suggestions of ocular protection.冠状病毒是可感染人类的一类常见呼吸道病毒，已知其是通过飞沫和密切接触在人与人之间传播。近期在世界范围内流行的2019新型冠状病毒（2019-nCoV）已造成数以千万人感染，在部分临床确诊患者的结膜分泌物和泪液中发现了2019-nCoV。本文就冠状病毒在眼表传染的解剖基础、其受体在眼部的表达特征、临床前动物研究结果以及患者临床表现，对文献进行总结和分析，以探讨眼部作为传播途径的可能性，同时提出眼部防护的思考和建议，以供眼科医师在临床工作中参考。

Acute liver injury (ALI) is associated with the Kupffer cells (KCs) inflammation and hepatocytes apoptosis. Previous studies have shown that miR-640 is a valid regulator of the Low-density lipoprotein receptor-related protein 1 (LRP 1) which expressed much lower in an inflammatory condition. However, it is unclear whether MiR-640 inhibition protects against ALI by the up-regulation of LRP 1. To explore the regulated mechanism of miR-640 on acute liver injury.We analyzed the expression of miR-640 in different times of acute injured liver tissues. Lipopolysaccharide (LPS) was employed in provoking the KCs inflammation to injure liver. We used miR-640 mimic or inhibitor to improve or resist the function of miR-640 to explore miR-640 function to ALI via the target of LRP1.We showed that the expression of miR-640 markedly increased in LPS-induced acute injured liver tissues. LPS promoted the progress of ALI, and the inhibition of miR-640 could reverse the injured effects of LPS. Moreover, WNT signaling pathway and LRP1 were significantly enhanced by miR-640 inhibition.These results suggested that miR-640 promotes KCs inflammation via restraining LRP 1 and WNT signaling pathway. But inhibiting miR-640 prevents inflammation damage and ameliorates ALI. MiR-640 inhibition may become a novel target for the therapy of ALI in the future.

Significance Analysis of synthetic lethal relationships identified in Saccharomyces cerevisiae was used to identify candidate therapeutic targets for cancers with BRCA1 or BRCA2 defects. Inhibition of one of these targets, flap endonuclease 1 (FEN1), with small-molecule inhibitors or siRNA knockdown of FEN1 expression preferentially killed BRCA1 and BRCA2 mutant cancer cell lines as well as some additional cancer cell lines without BRCA1 or BRCA2 defects. FEN1 inhibitors also preferentially reduced the growth of mouse xenografts established from FEN1 inhibitor-sensitive cancer cell lines. These studies support the use of S. cerevisiae genetics in identifying human therapeutic targets and establish FEN1 as a potential therapeutic target for further investigation and possible development., Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.

To evaluate the effects of water body environments on the microbial community ofSnails were caged water bodies in the Qianliang Lake marshland (Qianliang Lake regions) where natural extinction of snails was found and in the Junshan Park marshland (Junshan Park regions) in the eastern Dongting Lake for 30 days, and then all snails were collected and identified for survival or death. DNA sequencing of the fungi and bacteria was performed in snails before and after immersion in waters, and the biodiversity and abundance were analyzed.The survival rates ofThe water body environment affects the composition of the microbial community in

Pancreatic cancer is a malignant tumor of the digestive system with occult onset, low early diagnosis rate, rapid progression and poor prognosis. Perineural invasion (PNI) of pancreatic cancer is considered as an important factor which leads to the poor prognosis of pancreatic cancer. Neural invasion models of pancreatic cancer can simulate the occurrence and development of neural invasion of pancreatic cancer under the complex tumor microenvironment, which is an important carrier to study the molecular mechanism, early diagnosis and treatment improvement of PNI of pancreatic cancer. The current PNI model could be broadly divided into胰腺癌是一种恶性程度非常高的消化系统肿瘤，发病隐匿、早期诊断率低、进展快、预后差，胰腺癌的周围神经浸润(PNI)被认为是导致胰腺癌预后不良的重要因素。胰腺癌神经浸润模型可以在复杂的肿瘤微环境下模拟胰腺癌神经浸润的发生、发展过程，是研究胰腺癌PNI的分子机制、早期诊断和改良治疗方法的重要载体。PNI模型可以大致分为体内模型和体外模型，体内模型又可以分为异位模型和原位模型。近年来，在体外模型和体内模型基础上，又出现了基因工程小鼠模型、人源化肿瘤异种移植模型。文章分析了常用的胰腺癌神经浸润体外及体内模型的制备方法、临床用途和局限性。.

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal–mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene–transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress.

Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins.

Summary In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3 −/− mice, Stat3 is constitutively acetylated and naive CD4 + T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

LIF promotes self-renewal of mouse embryonic stem cells (mESCs), and in its absence, the cells differentiate. LIF binds to the LIF receptor (LIFR) and activates the JAK-STAT3 pathway, but it remains unknown how the receptor complex triggers differentiation or self-renewal. Here, we report that the LIFR cytoplasmic domain contains a self-renewal domain within the juxtamembrane region and a differentiation domain within the C-terminal region. The differentiation domain contains four SPXX repeats that are phosphorylated by MAPK to restrict STAT3 activation; the self-renewal domain is characterized by a 3K motif that is acetylated by p300. In mESCs, acetyl-LIFR undergoes homodimerization, leading to STAT3 hypo- or hyper-activation depending on the presence or absence of gp130. LIFR-activated STAT3 restricts differentiation via cytokine induction. Thus, LIFR acetylation and serine phosphorylation differentially promote stem cell self-renewal and differentiation.

The aim of this study was to investigate the effect of long non-coding ribonucleic acid (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) on sepsis-induced brain injury in mice through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).The mouse model of sepsis was established by cecal ligation and puncture induction. The relative expression levels of NEAT1 and NF-κB in brain tissues of mice in healthy group and sepsis group were determined via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting, respectively. Subsequently, the expression of NEAT1 was silenced by transfection of small interfering RNAs (siRNAs). Meanwhile, its effect on NF-κB expression was detected. To further explore the effect of sepsis on brain injury, the content of brain water and the expression levels of apoptosis-related proteins, including B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX), in mice of healthy group, sepsis group, and sepsis + si-NEAT1 group were measured. Furthermore, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to detect the proliferation and apoptosis of nerve cells.The relative expression levels of NEAT1 and NF-κB were significantly increased in the brain tissues of septic mice (p0.01). Si-NEAT1 transfection significantly decreased the expressions of NEAT1 and NF-κB in brain tissues of septic mice (p0.05). The content of brain water in mice of sepsis group was evidently increased (p0.05). However, si-NEAT1 treatment remarkably reduced this content (p0.05). In addition, sepsis markedly decreased the activity of nerve cells (p0.05). However, si-NEAT1 could significantly increase the activity of nerve cells in septic mice (p0.05). Moreover, si-NEAT1 notably decreased the expression of BAX (p0.05), whereas it increased the expression of Bcl-2 (p0.05). The results of apoptosis detection revealed that sepsis remarkably promoted the apoptosis of mouse nerve cells (p0.05). In addition, si-NEAT1 transfection could evidently alleviate the apoptosis of nerve cells in septic mice (p0.05).LncRNA NEAT1 promotes brain injury in septic mice by positively regulating NF-κB. However, si-NEAT1 transfection can reduce this injury.

Angiogenesis is a critical biological process essential for solid cancer growth and metastasis. It has been shown that microRNAs (miRNAs) play a vital role in a variety of biological processes in cancers. However, whether miR-130b is involved in prostate cancer angiogenesis remains ill-defined. We performed the miRNA microarray to analyze miRNA expression in human prostate cancer specimens. In vitro gain-of-function assays and loss-of-function assays were conducted to explore the potential functions of miR-130b in human prostate cancer cells. Correlation analysis and dual-luciferase reporter assay were performed to validate whether tumor necrosis factor-α (TNF-α) was a direct target of miR-130b. The Matrigel plug and tumor vascular imaging assays were performed to confirm the anti-angiogenic activity of miR-130b in nude mice. We found that miR-130b was one of the miRNAs being most significantly downregulated. Subsequently, we found that miR-130b expression was markedly downregulated in human prostate cancer cell lines. Down-regulation of miR-130b in prostate cancer cells significantly promoted the proliferation, invasion and tubule formation of human umbilical vein endothelial cells (HUVECs), while ectopic expression of miR-130b blocked prostate cancer angiogenesis in vitro and in vivo. Mechanistic analyses indicated that tumor necrosis factor-α (TNF-α) was regulated by miR-130b directly. MiR-130b attenuated nuclear factor-κB (NF-κB) signaling and its downstream gene vascular endothelial growth factor-A (VEGFA) by directly inhibiting TNF-α expression. Additionally, subsequent investigations identified that the ectopic level of VEGFA markedly abrogated the anti-angiogenic effect induced by miR-130b. Interestingly, VEGFA could in turn decrease the expression of miR-130b, thus forming a negative feedback loop that drives the angiogenesis of prostate cancer. These findings show that miR-130b/TNF-α/NF-κB/VEGFA feedback loop is significantly correlated with angiogenesis in prostate cancer and miR-130b could be regarded as potential therapeutic target for prostate cancer anti-angiogenesis treatment.

Background With advances in immunosuppressive therapy, heart transplantation is currently recommended as the only established surgical treatment for refractory heart failure. However, chronic immunosuppression increases the risk for malignancy. Everolimus (EVR) is a potent mammalian target of rapamycin inhibitor that is used after transplantation and to treat advanced malignancies, as we have done in Taiwan after heart transplantation since 2004. Mycophenolate mofetil (MMF) and EVR are frequently used as cell-cycle inhibitors to optimize post-transplantation outcomes. Methods We retrospectively analyzed the characteristics and outcomes of 454 patients who received either MMF (n = 232) or EVR (n = 222) after heart transplantation at the National Taiwan University Hospital from March 1, 1990, to March 1, 2015. Patient characteristics and Kaplan-Meier survival curves were compared between groups. Results During a median follow-up of 69.2 months, malignancy was diagnosed in 27 patients receiving MMF (n = 23) or EVR (n = 4). There was a significant difference in malignancy risk between groups (9.91% vs 1.80%, P = .001). The most common malignancies were non-Hodgkin lymphoma, skin cancers, and lung squamous cell carcinoma. The 2-year overall survival after malignancy was 50% in the EVR group and 47% in the MMF group (P = .745). Conclusions EVR treatment after heart transplant is associated with a lower risk of malignancy than is MMF treatment. The 2-year survival rate after malignancy was similar between EVR and MMF groups.

Epithelial cells aggregate into spheroids when deprived of matrix, and the proclivity for spheroid formation and survival is a hallmark of normal and tumorigenic mammary stem cells. We show here that Nuclear Respiratory Factor 1 (NRF1) is a spheroid promoter by in silico identification of this transcription factor as highly connected to top shRNA-hits deduced from re-iterative selections for shRNAs enriched in MCF10A spheroids. NRF1-promoted spheroid survival is linked to its stimulation of mitochondrial OXPHOS, cell migration, invasion, and mesenchymal transition. Conversely, NRF1 knockdown in breast cancer MDA-MB-231 cells reduced spheroids, migration, invasion, and mesenchymal marker expression. NRF1 knockdown also reduced tumor burden in mammary fat pads and lungs of orthotopic- or tail vein-transplanted mice. With the Luminal A subtype of breast cancer, higher NRF1 expression is associated with lower survival. These results show that NRF1, an activator of mitochondrial metabolism, supports mammary spheroid survival and tumor development.

The purpose of this study was to investigate how miR-203 promotes osteogenic differentiation of bone marrow mesenchymal cells (BMSCs) by regulating its target gene DKK1, thereby inhibiting the occurrence of osteoporosis.A total of 60 cases with postmenopausal osteoporosis and 40 cases of normal individuals were recruited. The expression of miR-203 in serum of all cases was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The capacity of osteogenesis and adipogenic differentiation of MSCs was determined by alizarin red staining and oil red staining, respectively. Transfection of miR-203 mimics and miR-203 inhibitor were mediated by Liposomes, and then the MSCs were induced osteogenic and adipogenic differentiation. MiR-203 mimic was co-transfected with wild-type or mutant DKK1 for luciferase reporter gene detection. In the osteoporosis model of rats, the tibia was taken for micro-CT examination of bone mineral density (BMD) and bone volume/structural parameters (BV/TV), while the femur was taken for the measurement of absorption parameters (Ob.S)./BS) and the number of osteoclasts per circumference of bone (N.Oc/B.Pm).The expression level of miR-203 was significantly lower in patients with postmenopausal osteoporosis than that in normal individuals. The osteogenic capacity of BMSCs in these patients was reduced, while their adipogenic capacity was enhanced. MiR-203 promoted the expression of osteogenic genes and inhibited that of adipogenic genes. Knockdown of miR-203 decreased the level of osteogenic related genes but increased that of adipogenic related genes, while overexpression of miR-203 led to the opposite results. Furthermore, miR-203 inhibited the protein expression of DKK1. In addition, bone density and bone volume/structural parameters were lower in ovariectomized rats than those in normal rats. Meanwhile, bone resorption parameters and the number of osteoclasts per bone circumference in ovariectomized rats were higher than those in normal rats.MiR-203 can promote osteogenic differentiation of mesenchymal stem cells by downregulating the gene expression of DKK1.

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity.

To investigate the influences of repeated propofol anesthesia on the hippocampal apoptosis and the long-term learning and memory abilities of neonatal rats.In the study, 45 male Sprague-Dawley rats, aged 7 days, were randomly divided into 3 group (n=15 each): control group (C group) that received intralipid 7.5 mL/kg intraperitioneal (IP) once a day×7 days; group propofol 1 (P1) that received propofol 75 mg/kg IP once a day×7 days; group propofol 2 (P2) that received intralipid 7.5 mL/kg IP once a day×6 days+propofol 75 mg/kg IP on the 7th day. In each group, 5 animals were chosen and arterial blood samples were obtained immediately after the animals were fully awake for blood gas analysis. Learning and memory abilities were assessed using Morris water maze when the other rats were 4 weeks old. The animals were decapitated after the tests. The hippocampi were isolated for detection of neuron-specific nucleoprotein (NeuN) expression by immunohistochemistry method and the expression of caspase-3 using the Western blot.There was no significant difference in the indexes of blood gas analysis among the 3 groups. Morris water maze test: compared with group C, the escape latency and the length of searching on the 5th day were significantly prolonged, and the searching time in target quadrant and platform crossing on the 6th day were significantly decreased in group P1 (P0.05) but not in group P2. Compared with group C, NeuN-positive neurons were decreased, and the expression of caspase-3 was increased in the rats of group P1 (P0.05) but not in group P2.Repeated propofol anesthesia may destroy long-term learning and memory abilities by inducing apoptosis of hippocampal neurons in neonatal rats, while single dose of propofol has no obvious effect on the hippocampal apoptosis and long-term learning and memory of neonatal rats.

Plasma adiponectin (APN) levels are decreased in diabetic patients. Dysfunctional mitochondrial biogenesis is involved in type 2 diabetes (T2DM) pathogenesis, by unclear mechanisms. The present study determined (1) whether myocardial mitochondrial biogenesis was impaired in cardiomyocytes exposed to a high glucose/high fat (HGHF) medium (a T2DM in vitro model), (2) the effects of APN administration upon mitochondrial biogenesis in cardiomyocytes affected by HGHF incubation, and 3) the involved underlying mechanisms.Neonatal rat ventricular myocytes (NRVMs) were isolated and incubated in HGHF medium. Mitochondrial function was assessed by ATP content, and fluorescent microscopic analysis of myocardial apoptosis was determined by TUNEL staining and caspase-3 activity.HGHF treatment reduced mitochondrial biogenesis, altered mitochondrial structure, and induced mitochondrial dysfunction in NRVMs. Administration of APN partially rescued these effects. However, siRNA-mediated knockdown of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) significantly blocked the beneficial effects of APN in mitochondria and cardiomyocytes subjected to hypoxia/reoxygenation injury.In the current study, we have provided the direct in vitro evidence that APN partially rescues HGHF-induced impairment of mitochondrial biogenesis and function via PGC-1α-mediated signaling.

The mammalian ABL1 gene encodes the ubiquitously expressed nonreceptor tyrosine kinase ABL. In response to growth factors, cytokines, cell adhesion, DNA damage, oxidative stress, and other signals, ABL is activated to stimulate cell proliferation or differentiation, survival or death, retraction, or migration. ABL also regulates specialized functions such as antigen receptor signaling in lymphocytes, synapse formation in neurons, and bacterial adhesion to intestinal epithelial cells. Although discovered as the proto-oncogene from which the Abelson leukemia virus derived its Gag-v-Abl oncogene, recent results have linked ABL kinase activation to neuronal degeneration. This body of knowledge on ABL seems confusing because it does not fit the one-gene-one-function paradigm. Without question, ABL capabilities are encoded by its gene sequence and that molecular blueprint designs this kinase to be regulated by subcellular location-dependent interactions with inhibitors and substrate activators. Furthermore, ABL shuttles between the nucleus and the cytoplasm where it binds DNA and actin--two biopolymers with fundamental roles in almost all biological processes. Taken together, the cumulated results from analyses of ABL structure-function, ABL mutant mouse phenotypes, and ABL substrates suggest that this tyrosine kinase does not have its own agenda but that, instead, it has evolved to serve a variety of tissue-specific and context-dependent biological functions.

Cytoplasmic STAT3, after activation by growth factors, translocates to different subcellular compartments, including nuclei and mitochondria, where it carries out different biological functions. However, the precise mechanism by which STAT3 undergoes mitochondrial translocation and subsequently regulates the tricarboxylic acid (TCA) cycle-electron transport chain (ETC) remains poorly understood. Here, we clarify this process by visualizing STAT3 acetylation in starved cells after serum reintroduction or insulin stimulation. CBP-acetylated STAT3 undergoes mitochondrial translocation in response to serum introduction or insulin stimulation. In mitochondria, STAT3 associates with the pyruvate dehydrogenase complex E1 (PDC-E1) and subsequently accelerates the conversion of pyruvate to acetyl-CoA, elevates the mitochondrial membrane potential, and promotes ATP synthesis. SIRT5 deacetylates STAT3, thereby inhibiting its function in mitochondrial pyruvate metabolism. In the A549 lung cancer cell line, constitutively acetylated STAT3 localizes to mitochondria, where it maintains the mitochondrial membrane potential and ATP synthesis in an active state.

The p53 protein has not only important tumor suppressor activity but also additional immunological and other functions, whose nature and extent are just beginning to be recognized. In this article, we show that p53 has a novel inflammation-promoting action in the intestinal tract, because loss of p53 or the upstream activating kinase, ATM, protects against acute intestinal inflammation in murine models. Mechanistically, deficiency in p53 leads to increased survival of epithelial cells and lamina propria macrophages, higher IL-6 expression owing to enhanced glucose-dependent NF-κB activation, and increased mucosal STAT3 activation. Blockade or loss of IL-6 signaling reverses the protective effects of p53 deficiency. Conversely, IL-6 treatment protects against acute colitis in a manner dependent on STAT3 signaling and induction of cytoprotective factors in epithelial cells. Together, these results indicate that p53 promotes inflammation in the intestinal tract through suppression of epithelium-protective factors, thus significantly expanding the spectrum of physiological and immunological p53 activities unrelated to cancer formation.

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Ablμ/μmice. When injected with cisplatin, we found similar levels of platinum in the Abl+/+and the Ablμ/μkidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Ablμ/μkidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl+/+but not in the Ablμ/μkidneys. The residual apoptosis in the Ablμ/μmice was not further reduced in the Ablμ/μ; p53-/-double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl+/+and the Ablμ/μkidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis. © 2013 Macmillan Publishers Limited.

Tumor necrosis factor (TNF)-α promotes tumor development under chronic inflammation. Because TNF also activates caspase-8, selective inhibition of TNF-induced extrinsic apoptosis would be required for inflammation-associated tumor growth. In a mouse model of inflammation-associated colon carcinogenesis, we found nuclear expression of β-catenin in tumors of wild-type, but not mutant, mice that were made resistant to TNF-induced apoptosis by a germline mutation blocking caspase cleavage of the retinoblastoma (RB) protein, despite similar frequencies of β-catenin exon-3 mutations in these two genetic backgrounds. TNF-induced apoptosis was also attenuated in human colon cancer cell lines with genetically activated β-catenin. However, we found that HCT116 cells, which contain an activated allele of β-catenin but do not express nuclear β-catenin, were sensitive to TNF-induced apoptosis. In HCT116 cells, TNF stimulated efficient RB cleavage that preceded chromatin condensation. In contrast, TNF did not induce RB cleavage in colon cancer cells expressing nuclear β-catenin and these cells could be sensitized to basal and/or TNF-induced apoptosis by the knockdown of β-catenin or RB. In the apoptosis-resistant colon cancer cells, knockdown of β-catenin led to a reduction in the RB protein without affecting RB mRNA. Furthermore, ectopic expression of the caspase-resistant, but not the wild-type, RB re-established resistance to TNF-induced caspase activation in colon cancer cells without β-catenin. Together, these results suggest that nuclear β-catenin–dependent RB stabilization suppresses TNF-induced apoptosis in caspase-8–positive colon cancer cells. Visual Overview: http://mcr.aacrjournals.org/content/11/3/207/F1.large.jpg. Mol Cancer Res; 11(3); 207–18. ©2012 AACR.

The aim of the study is to investigate the radiosensitivity of CD44+/CD24+ cervical cancer cells and to explore its mechanism of radiotherapy resistance. Moreover, we further to test whether the CD44+/CD24+ cervical cancer cells had the characteristics of stem cells.The human squamous cell carcinoma SiHa cells were cultured in vitro, and CD44+/CD24+ SiHa cells were sorted by FACS analysis. CD44+/CD24+ SiHa cells and the parental SiHa cells were given several fractionated irradiation at a cumulative dose of 8 Gy, 16 Gy, 30 Gy, respectively. Survival curves were obtained and fitted using clonogenic assays, and the radiosensitivity of tumor cells was compared according to the radiobiological parameters, including Do, Dq, N and SF 2. Morphological changes of cell apoptosis were determined using Hoechst 33258 fluorescence staining. The ultrastructural changes in cells with apoptosis were observed by transmission electron microscopy. Cell apoptosis rate was determined by FCAS analysis. DNA "ladder" in apoptotic cells was detected by gel electrophoresis. The mRNA levels of cell apoptosis-related genes were detected by RT-PCR assay. Balling capacities of CD44+/CD24+ SiHa cells and parental SiHa cells were detected by suspension culture without FBS. The in vivo tumorigenicity was detected by inoculating CD44+/CD24+ SiHa and parental SiHa cells into nude mice.The FACS analysis results demonstrated that there was a concomitant increase in the percentage of CD44+/CD24+ cells as the increasing irradiation doses. Colony formation assay results showed that the colony formation rate of CD44+/CD24+ SiHa cells was significantly higher than that of parental SiHa cells (p0.05). Moreover, the data from Hoechst 33258 staining, DNA fragment gel electrophoresis, transmission electron microscopy and FACS analysis showed that CD44+/CD24+ SiHa cells had no cell apoptosis after irradiation treatment. RT-PCR results showed that the mRNA levels of bcl-2, surviving and OCT4 were significantly higher in CD44+/CD24+ SiHa cells than that of parental SiHa cells (p0.01). CD44+/CD24+ SiHa cells could form more compact cell spheres with a larger volume than that of parental SiHa cells (p0.05). CD44+/CD24+ cervical cancer cells had more potent tumorigenicity than that of parental cervical cancer cells.CD44+/CD24+ cervical cancer resist cell apoptosis induced by irradiation therapy and possessed the characteristics of stem cells.

KCNQ1 (KvLQT1) is the pore-forming a-subunit of the potassium channel. To uncover its role in electrolyte metabolism, we investigated the effects of KCNQ1 A340E, a loss-of-function mutant, on J343 mice. Compared with the normal controls (C57BL/6J mice) bearing the wild-type KCNQ1 gene, J343 mice bearing KCNQ1 A340E demonstrated a much higher 24-h intake of electrolytes (potassium, sodium, and chloride). However, they suffered from significant electrolyte loss through both the feces and urine during a period of 24 h. Unbalance in electrolyte metabolism disrupted the electrolyte homeostasis in the J343 mice, which was characterized by the comparatively lower level of serum potassium (J343 vs C57BL/6J: 12.06 ± 1.47 vs 14.44 ± 3.58 mM, P = 0.01) and higher levels of serum sodium (J343 vs C57BL/6J: 148.05 ± 4.47 vs 115.15 ± 17.25 mM, P = 4.20 x 10(-4)) and chloride (J343 vs C57BL/6J: 118.0 ± 4.47 vs 85.21 ± 11.90 mM, P = 2.47 x 10(-5)). Between the J343 and C57BL/6J mice, there was no statistically significant difference in KCNQ1 expression in the gastrointestinal tract and kidney. Normal concentrations of plasma renin, angiotensin I, and aldosterone were also detected in both lines of mice. KCNQ1, therefore, is suggested to play a central role in electrolyte metabolism. KCNQ1 A340E, with the loss-of-function phenotype, may dysregulate electrolyte homeostasis in mice independently of the activity of the renin-angiotensin-aldosterone system.

Body weight is one of the most important economic traits in the poultry industry. In the present study, a custom SNP Beadchip was used to analyze the association between those 15 SNPs and 12 growth traits of Jinghai yellow chickens, and other important genetic parameters were also calculated and analyzed. The results indicated that nine of the 15 SNPs were associated with growth traits in Jinghai yellow chickens (P < 0.05), and the identified SNPs were also in linkage disequilibrium. Five of the nine identified SNPs were mainly associated with all of the growth traits, which indicated that those five SNPs might have significant influence on Jinghai yellow chicken growth traits. Polymorphism information content (PIC) analyses indicated that five of the nine SNPs exhibited moderate polymorphism (0.25 < PIC < 0.5), which reflected intermediate genetic diversity. Six candidate genes surrounding the significant SNPs were obtained and subjected to Gene Ontology annotation analyses and pathway analyses. The functions of six important candidate genes (SETDB2, ATP7B, INTS6, KPNA3, DLEU7, and FOXO1A) were discussed. The present study provided basic data for marker-assisted selection in Jinghai yellow chickens.

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.

The LXCXE peptide motif facilitates interaction between the RB tumor suppressor and a large number of cellular proteins that are expected to impinge on diverse biological processes. In vitro and in vivo analyses demonstrated that LXCXE binding function is dispensable for RB promoter association and control of basal gene expression. Dependence on this function of RB is unmasked after DNA damage, wherein LXCXE binding is essential for exerting control over E2F3 and suppressing cell-cycle progression in the presence of genotoxic stress. Gene expression profiling revealed that the transcriptional program coordinated by this specific aspect of RB is associated with progression of human hepatocellular carcinoma and poor disease outcome. Consistent with these findings, biological challenge revealed a requirement for LXCXE binding in suppression of genotoxin-initiated hepatocellular carcinoma in vivo. Together, these studies establish an essential role of the LXCXE binding motif for RB-mediated transcriptional control, response to genotoxic insult, and tumor suppression.

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

Using ulnar nerve as donor and musculocutaneous nerve as recipient we recently demonstrated that end-to-end neurorrhaphy in young adult male Wistar rats resulted in good recovery following protracted survival. Here we explored whether anti-inflammatory drug- methylprednisolone, regeneration/myelination-enhancing agent- methylcobalamin and neurite growth-enhancing and angiogenic factor- pleiotrophin accelerated its recovery. Methylprednisolone suppressed the perineuronal microglial reaction and periaxonal ED-1 expression while pleiotrophin increased the blood vessel density and nerve fiber densities in the reconnected nerve as expected. Neither methylprednisolone nor methylcobalamin altered the expression of growth associated protein 43 in the neurons examined suggesting that they did not interfere with axonal regeneration attempt. Surprisingly methylcobalamin enhanced the recovery of compound muscle action potentials and motor end plate innervation and the performance on sticker removal grooming test and augmented the diameters and myelin thicknesses of regenerated axons dramatically while enhancing S-100 expression in Schwann cells; remarkable recovery was achieved 1 month following neurorrhaphy. Simultaneous methylcobalamin and pleiotrophin treatment resulted in quick and persistent supernumerary reinnervation but failed to enhance the recovery over that of the former alone. Methylprednisolone transiently suppressed the enumeration of regrowing axons. In conclusion, methylcobalamin may be preferred over methylprednisolone to facilitate the recovery of peripheral nerves following end-to-end neurorrhaphy. The long-term effect of this treatment however remains to be clarified.

Head trauma and acute disorders often instantly compress the cerebral cortex and lead to functional abnormalities. Here we used rat epidural bead implantation model and investigated the immediate changes following acute compression. The dendritic arbors of affected cortical pyramidal neurons were filled with intracellular dye and reconstructed 3-dimensionally for analysis. Compression was found to shorten the apical, but not basal, dendrites of underlying layer III and V cortical pyramidal neurons and reduced dendritic spines on the entire dendritic arbor immediately. Dendrogram analysis showed that in addition to distal, proximal apical dendrites also quickly reconfigured. We then focused on apical dendritic trunks and explored how proximal dendrites were rapidly altered. Compression instantly twisted the microtubules and deformed the membrane contour of dendritic trunks likely a result of the elastic nature of dendrites as immediate decompression restored it and stabilization of microtubules failed to block it. Subsequent adaptive remodeling restored plasmalemma and microtubules to normal appearance in 3 days likely via active mechanisms as taxol blocked the restoration of microtubules and in addition partly affected plasmalemmal reorganization which presumably engaged recycling of excess membrane. In short, the structural dynamics and the associated mechanisms that we revealed demonstrate how compression quickly altered the morphology of cortical output neurons and hence cortical functions consequently.

The cDNA encoding noggin2 protein was obtained from the brain and spinal cord cDNA library of Gekko japonicus. The size of the noggin2 transcript and its expression in different tissues were analyzed by Northern blot analysis. In situ hybridization revealed positive hybridization signals in both gray and white matter of the spinal cord. Changes in noggin2 expression in the spinal cord after tail amputation were examined by real-time PCR. The noggin2 was expressed in the normal spinal cord and down-regulated three days after tail amputation, reaching the lowest level at two weeks, during the time course when we followed the expression levels. We concluded that the expression of noggin2 is affected by the process of spinal cord injury and regeneration.

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I- Sce I endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

The success of imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing imatinib therapy almost invariably relapse, demonstrating that an imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the beta-catenin pathway. Characterization of these cells and evaluation of their sensitivity to imatinib is critical to our understanding and treatment of CML blast crisis.

During blast crisis of chronic myelogenous leukemia (CML), abnormal granulocyte macrophage progenitors (GMP) with nuclear β-catenin acquire self-renewal potential and may function as leukemic stem cells (Jamieson et al. N Engl J Med , 2004). To develop a mouse model for CML-initiating GMP, we expressed p210 BCR-ABL in an established line of E2A -knockout mouse BM cells that retain pluripotency in ex vivo culture. Expression of BCR-ABL in these cells reproducibly stimulated myeloid expansion in culture and generated leukemia-initiating cells specifically in the GMP compartment. The leukemogenic GMP displayed higher levels of β-catenin activity than either the nontransformed GMP or the transformed nonGMP, both in culture and in transplanted mouse BM. Although E2A -deficiency may have contributed to the formation of leukemogenic GMP, restoration of E2A-function did not reverse BCR-ABL-induced transformation. These results provide further evidence that BCR-ABL-transformed GMP with abnormal β-catenin activity can function as leukemic stem cells.

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2 -deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

DNA damage can, but does not always, induce cell death. While several pathways linking DNA damage signals to mitochondria-dependent and -independent death machineries have been elucidated, the connectivity of these pathways is subject to regulation by multiple other factors that are not well understood. We have proposed two conceptual models to explain the delayed and variable cell death response to DNA damage: integrative surveillance versus autonomous pathways. In this review, we discuss how these two models may explain the in vivo regulation of cell death induced by ionizing radiation (IR) in the developing central nervous system, where the death response is regulated by radiation dose, cell cycle status and neuronal development.

The nonreceptor Abl tyrosine kinase stimulates F-actin microspikes and membrane ruffles in response to adhesion and growth factor signals. We show here that induced dimerization of Abl-FKBP, but not the kinase-defective AblKD-FKBP, inhibits cell spreading on fibronectin. Conversely, knockdown of cellular Abl by shRNA stimulates cell spreading. The Abl kinase inhibitor, imatinib, also stimulates cell spreading and its effect is overridden by the imatinib-resistant AblT315I. Expression of Abl but not AbkKD in Abl/Arg-deficient cells again inhibits spreading. Furthermore, Abl inhibits spreading of cells that express the activated Rac, RacV12, correlating with RacV12 localization to dorsal membrane protrusions. Ectopic expression of CrkII, a Rac activator that is inactivated by Abl-mediated tyrosine phosphorylation, antagonizes Abl-mediated dorsal membrane localization of RacV12. Ectopic expression of a dynamin-2 mutant, previously shown to induce Rac-GTP localization to the dorsal membrane, abolishes the stimulatory effect of imatinib on cell spreading. These results suggest that Abl tyrosine kinase, through CrkII phosphorylation and in collaboration with dynamin-2 can regulate the partitioning of Rac-GTP to favor dorsal ruffles during cell spreading. The Abl-dependent dorsal membrane localization of activated Rac explains its positive role in ruffling and negative role in cell spreading and migration.

Tumor necrosis factor-alpha (TNF) activates caspase-8 to cleave effector caspases or Bid, resulting in type-1 or type-2 apoptosis, respectively. We show here that TNF also induces caspase-8-dependent C-terminal cleavage of the retinoblastoma protein (Rb). Interestingly, fibroblasts from Rb(MI/MI) mice, in which the C-terminal caspase cleavage site is mutated, exhibit a defect in Bid cleavage despite caspase-8 activation. Recent results suggest that TNF receptor endocytosis is required for the activation of caspase-8. Consistent with this notion, inhibition of V-ATPase, which plays an essential role in acidification and degradation of endosomes, specifically restores Bid cleavage in Rb(MI/MI) cells. Inhibition of V-ATPase sensitizes Rb(MI/MI) but not wild-type fibroblasts to TNF-induced apoptosis and stimulates inflammation-associated colonic apoptosis in Rb(MI/MI) but not wild-type mice. These results suggest that Rb cleavage is required for Bid cleavage in TNF-induced type-2 apoptosis, and this requirement can be supplanted by the inhibition of V-ATPase.

The DNA damage response network stimulates microRNA (miRNA) biogenesis to coordinate repair, cell cycle checkpoints, and apoptosis. The multistep process of miRNA biogenesis involves the cleavage of primary miRNAs by the microprocessor complex composed of the ribonuclease Drosha and the RNA binding protein DGCR8. We found that the tyrosine kinase ABL phosphorylated DGCR8, a modification that was required for the induction of a subset of miRNAs after DNA damage. Focusing on the miR-34 family, ABL stimulated the production of miR-34c, but not miR-34a, through Drosha/DGCR8-dependent processing of primary miR-34c (pri-miR-34c). This miRNA-selective effect of ABL required the sequences flanking the precursor miR-34c (pre-miR-34c) stem-loop. In pri-miRNA processing, DGCR8 binds the pre-miR stem-loop and recruits Drosha to the miRNA. RNA cross-linking assays showed that DGCR8 and Drosha interacted with pri-miR-34c, but we found an inverse correlation between ABL-stimulated processing and DGCR8 association with pri-miR-34c. When coexpressed in HEK293T cells, ABL phosphorylated DGCR8 at Tyr(267). Ectopic expression of a Y267F-DGCR8 mutant reduced the recruitment of Drosha to pri-miR-34c and prevented ABL or Drosha from stimulating the processing of pri-miR-34c. In mice engineered to express a nuclear import-defective mutant of ABL, miR-34c, but not miR-34a, expression was reduced in the kidney, and apoptosis of the renal epithelial cells was impaired in response to cisplatin. These results reveal a new pathway in the DNA damage response wherein ABL-dependent tyrosine phosphorylation of DGCR8 stimulates the processing of selective primary miRNAs.

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors--imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Genotoxic agents or inflammatory cytokines activate cellular stress responses and trigger programmed cell death. We have identified a signal transduction module, including three nuclear proteins that participate in the regulation of cell death induced by chemotherapeutic agents and tumor necrosis factor (TNF). In this nuclear signaling module, retinoblastoma protein (Rb) functions as an inhibitor of apoptotic signal transduction. Inactivation of Rb by phosphorylation or caspase-dependent cleavage/degradation is required for cell death to occur. Rb inhibits the Abl tyrosine kinase. Thus, Rb inactivation is a pre-requisite for Abl activation by DNA damage or TNF. Activation of nuclear Abl and its downstream effector p73 induces mitochondriadependent cell death. The involvement of these nuclear signal transducers in TNF induced apoptosis, which does not require new gene expression, indicates that nuclear events other than transcription can contribute to extrinsic apoptotic signal transduction.

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.

Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine that coordinates the systemic response to infection and injury. TNF-α interacts with two types of cell surface receptors (types I and II) to regulate various cell-type-specific responses (8). Binding of TNF-α to the type I receptor (TNFRI) can activate the NF-κB survival pathway (23) or caspase-dependent cell death (24, 38). Activated TNFRI recruits TRADD (TNFR-associated death domain) (25), which in turn recruits TRAFs (TNFR-associated factors) and RIP to activate NF-κB. TNFRI also stimulates the formation of a cytoplasmic TRADD complex, containing FADD (FAS-associated death domain) and pro-caspase 8, leading to the activation of caspase 8 and the initiation of an apoptotic signaling cascade (24, 25, 38). Activated caspase 8 can directly cleave and activate executioner caspases (11). Caspase 8 also cleaves a BH3-only protein, BID (32, 35). The truncated BID, in collaboration with BAX and BAK, causes the release of apoptogenic factors from mitochondria to activate the apoptosome and downstream caspases (58). Cells derived from Bid−/− mice show delayed caspase activation and reduced apoptosis in response to TNF-α (63). Thus, TNFRI-induced apoptosis is mediated through mitochondrion-independent and mitochondrion-dependent mechanisms. It was previously shown that degradation of the retinoblastoma tumor suppressor protein (RB) is required for TNF-α to activate mitochondrion-dependent apoptosis (6). Upon the activation of TNFRI, RB is cleaved by caspase and then degraded. We have created in the mouse germ line an Rb-MI allele, which contains two substitutions at a C-terminal caspase cleavage site (DEADG to DEAAE) (6). Rb-MI is not degraded upon the activation of TNFRI; cells expressing Rb-MI maintain their mitochondrial integrity and display a protracted death response to TNF-α (6). Rb-MI is sensitive to degradation when TNFRI and TNFRII are both activated (6). As a result, cells expressing Rb-MI are sensitive to death induced by the costimulation of TNFRI and TNFRII (6). These and other results strongly suggest that RB has an apoptosis suppression function (7). It has been previously shown that RB binds to nuclear c-ABL tyrosine kinase and inhibits its activity (59, 60). The activation of nuclear c-ABL tyrosine kinase by DNA damage is associated with the induction of apoptosis (57). In addition to DNA damage, nuclear c-ABL kinase could also be activated by TNF-α (12, 57), and STI-571, a chemical inhibitor of c-ABL kinase, reduces the apoptotic response to TNF-α (12). It was found that z-VAD, a general inhibitor of caspase, blocks TNF-α-induced activation of c-ABL (12). While caspase can cleave c-ABL protein, cleavage does not activate the tyrosine kinase activity but instead promotes the nuclear accumulation of c-ABL (3). In this study, we show that caspase-dependent removal of RB is required for TNF-α to activate the nuclear c-ABL tyrosine kinase, thus explaining the inhibitory effect of z-VAD on c-ABL activation. c-ABL tyrosine kinase regulates the function of p53 and p73, which play essential roles in DNA damage-induced apoptosis (17). The human c-ABL protein may interact directly with p53 to activate its function (18). Overproduction of c-ABL can also interfere with MDM2-mediated degradation of p53 (19, 46). However, activated nuclear c-ABL kinase can induce apoptosis in p53-deficient cells (52, 53). By contrast, activated nuclear c-ABL kinase does not induce apoptosis in p73-deficient cells (52). The c-ABL kinase can directly phosphorylate p73 (1, 64), increase the half-life of p73 (21), and promote the acetylation of p73 by p300 (10). Coexpression of activated nuclear c-ABL kinase with p73 synergistically activates cell death (52). Thus, c-ABL and p73 cooperate to promote apoptosis. In this study, we show p73 to also be required for TNF-α-induced cell death and RB to be a suppressor of the p73 apoptotic function.