ABSTRACT
Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI). Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence. In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death. Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.
Subject(s)
Autophagy , DNA Damage , Lamin Type B , Neurons , Spinal Cord Injuries , Sumoylation , Ubiquitin-Conjugating Enzymes , Animals , Mice , Cell Nucleus/metabolism , Lamin Type B/metabolism , Lamin Type B/genetics , Neurons/metabolism , Neurons/pathology , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/genetics , Spinal Cord Injuries/pathology , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Mice, Inbred C57BL , Cell Line, TumorABSTRACT
OBJECTIVE: To assess the role of XPG, XPC, CCNH and MMS19L polymorphisms response to chemotherapy in osteosarcoma, and the clinical outcome of osteosarcoma. METHODS: One hundred and sixty eight osteosarcoma patients who were histologically confirmed were enrolled in our study between January 2007 and March 2009. Genotyping of XPG, XPC, CCNH and MMS19L was performed in a 384-well plate format on the MassARRAY® platform. RESULTS: Individuals with rs2296147 TT genotype showed a better response as compared with CC genotype, with the OR (95% CI) of 3.89(1.49-10.95). Those carrying rs29001322 TT genotype presented better response to chemotherapy, and the OR (95% CI) was as high as 12.25(2.63-121.84). Patients carrying TT genotype of XPG rs2296147 and MMS19L rs29001322 showed a significantly longer overall survival than CC genotype, they had 0.37 and 0.31-fold risk of death when compared with wide-type of this gene. CONCLUSIONS: XPG rs2296147 and MMS19L rs29001322 are correlated with response to chemotherapy and prognosis of osteosarcoma. Our findings would provide important evidence for prognostic and therapeutic implications in osteosarcoma.
ABSTRACT
INTRODUCTION: Fluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear. METHODS: Cell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays. RESULTS: Low-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA. CONCLUSION: NaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.
ABSTRACT
Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Subject(s)
Acrylamides/pharmacology , Cryopreservation , Heart Transplantation/methods , Histone Deacetylase Inhibitors/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Phenylenediamines/pharmacology , Animals , Apoptosis/drug effects , Disaccharides/pharmacology , Electrolytes/pharmacology , Glutamates/pharmacology , Glutathione/pharmacology , Heart/drug effects , Heart/physiology , Hepatocyte Growth Factor/antagonists & inhibitors , Histidine/pharmacology , Male , Mannitol/pharmacology , Oxidative Stress/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , YAP-Signaling ProteinsABSTRACT
This study aims to explore the potential pathways and molecular characteristics of fluorine-induced osteoblast apoptosis. In vitro fluorine-induced model was established with an osteogenesis sarcoma cell line Saos-2. Then flow cytometry was used to determine the mitochondrial membrane potential at 24 h after the intervention. 84 apoptosis-related genes in the cells were determined using the functional polymerase chain reaction (PCR) chip and part of the differentially expressed genes was verified with immune blotting. When the stimulated concentration of sodium fluoride were 20 mg/L, 40 mg/L and 80 mg/L, the mitochondrial membrane potential of the osteoblast cells were 27.0%, 28.8% and 38.6%, respectively, significantly higher than that in the blank control group (P<0.05). The PCR chip detection found 13 up-regulating genes and 15 down-regulating genes, among which the expression of Bim, Caspase 9, Caspase 14, B-cell lymphoma-2 (BCL2) and BAX increased with the doses of sodium fluoride, while the expression of Caspase 3 down-regulated in 5 mg/L sodium fluoride but up-regulated at the concentration of sodium fluoride more than 10 mg/L. Caspase 7 expression showed no obvious difference between the different concentration groups. However, Caspase 10 decreased with the increasing doses of sodium fluoride. Fluoride-induced osteoblast apoptosis may be through the mitochondrial pathway (including endoplasmic reticulum stress pathway) and death receptor pathway.
ABSTRACT
We aimed to assess the role of XPG, XPC and MMS19L polymorphisms on response to chemotherapy in osteosarcomas, and the clinical outcomes. One hundred and eighty five osteosarcoma patients who were histologically confirmed were enrolled in our study between January 2007 and December 2009. Genotyping of XPG, XPC and MMS19L was performed in a 384-well plate format on the MassARRAY® platform. Individuals with XPG TT genotype and T allele were more likely to be better response to chemotherapy than CC genotype, with the OR (95% CI) of 4.17 (1.64-11.54) and 2.66 (1.39-5.11), respectively. Those carrying MMS19L TT genotype and T allele showed better response to chemotherapy, with ORs (95% CI) of 4.8 (1.56-17.7) and 2.3 (1.22-4.36), respectively. Patients carrying TT genotype of XPG and MMS19L showed a significantly longer overall survival than CC genotype, with a 0.47 and 0.30-fold risk of death when compared with the wild-type of the gene. XPG and MMS19L are correlated with response to chemotherapy and prognosis of osteosarcoma, so that they could be used as predictive markers for prognosis.