The Role Of Ubiquitination And Acetylation Of Histone In DNA Damage Repair

Objiectives:Neurodegeneration is caused by the progressive loss of the neuronal of brain and spinal cord.As time goes on,the deterioration of neurodegeneration leads to a variety of neurological dysfunction.The failure of DNA damage repair can lead to apoptosis or gene mutation.The lack of a variety of DNA damage related proteins have been found to contribute to neurodegeneration.The RNF8 is a 485 residue nuclear polypeptide which contains a C-terminal RING domain with the activity of E3 and an N-terminal FHA domain which plays an important role in DNA damage repair.Although the ubituitin ligase RNF8 involves in DNA double-strand break(DSBs)repair by ubiquitination of histones,but whether the defect of RNF8 can lead to neurodegeneration remains to be seen.The aim of the study is to identify whether RNF8 involves in neuronal DNA damage repair and contributes to neurodegeneration by study the RNF8 knockout mice.Methods:RNF8+/-mice were intercrossed to generate RNF8-/-mice which were identified by genomic DNA PCR.Wild-type littermates were used as controls.We detected the weight of individual mice of the RNF8-/-mice and WT genotypes from weaning to 12 months.The 4months mice were divided into two groups:One was control group;the other was treated with IR(2Gy).After recovered for 6hours,brains were fixed by perfusing mice with paraformaldehyde(4%),cutting into frozen sections.The cerebral cortex and hippocampus of the rest brains were used to extract the histone for western blots.Immunofluorescence staining detected the expression of y-H2AX,MDC1,53BP1 and BRCA1 in neurons.HT22' cells knockout RNF8 by siRNA.Western blots detected the expression of ub-H2A and ub-H2B in mice,HT22 cells and MEFs.Neutral single cell gel electrophesis detected the DNA DSBs.Immunohistochemical staining detected the expression of GFAP.The Fluoro-Jade C dyeing detected the degeneration of neurons.TUNEL staining detected neuronal apoptosis.The Cresyl Violet staining and silver staining demonstrated the morphological changes on nerve.The mice were divided into two groups:The one was control group;the other was treated with IR(2Gy).And then III those were bred for several months.Before being executed,the open field exploring test and the step-down avoidance test were used to investigate the neurological dysfunction.Results:The RNF8-/-mice seemed to smaller in size at birth in the same litters.The RNF8-/-mice were smaller and generally stunted growth.The RNF8-/? mice displayed premature death and sensitive to radiation.In addition,RNF8 knockout mice also demonstrated hair mats,slower movement and the decline of motor ability and coordination ability.Immunofluorescence staining displayed that after being treated with IR(2Gy),y-H2AX and MDC1 were expressed,but 53BP1 and BRCA1 were not expressed in RNF8-/-mice.Western Blots showed that after being treated with IR(2Gy),the expression of ub-H2A and ub-H2B were no significant difference in RNF8-/-mice and SiRNF8 comparing to unirradiated those.Neutral single cell gel electrophesis demonstrated that,comparing to unirradiated RNF8-/-mice neurons,the tail DNA%(TDNA%)and tail moment(TM)in RNF8-/-mice treated with IR(2Gy)increased(P<0.05);And also the olive tail moment(OTM)displayed a sharply increase(P<0.01).Immunohistochemistry showed that the expression of GFAP in RNF8-/-mice increased significantly after the treatment(2Gy).Fluoro-Jade C staining revealed that sporadic degeneration of neurons was found in RNF8-/-mice,after being treated with IR(2Gy),more denatured neurons were found in RNF8-/-mice(P<0.01).TUNEL staining showed that apoptotic cells were appeared in RNF8-/-mice,the apoptotic cells increased sharply(P<0.01)after being treated with IR(2Gy).Cresyl Violet staining demonstrated that the number of pyramidal cells in cerebral cortex of RNF8-/-mice treated with IR(2Gy)vacuoles,nuclear concentration in margin and perineural phenomenon were found in the cortex and hippocampus of RNF8-/-mice.Silver staining displayed that some abnormal neurons which were sporadic,dark and irregular shape were found in the hippocampus of RNF8-/-mice?Being treated with IR(2Gy),axonal degeneration of neurons were found in the cortex and hippocampus of RNF8-/-mice(P<0.01),showed that the axonal were spirally twisted,swelling and even ruptured.In addition,in the hippocampus of RNF8-/?mice revealed some dark plaque surrounding by particulates and some neurons with unconspicuous dyeing.Open field exploring test showed that,after 2 and 4 months for being treated with IR(2Gy),the horizontal activity of the RNF8-/-mice decreased significantly(P<0.01);After 6months,the horizontal activity also decreased(P<0.05).After being treated with IR(2Gy)for 2 and 4months,the central residence time decreased(P<0.05)in RNF8-/-mice;For 6months,the central residence time decreased significantly(P<0.01).After 3 months for being treated with IR(2Gy),the step-down avoidance test demonstrated the increased incidence of errors and decreased step down latency(SDL)in RNF8-/? mice(P<0.05);After 6months,the incidence of errors and SDL displayed significant difference(P<0.01).Conclusion:RNF8 is important to maintain the genetic integrity of neurons by mediating ubiquition of histone involving in DSBs repair.RNF8 defects in neurons of RNF8-/-mice impacts the ability of DNA damage repair.It is more easily for RNF8-/-mice to induce the apoptosis by DNA damage.The RNF8-/-mice display neuropathology,showing the decline in the cognitive ability,motor ability,exploring ability and short-memory.Objectives: Histone deacetylase inhibitors(HDACIs)are a promising class of drugs that act as anti-proliferative agents by promoting differentiation and inducing apoptosis.Valproic acid(VPA)is an HDACI that has been widely used as an anti-convulsant and shows promise as a DNA damage drug for a number of tumor cells.The present study aimed to investigate the inhibitory effect of VPA on the viability of bladder cancer cells and its synergistic effect with DNA damage drugs in vitro and in vivo.Methods: MTT assay was used to detected the inhibition of VPA on T24?BIU87and 5637 cells.Hoechst 33268 staining was used to visualize the apoptotic cells in the bladder cancer cell lines and cancerous tissues from the N-methyl-N-nitrosourea(MNU)-induced bladder cancer rats.The MTT assay was performed to analyze synergistic inhibition of the drug combinations(VPA and DDP or MMC or ADM).Flow cytometry of apoptosis by annexin V and propidium iodide(PI)double staining,following 72 hr of VPA and/or DDP treatment.Western blot was used to detect the expression of survivin and acetylated histone H3 in T24 cells.Animal studies of the effects of VPA in combination with DDP on MNU-induced bladder cancer through the HE staining.Results: VPA inhibits T24?BIU87 and 5637 cells proliferation.The cell viability decreased in a dose-dependent manner in all three cell lines.Hoechst 33258 staining revealed that the viable cells displayed diffuse fluorescence in the cellular nuclei,but the apoptotic cells demonstrated concentrated dense granular fluorescence.Numerous apoptotic cells were observed in the group that was treated with VPA or VPA combined with DDP.The MTT assay revealed the synergistic inhibition on the survival of bladder cancer cells by VPA combined with DDP,MMC and ADM.Each of the coefficient of drug interaction(CDI)value was <1.Annexin V and PI double staining was performed and detected using flow cytometry following VPA and /or DDP treatment,the percentage of apoptotic cells increased and the percentage of living cells decreased significantly.Western blots showed that the expression of survivin in the T24 cells was down-regulation,and the expression of histone H3 was up-regulation.Intravesical instillation VPA was able to prevent the progression of bladder cancer(P<0.05).Improved results were achieved using VPA combined with DDP in treating bladder cancer(P<0.01).Conclusions: VPA exhibited anti-proliferative activity and potently induced apoptosis in the human bladder cancer cells without apparent toxic side-effects.Furthermore,VPA was able to down-regulate the expression of survivin and increase the sensitivity of bladder cancer to chemotherapeutic dugs.The intravesical instillation of VPA and VPA combined with DDP was able to prevent tumor progression in rats with MNU-induced bladder cancer.These findings raise the possibility that VPA may prove particularly effective in treating bladder cancers when combined with DNA damage drugs.