ABSTRACT
The aim of this study was to investigate the relationship between the effects of different doses of X-rays on DNA damage and JAK/STAT signaling pathway activation in A549 cells. The A549 cells were radiated with X-rays at doses of 2, 4, and 8 Gy. The proliferation of A549 cells was detected by CCK8 method. The content of interleukin 6 (IL-6) in culture medium at different time points after irradiation was detected by enzyme-linked immunoassay, and the expression levels of IL-6 receptor (IL-6R) and p53 binding protein 1 (53BP1) were detected by immunofluorescent staining. The expression levels of JAK2, p-JAK2, STAT3 and p-STAT3 were detected by Western blot. The results showed that, compared with the control group, X-ray irradiation reduced the cellular proliferation, up-regulated the expression of 53BP1, increased the IL-6 content in the medium supernatant, and up-regulated the protein expression levels of IL-6R, JAK2, p-JAK2, STAT3, and p-STAT3. The above effects of X-ray irradiation were dose-dependent. These results suggest that the mechanism by which X-rays cause DNA damage in A549 cells may involve activation of the JAK/STAT signaling pathway.
Subject(s)
Humans , A549 Cells , DNA Damage , Radiation Effects , Janus Kinase 2 , Metabolism , Receptors, Interleukin-6 , Metabolism , STAT3 Transcription Factor , Metabolism , Signal Transduction , Tumor Suppressor p53-Binding Protein 1 , Metabolism , X-RaysABSTRACT
DNA damage response (DDR) is essential for maintaining genome stability and protecting cells from tumorigenesis. Ubiquitin and ubiquitin-like modifications play an important role in DDR, from signaling DNA damage to mediating DNA repair. In this report, we found that the E3 ligase ring finger protein 126 (RNF126) was recruited to UV laser micro-irradiation-induced stripes in a RNF8-dependent manner. RNF126 directly interacted with and ubiquitinated another E3 ligase, RNF168. Overexpression of wild type RNF126, but not catalytically-inactive mutant RNF126 (CC229/232AA), diminished ubiquitination of H2A histone family member X (H2AX), and subsequent bleomycin-induced focus formation of total ubiquitin FK2, TP53-binding protein 1 (53BP1), and receptor-associated protein 80 (RAP80). Interestingly, both RNF126 overexpression and RNF126 downregulation compromised homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Taken together, our findings demonstrate that RNF126 negatively regulates RNF168 function in DDR and its appropriate cellular expression levels are essential for HR-mediated DSB repair.
Subject(s)
Humans , Carrier Proteins , Metabolism , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Repair , Genetics , DNA-Binding Proteins , Metabolism , Genomic Instability , HeLa Cells , Histones , Metabolism , Nuclear Proteins , Metabolism , RNA Interference , RNA, Small Interfering , Genetics , Signal Transduction , Tumor Suppressor p53-Binding Protein 1 , Metabolism , Ubiquitin , Ubiquitin-Protein Ligases , Genetics , Metabolism , UbiquitinationABSTRACT
<p><b>OBJECTIVE</b>To study the incidence of 53BP1 gene mutations in prostatic adenocarcinoma and benign prostatic hypertrophy, and to analyze the relationship between 53BP1 mutations and prostatic adenocarcinoma.</p><p><b>METHODS</b>Genomic DNA extraction, PCR amplification and gene sequencing were used to detect the occurrence of 53BP1 gene mutations in 50 cases of prostatic adenocarcinoma. Ten cases of benign prostatic hypertrophy were included as controls.</p><p><b>RESULTS</b>Amongst the 50 cases of prostatic adenocarcinoma studied, 15 showed genetic alterations of 53BP1, including 4 cases with single nucleotide polymorphism. The mutation rate was 24.0% (12/50). Seven of the 53BP1 mutations detected represented missense mutations and none of them were situated in functionally important domains. The other 4 were synonymous mutations, in which c. 4760G > T was situated in Tudor domain. There was no obvious correlation between 53BP1 gene mutations and the various clinicopathologic parameters of prostate adenocarcinoma (P>0.05).</p><p><b>CONCLUSION</b>Certain percentage of prostatic adenocarcinoma harbors 53BP1 mutations which may be involved in the carcinogenesis.</p>
Subject(s)
Aged , Humans , Male , Middle Aged , Adenocarcinoma , Genetics , Pathology , Exons , Intracellular Signaling Peptides and Proteins , Genetics , Mutation , Mutation Rate , Mutation, Missense , Polymorphism, Single Nucleotide , Prostatic Hyperplasia , Genetics , Prostatic Neoplasms , Genetics , Pathology , Tumor Suppressor p53-Binding Protein 1ABSTRACT
<p><b>AIM</b>To investigate whether adriamycin induces DNA damage and the formation of gammaH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa.</p><p><b>METHODS</b>Human spermatozoa were treated with adriamycin at different concentrations. gammaH2AX was analyzed by immunofluorescent staining and flow cytometry and double-strand breaks (DSB) were detected by the comet assay.</p><p><b>RESULTS</b>The neutral comet assay revealed that the treatment with adriamycin at 2 microg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 microg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of gH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with gammaH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with gammaH2AX.</p><p><b>CONCLUSION</b>Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.</p>
Subject(s)
Humans , Male , Androstadienes , Pharmacology , Antibiotics, Antineoplastic , Pharmacology , Cells, Cultured , Comet Assay , DNA Breaks, Double-Stranded , DNA Damage , DNA Repair Enzymes , Metabolism , DNA-Binding Proteins , Metabolism , Doxorubicin , Pharmacology , Drug Interactions , Flow Cytometry , Histones , Metabolism , Intracellular Signaling Peptides and Proteins , Metabolism , Phosphorylation , Protein Kinase Inhibitors , Pharmacology , Spermatozoa , Cell Biology , Metabolism , Tumor Suppressor p53-Binding Protein 1ABSTRACT
<p><b>OBJECTIVE</b>To observe the expression of DNA damage checkpoint mediator 1 (MDC1) and p53-binding protein 1 (53BP1) at both mRNA and protein levels and their significance in different human esophageal cancer cell lines.</p><p><b>METHODS</b>In 3 human esophageal carcinoma cell lines, TE-1, TE-13 and Eca109 cells, the expressions of MDC1 and 53BP1 mRNA were detected with RT-PCR, and MDC1 and 53BP1 protein expressions were measured with immunohistochemistry, indirect immunofluorescence and Western blotting, respectively.</p><p><b>RESULTS AND CONCLUSIONS</b>MDC1 and 53BP1 expressions were observed for the first time in human esophageal carcinoma cell lines TE-1,TE-13 and Eca109 cells, at both the mRNA and protein levels. The expressions of MDC1 and 53BP1 proteins may be implicated in the radiosensitivity of human esophageal carcinoma.</p>