OBJECTIVETo detect and verify activator protein-1 (AP-1) binding to p53 gene promoter region in A549 cell lines in vitro.

METHODSAP-1 binding to p53 gene promoter region was detected by chromatin immunoprecipitation (ChIP) assay using c-jun specific antibody, and PCR amplified its gene specific DNA fragment.

RESULTSThe p53 gene specific fragment was found in the DNA fragment immunoprecipitated by c-jun antibody.

CONCLUSIONAP-1 binds to p53 gene promoter region in A549 cells, and regulates its expression.

Cell Line, Tumor ; Chromatin Immunoprecipitation ; Gene Expression Regulation, Neoplastic ; Humans ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Transcription Factor AP-1 ; genetics ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo study effects of alternative transcripts of ING1 transfection on human cancer cell lines.

METHODSp47/ING1A and p33/ING1B expression vehicles were constructed and introduced into a human breast cancer cell line MCF-7 and a human lung cancer cell line PAa, both expressing wild-type p53 protein. Growth characteristics of the transfectants and potentially related genes were analyzed.

RESULTSThe levels of p47/ING1A and p33/ING1B protein elevated respectively in tumor cells of MCF-7 and PAa after transfected with p47/ING1A and p33/ING1B, and the latter was much higher than that of the former. Ectopic overexpression of p33/ING1B effectively blocked tumor cell growth and arrested cells in the G(0) approximately G(1) phase of the cell cycle (P < 0.01), while p47/ING1A gave no effect on cell growth or cell cycle. Tumor cells overexpressing p33/ING1B contained more p21(WAF1) protein than that of the control cells, with undisturbed p53 protein level.

CONCLUSIONSExpression of two different transcripts of ING1 may have different effects on tumor cell growth. p33/ING1B may cooperate with p53 in stimulating expression of p21(WAF1) gene, thus to arrest cell cycle and to inhibit tumor cell growth. p33/ING1B may be considered to be a candidate as a partner of p53 in gene therapy.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Alternative Splicing ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Cycle Proteins ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; DNA-Binding Proteins ; Genes, Tumor Suppressor ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Nuclear Proteins ; Protein Biosynthesis ; Proteins ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; Tumor Suppressor Proteins

OBJECTIVETo explore the relationship between mutant p53 and multidrug resistance in gastric cancer.

METHODSMutant p53 (mp53) and mp53+sv40Tag were transferred to gastric cancer cell line SGC-7901. The MDR-1 mRNA was examined using RT-PCR, and the difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 was compared with those with mp53+sv40Tag and controls by MTT method.

RESULTSSGC-7901 cells with mutant p53 showed higher MDR-1 mRNA than that of other two groups. SGC-7901 cells with mutant p53 showed higher chemotherapeutic sensitivity to 5-Fu than that with mp53+sv40Tag and control (P<0.05), but no difference between those with mp53+sv40Tag and control (P>0.05). SGC-7901 cells with mutant p53 and those with mp53+sv40Tag showed higher chemotherapeutic sensitivity to ADM than control (P<0.05), but no difference between those with mp53 and with mp53+sv40Tag (P>0.05). There was no difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 compared with those with mp53+sv40Tag and control to CDDP (P>0.05).

CONCLUSIONMutante p53 genes relates to multidrug resistance of gastric cancer.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adenocarcinoma ; genetics ; Drug Resistance, Multiple ; genetics ; Humans ; Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; genetics

BACKGROUNDThis study aimed to investigate multi-slice CT contrast-enhanced presentation of gastric cancer and its correlation with histo-differentiation and p53 and P-glycoprotein (P-gp) expression.

METHODSSixty-six patients with gastric cancer in the present study underwent a multi-slice CT preoperative routine and dual-phase contrast-enhanced examination of the upper abdomen; postoperative specimens were used to determine histo-differentiation and the expression of p53 and P-gp. The correlation of multi-slice CT contrast-enhanced presentation with histo-differentiation and expression of p53 and P-gp was analyzed.

RESULTSThe dual-phase contrast-enhanced ratio (CER) was not correlated with the histo-differentiation of gastric cancer (P > 0.05). Positive expression of p53 and P-gp was significantly higher in the cases of layered or heterogeneous enhancement than in the cases of homogenous enhancement (P < 0.05). Positive expression of p53 was also correlated with the arterial phase CER, tumor size and lymph node metastasis (P < 0.05), but not with infiltration thickness of the gastric wall, nor was it correlated with the portal phase CER (P > 0.05). Positive expression of P-gp was only correlated with the portal phase CER (P = 0.005).

CONCLUSIONSDifferently enhanced pattern and CER of the arterial and portal phase in gastric cancer correlate with its different histo-differentiation and expression of p53 and P-gp respectively. In addition, tumor size and lymph node metastasis of gastric cancer relate to the expression of p53.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Stomach Neoplasms ; diagnostic imaging ; metabolism ; pathology ; Tomography, X-Ray Computed ; methods ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo 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.

METHODSGenomic 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.

RESULTSAmongst 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).

CONCLUSIONCertain percentage of prostatic adenocarcinoma harbors 53BP1 mutations which may be involved in the carcinogenesis.

Adenocarcinoma ; genetics ; pathology ; Aged ; Exons ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Middle Aged ; Mutation ; Mutation Rate ; Mutation, Missense ; Polymorphism, Single Nucleotide ; Prostatic Hyperplasia ; genetics ; Prostatic Neoplasms ; genetics ; pathology ; Tumor Suppressor p53-Binding Protein 1

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.
A549 Cells ; DNA Damage ; radiation effects ; Humans ; Janus Kinase 2 ; metabolism ; Receptors, Interleukin-6 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Tumor Suppressor p53-Binding Protein 1 ; metabolism ; X-Rays

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.
Carrier Proteins ; metabolism ; Cell Line, Tumor ; DNA Breaks, Double-Stranded ; DNA Repair ; genetics ; DNA-Binding Proteins ; metabolism ; Genomic Instability ; HeLa Cells ; Histones ; metabolism ; Humans ; Nuclear Proteins ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Signal Transduction ; Tumor Suppressor p53-Binding Protein 1 ; metabolism ; Ubiquitin ; Ubiquitin-Protein Ligases ; genetics ; metabolism ; Ubiquitination

AIMTo investigate whether adriamycin induces DNA damage and the formation of gammaH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa.

METHODSHuman 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.

RESULTSThe 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.

CONCLUSIONHuman mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

Androstadienes ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Cells, Cultured ; Comet Assay ; DNA Breaks, Double-Stranded ; drug effects ; DNA Damage ; DNA Repair Enzymes ; metabolism ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; pharmacology ; Drug Interactions ; Flow Cytometry ; Histones ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Spermatozoa ; cytology ; drug effects ; metabolism ; Tumor Suppressor p53-Binding Protein 1

OBJECTIVETo 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.

METHODSIn 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.

RESULTS AND CONCLUSIONSMDC1 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.

Animals ; Blotting, Western ; Cell Line, Tumor ; DNA Damage ; genetics ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; metabolism ; Tumor Suppressor p53-Binding Protein 1

OBJECTIVETo study the expressions and activities of Rho GTPases in hypoxia and its relationship with tumor angiogenesis.

METHODSThree tumor cell lines were used in this study: gastric cancer cell lines AGS, SGC7901 and hepatocellular carcinoma cell line HepG2. Expression level of Rac1 mRNA was detected by semi-quantitative RT-PCR. Activity of Rac1 was determined by pull-down assay and expression of HIF-1alpha, VEGF, p53 and PTEN protein was detected by Westernblot.

RESULTSThe expression level of Rac1 mRNA was significantly increased in hypoxia compared to normoxia. Pull-down assay showed that hypoxia-induced activity of Rac1 was elevated in a time-dependent manner and climaxed at 3 hours. The expressions of HIF-1alpha and VEGF protein were up-regulated, while those of PTEN and p53 protein were down-regulated.

CONCLUSIONThese results indicate that hypoxia enhances Rac1 expression which might be involved in tumor angiogenesis by reacting with hypoxia-responsive genes.

Cell Hypoxia ; Cell Line, Tumor ; Hepatoblastoma ; blood supply ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Liver Neoplasms ; blood supply ; metabolism ; pathology ; Neovascularization, Pathologic ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; blood supply ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics ; rac1 GTP-Binding Protein ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics