RAD51 Research Articles

SWAT model calibration for hydrological modeling using concurrent methods, a case of the Nile Nyabarongo River basin in Rwanda

The Nile Nyabarongo, which is Rwanda's largest river, is facing stress from both human activities and climate change. These factors have a substantial contribution to the water processes, making it difficult to effectively manage water resources. To address this issue, it is important to find out the most accurate techniques for simulating hydrological processes. This study aimed to calibrate the SWAT model employing various algorithms such as GLUE, ParaSol, and SUFI-2 for the simulation of hydrology in the basin of the Nile Nyabarongo River. Different data sources, such as DEM, Landsat images, soil data, and daily meteorological data, were utilized to input information into the SWAT modeling process. To divide the basin area effectively, 25 sub-basins were created, with due consideration of soil characteristics and the diverse land cover. The outcomes point out that SUFI-2 outperformed the other algorithms for SWAT calibration, requiring fewer computing model runs and producing the best results. ParaSol established residing the least effective algorithm. After calibration with SUFI-2, the most sensitive parameters for modeling were revealed to be (1) the Effective Channel Hydraulic Conductivity (CH K2) measuring how well water can flow through a channel, with higher values indicating better conductivity, (2) Manning's n value (CH N2) representing the roughness or resistance to flow within the channel, with smaller values suggesting a smoother channel, (3) Surface Runoff Lag Time (SURLAG) quantifying the delay between rainfall and the occurrence of surface runoff, with shorter values indicating faster runoff response, (4) the Universal Soil-Loss Equation (USLE P) estimating the amount of soil loss. The average evapotranspiration within the basin was calculated to be 559.5 mma-1. These calibration results are important for decision-making and updating policies related to water balance management in the basin.

Time-Dependent Induction of the Nucleotide Excision Repair Gene XPA and RAD51 in Homologous Recombination in Human Lymphocytes Exposed to Low Doses of Ionizing Radiation

Introduction: The aim of the present study was to understand the effect of low-doses of ionizing radiation (LDIR) on repair genes expression in blood samples that were taken from healthy donors. The next purpose was to examine the time-effect on the modified gene expression caused by low-doses of ionizing radiation. Material and method: The RNA of peripheral blood lymphocytes (PBLs) taken from four healthy donors was isolated at different time points after exposure included 4, 24, 48, 72, and 168 hours and then cDNA was synthesized. Modification of XPA and RAD51 expression levels due to LDIR (2, 5, 10 cGy) were evaluated by relative quantitative reverse transcription-polymerase chain reaction.Results: Significant up-regulation of both repair genes was observed at the 4 and 168 h following to 10 cGy. Also, this dose could increase expression levels of RAD51 at 48 and 72 h after radiation. For lower doses at 5 cGy, only XPA levels were significantly up-regulated after 168 h. A significant regression was found between the XPA levels and the dose, at 168 h after irradiation to PBLs that can represent a new potential biomarker for biological dosimetry purposes.Conclusion: The results of this study could support the hypothetical role of the different DNA repair pathways in response to LDIR. This led us to propose a molecular biodosimetry method for ionizing radiation in the range of LDIR.

Arsenic trioxide upregulates UL16 binding protein 1 expression in KG1a cells through ataxia telangiectasia mutated and RAD3-related kinase pathway

Objective To observe the effect of arsenic trioxide (ATO) on the expression of NKG2D ligand UL16 binding protein 1(ULBP1) in acute myeloid leukemia KG1a cells, and explore the molecular mechanism for its regulation of ULBP1 expression. Methods KG1a cells were cultured in vitro.Then, the inhibition of KG1a cell proli-feration by different concentrations of ATO was detected by cell counting kit-8(CCK8) assay, and the expression of ULBP1 mRNA and surface protein in KG1a cells were examined by real-time RT-PCR and flow cytometry, respectively.After that, the blocking effects of ataxia telangiectasia mutated and RAD3-related kinase (ATM/ATR) inhibitor caffeine on ATO-upregulated expression of ULBP1 mRNA and surface protein expressions were investigated, and the effects of ATO on the expression of CHK1 and CHK2 proteins and their phosphorylation in KG1a cells were observed by Western blot method. Results Different concentrations (1, 2, 3, 4, 5 μmol/L) of ATO could inhibit the proliferation of KG1a cells, which was concentration dependent, and the half inhibitory (IC50) concentration to KG1a cells was 2.7 μmol/L.The expression of ULBP1 mRNA on KG1a cells were increased when incubated with ATO at concentration 1, 2, 3, 4, 5 μmol/L, compared without ATO group, ULBP1 mRNA expression level relatively increased respectively to (1.86±0.30) times, (3.02±0.71) times, (3.16±0.75) times, (4.80±0.70) times and (3.70±0.89) times, and the differences were statistically significant (all P<0.05). Furthermore, ATO (1, 2, 3, 4 and 5 μmol/L) upregulated ULBP1 protein expression on KG1a cells compared with that in the group without caffeine, and the differences were statistically significant (all P<0.05). After caffeine pretreat KG1a cell 2 h and ATO incubate KG1a cell 24 h, ULBP1 mRNA and protein expression levels were significantly reduced.When caffeine concentration was 8 mmol/L, ULBP1 mRNA expression level relatively reduces from (9.55±0.38) times to (6.36±0.93) times compared with that in the group without caffeine, and the difference was statistically significant (P<0.05). When caffeine concentration was 2, 4 and 8 mmol/L respectively, the expression of ULBP1 protein was reduced from that in the group without caffein treatment (3.50±0.08) times to (2.17±0.07) times, (2.02±0.06) times and (1.75±0.06) times, respectively, and the differences were statistically significant (all P<0.05). The expression of CHK1 and CHK2 proteins decreased with the increase of ATO concentration, while p-CHK1 and p-CHK2 are increased as ATO. Conclusions ATO upregulate the expression of ULBP1 mRNA and protein in KG1a cells, and the ATM/ATR-CHK1/CHK2 pathway may be involved in it. Key words: Arsenic trioxide; KG1a cells; Natural killer cell; UL16 binding protein 1; Ataxia telangiectasia mutated and RAD3-related kinase pathway

The miR-205-5p/BRCA1/RAD17 Axis Promotes Genomic Instability in Head and Neck Squamous Cell Carcinomas.

Defective DNA damage response (DDR) is frequently associated with tumorigenesis. Abrogation of DDR leads to genomic instability, which is one of the most common characteristics of human cancers. TP53 mutations with gain-of-function activity are associated with tumors under high replicative stress, high genomic instability, and reduced patient survival. The BRCA1 and RAD17 genes encode two pivotal DNA repair proteins required for proper cell-cycle regulation and maintenance of genomic stability. We initially evaluated whether miR-205-5p, a microRNA (miRNA) highly expressed in head and neck squamous cell carcinoma (HNSCC), targeted BRCA1 and RAD17 expression. We found that, in vitro and in vivo, BRCA1 and RAD17 are targets of miR-205-5p in HNSCC, leading to inefficient DNA repair and increased chromosomal instability. Conversely, miR-205-5p downregulation increased BRCA1 and RAD17 messenger RNA (mRNA) levels, leading to a reduction in in vivo tumor growth. Interestingly, miR-205-5p expression was significantly anti-correlated with BRCA1 and RAD17 targets. Furthermore, we documented that miR-205-5p expression was higher in tumoral and peritumoral HNSCC tissues than non-tumoral tissues in patients exhibiting reduced local recurrence-free survival. Collectively, these findings unveil miR-205-5p’s notable role in determining genomic instability in HNSCC through its selective targeting of BRCA1 and RAD17 gene expression. High miR-205-5p levels in the peritumoral tissues might be relevant for the early detection of minimal residual disease and pre-cancer molecular alterations involved in tumor development.

Induction of apoptosis and modulation of homologous recombination DNA repair pathway in prostate cancer cells by the combination of AZD2461 and valproic acid.

Cancer therapies using defects in homologous recombination (HR) DNA repair pathway of tumor cells are not yet approved to be applicable in patients with malignancies other than BRCA1/2-mutated tumors. This study was designed to determine the efficacy of combination therapy of a histone deacetylase inhibitor, valproic acid (VPA) and a novel PARP inhibitor AZD2461 in both PC-3 (PTEN-mutated) and DU145 (PTEN-unmutated) prostate cancer cell lines. The Trypan blue dye exclusion assay and the tetrazolium-based colorimetric (MTT) assay were performed to measure the cytotoxicity while combination effects were assessed based on Chou-Talalay's principles. Flow-cytometric assay determined the type of cell death. The real-time PCR analysis was used to evaluate the alterations in mRNA levels of HR-related genes while their protein levels were measured using the ELISA method. γ-H2AX levels were determined as a marker of DNA damage. We observed a synergistic relationship between VPA and AZD2461 in all affected fractions of PC-3 cells (CI<0.9), but not in DU145 cells (CI>1.1). Annexin-V staining analysis revealed a significant induction of apoptosis when PC-3 cells were treated with VPA+AZD2461 (p<0.05). Both mRNA and protein levels of Rad51 and Mre11 were significantly decreased in PC-3 cells co-treated with VPA+AZD2461 while enhanced H2AX phosphorylation was found in PC-3 cells after 12 and 24 hours of co-treatment (p<0.05). Our findings established a preclinical rationale for selective targeting of HR repair pathways by a combination of VPA and AZD2461 as a mechanism for reducing the HR pathway sufficiency in PTEN-mutated prostate cancer cells.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

Helicobacter pylori virulence factor vacuolating cytotoxin A (VacA) promotes homologous recombination repair in GES-1 cells

Objective To analyze the impact of Helicobacter pylori standard strain (Hp P12) and its virulence factor vacuolating cytotoxin A (VacA) on DNA damage and homologous recombination (HR) repair in a human gastric epithelial cell line (GES-1). Methods Strains of Hp P12 and vacA gene knockout Hp P12 (Hp P12 ΔvacA) were respectively used to infect GES-1 cells at a multiplicity of infection of 100. GES-1 cells treated with etoposide (50 μmol/L) or mitomycin (0.5 μg/ml) for 2 h were used as positive control. Western blot and immunofluorescence were performed to detect the expression of DNA damage marker protein γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) and the recruitment of them at DNA damage sites. Human embryonic kidney HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA strains to verify the impact of VacA on HR repair efficiency. Results The expression and recruitment of γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) were increased in Hp P12-infected cells as compared with that in uninfected and Hp P12 ΔvacA-infected cells (all P<0.05). To evaluate the HR repair efficiency, I-SceⅠ plasmid-transfected HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA and the results showed that green fluorescent protein (GFP)-positive cells were decreased after infection, especially in Hp P12 ΔvacA-infected cells (both P<0.05). Conclusions Hp P12 infection could cause DNA damage and promote HR repair in GES-1 cells, in which the virulence factor VacA played an important role. Key words: Helicobacter pylori; Homologous recombination repair; GES-1 cell; Vacuolating cytotoxin A (VacA)

Research of radiation resistance delivery through paracrine effects on non-small cell lung cancer

Objective To investigate the role and possible mechanisms of paracrine in radiation tolerance of non-small cell lung cancer (NSCLC) by observing the effects of radiation-resistant NSCLC H460R cell secretion on the radiosensitivity of parental H460 cells. Methods H460 cells were inoculated and cultured, and then divided into control group, conditional culturing group, irradiation group and irradiation combined conditional culturing group. One-time irradiation with 137Cs γ-rays was conducted with a dose rate of 1.02 Gy/min. Effect of H460R conditioned media on the radiation sensitivity of H460 cells was observed by clonogenic assay. The percentage of phospho-histone histone H2AX (γH2AX) positive cells was detected by immunofluorescence staining. The cyclical changes of the cells were detected by flow cytometry. The expression of DNA damage-associated proteins γH2AX and Rad51 were detected by Western Blot. Results The results showed that compared with the single γ-rays irradiation, the number of cell clones was significantly increased in the H460R cells after γ-rays irradiation combined with conditional culturing treatment at different doses of 2, 4, and 6 Gy, and the differences were statistically significant (2,4 Gy: all P<0.05; 6 Gy: P<0.01). The results of immunofluorescence staining showed that the percentage of γH2AX positive cells in the conditional culturing group was higher than that in the control group [(39.40±2.51)% vs. (25.21±2.05)%], and the difference was statistically significant (P<0.01). This value in the irradiation combined conditional culturing group was lower than that in irradiated group [(60.48±2.79)% vs. (80.65±2.05)%], and the difference was statistically significant (P<0.001). The flow cytometry analysis showed that the percentage of cells in G2/M phase for the irradiation (4 Gy) combined conditional culturing group was higher than that of irradiated group [(26.83±1.42)% vs. (15.73±1.29)%], and the difference was statistically significant (P<0.001). The Western Blot results showed that γH2AX and Rad51 protein expression in the irradiation (4 Gy) combined conditional culturing group respectively was lower and higher than that in irradiated group. Conclusion In the tumor microenvironment, radiation-resistant H460R cells can enhance the radioresistance of H460 cells by paracrine, which may be related to the promotion of DNA repair ability after irradiation Key words: Non-small cell lung cancer; Paracrine; Radioresistance; DNA repair

A review on role of ATM gene in hereditary transfer of colorectal cancer.

Colorectal cancer found to be the most commonly occurring cancer worldwide which can be prevented by screening and its curable if diagnosed early. Lynch syndrome/HNPCC being an autosomal genetic disease and propensity in forming colorectal cancer is inherited wherein genomic instabilities and epigenetic changes are being the characteristic forms in hereditary cancers. It is very important to determine the polymorphism in several DNA repairing genes such as ATM, RAD51, XRCC2, XRCC3 and XRCC9 to study the risk exploring both the prognosis and the developing of colorectal cancer. The role of ATM gene has been studied which involves in the hereditary transfer of colorectal cancer associated with other related cancers such as stomach, lung and breast cancers. ATM found to be the mutation target and also a modifier gene with more risk of developing the disease by its polymorphism in variant of ATM D1853N. It was identified that ATM gene polymorphism did not drastically change HNPCC age of onset. ATM expression levels were studied and it has been concluded that the complete loss of ATM expression resulted in a propensity of worse survival and no better prognosis with increase in mortality rate. This ATM gene might be considered to be a predicted biomarker in colorectal cancer. (www.actabiomedica.it)

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.

Lentivirus mediated RNAi silence esophageal MDC1 Eca109 cell gene expression of the influence of nude mouse transplantation tumor radiosensitivity

Objective To investigate the effects of inhibition of MDC1 protein expression on xenografted tumors in nude mice, and to observe the histopathological and cellular changes in nude mice. Methods Three pairs of effective and control short hairpin RNA targeting MDC1 mRNA were designed and cloned into the pSIH1-H1-copGFP vector. Real-time PCR and Western blot were used to determine the mRNA and protein expression of MDC1. After selection by copGFP reporter gene, cells were divided into negative transfection group (ECA109-N) and MDC1 transfection group (ECA109-M). The transfected cells were injected into nude mice. The mice were divided into ECA109 group, ECA109-N group, and ECA109-M group. Each group was divided into irradiation subgroup and non-irradiation subgroup. The changes in tumor size after irradiation were evaluated in each group. Western blot was used to measure the expression of CHK1, CHK2, and CHK2T68 in xenografted tumors. Flow cytometry was used to analyze the cell cycle distribution and apoptosis of tumor cells in nude mice. The variance analysis was used to compare the mean of multiple groups, and the SNK-q test was used in the two two groups. Results The pMDC1-shRNA plasmid was successfully constructed and used to transfect ECA109 cells. ECA109-M cells were obtained by stable transfection with the recombinant plasmid. All inoculated nude mice survived with visible xenografted tumors at the underside of the paw in about one week. There was no swelling and wound in inoculation sites. There was no significant difference in tumor size between different groups (P>0.05). The tumor growth in the ECA109 group and the ECA109-N group significantly slowed down after irradiation with a dose of 15 Gy (P 0.05); however, the phosphorylation of CHK2T68 protein was significantly reduced after irradiation (P 0.05). Conclusions Inhibition of MDC1 protein expression by RNA interference can effectively inhibit the growth of xenografted tumors after irradiation in the nude mice by increasing their radiosensitivity. Key words: RNA interference; Radiosensitivity; MDC1 gene; Implanted tumor in nude mice, ECA109 cell line; Tumor growth delay

Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive ovarian carcinoma cell lines.

Although epithelial ovarian cancers (EOCs) are initially treated with platinum-based chemotherapy, EOCs vary in platinum responsiveness. Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive EOC cell lines is valuable for development of therapeutic strategies to avoid platinum inefficacy and to exploit platinum sensitivity. TOV-21G devoid of FANCF expression, OV-90 and SKOV-3 were employed as examples of platinum-sensitive, platinum-intermediate and platinum-resistant cell lines, respectively. Antineoplastic agents examined included mitomycin C, doxorubicin, etoposide, gemcitabine, chlorambucil, paclitaxel, triapine and X-rays. Their effectiveness against cell lines was analyzed by clonogenic assays. Cytotoxic profiles of mitomycin C and carboplatin were similar, with mitomycin C exhibiting greater potency and selectivity against TOV-21G than carboplatin. Cytotoxic profiles of doxorubicin, etoposide and X-rays overlapped with that of carboplatin, while OV-90 overexpressing Rad51 was more resistant to chlorambucil than SKOV-3. The efficacy of paclitaxel and triapine was independent of platinum sensitivity or resistance. Consistent with these cytotoxic profiles, cisplatin/mitomycin C, triapine, and paclitaxel differed in the capacity to induce phosphorylation of H2AX, and produced unique inhibitory patterns of DNA/RNA syntheses in HL-60 human leukemia cells. Paclitaxel and triapine in combination produced additive antitumor effects in M109 murine lung carcinoma. In conclusion, mitomycin C is potentially more effective against Fanconi anemia pathway-deficient EOCs than carboplatin. Doxorubicin and etoposide, because of their overlapping cytotoxic properties with carboplatin, are unlikely to be efficacious against platinum-refractory EOCs. Paclitaxel and triapine are effective regardless of platinum sensitivity status, and promising in combination for both platinum-sensitive and platinum-refractory EOCs.

Effect of RNA interference for MDC1 gene on cell cycle and expression of related proteins in esophageal carcinoma cells after X-ray radiation

Objective To apply RNA interference technique for reducing the expression of MDC1 gene in esophageal carcinoma cell line ECA109, observe the changes in cell cycle and radiosensitivity after radiation, and discuss related mechanisms. Methods Three pairs of effective interference sequences and negative control sequences were synthesized for MDC1 mRNA sequence, and a recombinant plasmid was constructed with the vector pSIH1-H1-copGFP. RT-PCR and Western blot were used to determine the expression levels of MDC1 mRNA and protein. Colony-forming assay was applied to measure radiosensitivity, flow cytometry to determine cell cycle, Western blot to determine the expression of CHK1 and CHK2 proteins, and laser scanning confocal microscope to observe the number of MDC1 blotches inside the nucleus. One-way analysis of variance was used to analyze the differences between groups. Results The pSIH1-H1-copGFP plasmid was constructed successfully and ECA109 cells were infected to obtain ECA109M cells with stable transfection. The expression levels of MDC1 mRNA and protein in ECA109M cells were lower than those in ECA109N and ECA109 cells (P=0.032 and 0.041, respectively). After 5-Gy radiation, ECA109M cells had a lower proportion of G2+ M cells than ECA109N and ECA109 cells (P=0.026). After 5-Gy radiation, ECA109, ECA109N, and ECA109M cells had similar expression levels of CHK1 and CHK2 proteins (P=0.345 and 0.451, respectively), and ECA109M cells had a lower expression level of CHK2 T68 protein than ECA109 and ECA109N cells (P=0.012). ECA109 cells had a D0 value of 3.06 Gy and an SF2 value of 0.91; the D0 values for ECA109N and ECA109M cells were 2.90 Gy and 1.88 Gy, respectively, and the SF2 values for them were 0.89 and 0.84, respectively (P=0.021 and 0.037, respectively). Conclusions RNA interference can reduce the expression levels of MDC1 protein and cell cycle-related proteins, release cell cycle arrest, and enhance radiosensitivity in esophageal carcinoma ECA109 cells. Key words: Esophageal carcinoma; RNA interference; cell cycle protein; MDC1; radiosensitivity

TODRA, a lncRNA at the RAD51 Locus, Is Oppositely Regulated to RAD51, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair.

Expression of RAD51, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand RAD51 regulation we functionally characterized a long non-coding (lnc) RNA, dubbed TODRA (Transcribed in the Opposite Direction of RA D51), transcribed 69bp upstream to RAD51, in the opposite direction. We demonstrate that TODRA is an expressed transcript and that the RAD51 promoter region is bidirectional, supporting TODRA expression (7-fold higher than RAD51 in this assay, p = 0.003). TODRA overexpression in HeLa cells induced expression of TPIP, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of RAD51. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes RAD51 expression, results in downregulation of TODRA. Moreover, TODRA overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.

Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

Genomic instability (IN) is a common feature of many human cancers. The TP53 tumour suppressor gene is mutated in approximately half of human cancers. Here, we show that BRCA1 and RAD17 genes, whose derived proteins play a pivotal role in DNA damage repair, are transcriptional targets of gain-of-function mutant p53 proteins. Indeed, high levels of mutp53 protein facilitate DNA damage accumulation and severely impair BRCA1 and RAD17 expression in proliferating cancer cells. The recruitment of mutp53/E2F4 complex onto specific regions of BRCA1 and RAD17 promoters leads to the inhibition of their expression. BRCA1 and RAD17 mRNA expression is reduced in HNSCC patients carrying TP53 mutations when compared to those bearing wt-p53 gene. Furthermore, the analysis of gene expression databases for breast cancer patients reveals that low expression of DNA repair genes correlates significantly with reduced relapse free survival of patients carrying TP53 gene mutations. Collectively, these findings highlight the direct involvement of transcriptionally active gain of function mutant p53 proteins in genomic instability through the impairment of DNA repair mechanisms.

Suppression of ATR reverses the cisplatin resistance in ovarian cancer SKOV3 cells

To explore the effect of ataxia telangiectasia mutated and RAD3 related protein (ATR) expression and ATR kinase activity on the sensitivity to cisplatin in ovarian cancer SKOV3 cells. SiRNA targeting ATR was transfected into SKOV3 cells for 48 h to reduce the ATR protein level, and ATR kinase inhibitor VE-821 was used for 12 h to inhibit the ATR pathway activity. The alteration of cell viability was examined by CCk-8 assay. Expression levels of ATR, p-ATR and γ-H2AX proteins were detected by Western blot. The DNA double strand breaks (DSB) marker γ-H2AX and homologous recombination repair key protein RAD51 and their co-localization in the cells were examined under the confocal microscope. The status of DNA double strand breaks (DSB) in single cells was visualized by alkaline comet assay. Finally, the cell cycle distribution was assessed using flow cytometry. DDP caused evident DNA double strands breaks and activated ATR kinase pathway. ATR-siRNA notably reduced ATR protein level, the 48 h IC(50) value of DDP was 72.12 µmol/L and 41.25 µmol/L, respectively, in the NC-siRNA and ATR-siRNA groups (P < 0.05). Confocal microscopic assay presented decreased recruitment of RAD51 at the DSB loci and comet assay showed enhanced DSB in the cells after ATR knocking down. After the inhibition of ATR kinase by VE-821, the 48 h IC(50) value of DDP was 75.32 µmol/L and 45.64 µmol/L, respectively, in the DMSO and VE-821 groups (P < 0.05 for both), confocal microscopic assay demonstrated reduced RAD51 recruitment, and comet assay showed increased DSB in cells after ATR kinase inhibition. Flow cytometry showed that percentage of cells distributed in G(0)/G(1), S and G(2)/M phases was 71.2%, 13.4% and 15.4%, repectively, after 40 µmol/L DDP treatment for 24 h. Compared with that of control group (G(0)/G(1): 54.2%, S: 21.3% and G(2)/M: 24.4%), DDP induced G(0)/G(1) phase arrest. DDP intervention resulted in the cell cycle status (G(0)/G(1): 43.2%, S: 20.4%, G(2)/M: 36.4%) in the ATR-siRNA group and (G(0)/G(1): 40.2%, S: 22.5%, G(2)/M: 37.3%) in the VE-821 group, indicating that the inhibition of ATR or ATR kinase could abrogate the effect of G(0)/G(1) phase arrest induced by DDP. Suppression of ATR can affect the homologous recombination repair in ovarian cancer cells, leading to accumulation of DNA double strand breaks in the cell nuclei as well as reduction of DDP-caused G(0)/G(1) phase arrest, finally enhances the sensitivity to cisplatin in the ovarian cancer SKOV3 cells.

RAD52 (RAD52 homolog (S. cerevisiae))

Review on RAD52, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

Expression of checkpoint kinase 1 in esophageal squamous cell carcinoma and its significance

Objective To investigate the expression of checkpoint kinase 1 (Chk1) in esophageal squamous cell carcinoma (ESCC) and its significance.Methods hnmunohistochemistry and in situ hybridization methods were used to detect the expression of Chk1 gene in 62 cases of ESCC,31 cases of adjacent dysplasia and 62 cases of normal esophageal mucosa.Results The expression of Chkl mRNA and protein was gradually increased from normal esophagus mucous tissue (protein:14.5％,mRNA:82.3％),adjacent atypical hyperplasia tissue (protein:38.7％,mRNA:35.5％) to ESCC tissue (pro-tein:88.7％,mRNA:8.1％),and there was significant difference among three groups (P ＜ 0.05).The expression levels of Chk1 mRNA and protein in the deep layer invasive groups (protein:94.5％,mRNA:85.5％) were considerably higher than those in infiltrating shallow layer group (protein:42.9％,mRNA:57.1％),and there was significant difference among the groups (P ＜ 0.05).The expression levels of Chk1 mRNA and protein in lymphatic metastasis groups (protein:100.0％,mRNA:95.0％) were significantly higher than those in non-lymphatic metastasis group (protein:83.3％,mRNA:76.2％),and there was significant difference among the groups (P ＜ 0.05).The expression levels of Chk1 mRNA and protein in poor differentiated ESCC tissues were markedly higher than those in well differentiated tissues (P ＜0.05).Conclusion The expression of Chk1 protein and mRNA was closely associated with the invasion and metastasis of ESCC. Key words: Esophageal squamous carcinoma; Checkpoint kinases 1 ; Infiltration; Metastasis

The effect of checkpoint kinase 1 small interfering RNA on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo

Objective Constructing the nude mouse esophageal cancer transplanted tumor model,to observe the effect of checkpoint kinase 1 (Chk1) small interfering RNA (siRNA) interference expression vector on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivo.Methods Group breeding tumor-burdened nude mouse,namely A groups:injection Sterile Saline; B group:injection Chk1 siRNA add cisplatin (5 mg/kg),C groups:injection cisplatin (5 mg/kg).Observing the transplantation tumor growth,using immunohistochemical to test the protein expression of Chk1 in the tumor tissues,using in situ hybridization to test the mRNA expression of Chk1 in the tumor tissues,using TdT-mediated dUTP nick end labeling (TUNEL) method to test the apoptosis situation of tumor tissues.Results The volume of transplanted tumor in the B group [(279.11 ± 27.23) mm3] was noticeably less than that in the C group [(512.41 ±35.18) mm3] and A group [(907.82 ±58.09) mm3],there were a statistical difference among three groups (P ＜ 0.01),the expression of Chk1 protein (26.87 ± 5.31) or mRNA (45.29 ± 6.47) in B group was significantly lower than that in the C group (protein:56.80 ± 7.00 ; mRNA:87.51 ± 8.38) and A group (protein:98.23 ± 15.67 ; mRNA:187.23 ± 18.55),there were a statistical difference among three groups (P ＜ 0.05),the resuhs of TUNEL method detection:the cells apoptosis index (AI) in group B [(21.03 ± 1.35) ％] is obviously higher than that in group C [(12.41 ± 1.23) ％] and group A [(1.34 ± 0.12) ％],there were a statistical difference among three groups (P ＜ 0.05).Conclusion Silencing Chk1 expression may strengthen the growth inhibition of nude mouse esophageal cancer transplantation tumor which cisplatin bring on,induce cell apoptosis of transplantation tumor. Key words: Esophageal squamous carcinoma; Checkpoint kinases 1 ; Cisplatin; Nud emice

Radiobiological characteristics and MRN complex expression of human nasopharyngeal carcinoma cell lines

To study the radiobiological characteristic of human nasopharyngeal carcinoma cell lines CNE1 and CNE2 and the changes in expression MRN (Mre11-Rad50-Nbs1) complex in the cell lines exposed to irradiation. CNE1 and CNE2 were irradiated by a linear accelerator. Radiobiological characteristics were detected by colony assay and MTT assay. MRN complex expression were examined by Western blot. Surviving fraction at 2 Gy (SF2), quasi-threshold Dose (Dq), and mean lethal dose (Do) of CNE1 were 0.56, 1.449 Gy and 1.480 Gy; SF2, Dq, and Do of CNE2 were 0.44, 0.776 Gy and 1.685 Gy, respectively. Survival fraction of CNE1 at the day 6 after 4 Gy irradiation was 0.59 and that of CNE2 was 0.79 when compared with control, with the up-regulated expressions of Rad50 in CNE1 and Mre11, Rad50 and Nbs1 in CNE2 (P < 0.05). CNE1 and CNE2 were sensitive to radiation, but there were radioresistance cells in CNE2. The expressions of some components of MRN complex were up-regulated to repair DNA lesions induced by radiation.