Bavachin induces apoptosis in colorectal cancer cells through Gadd45a via the MAPK signaling pathway / 中国天然药物

Bavachin is a dihydroflavonoid compound isolated from Psoralea corylifolia, and exhibits anti-bacterial, anti-inflammatory, anti-tumor and lipid-lowering activities. Recent attention has gradually drawn on bavachin-induced apoptosis in many human cancer cell lines. However, the anti-cancer effects and related mechanisms in colorectal cancer remain unknown. Here, we investigated the effects of bavachin on colorectal cancer in vivo and in vitro. The results showed that bavachin inhibited the proliferation of human colorectal cancer cells and induce apoptosis. These changes were mediated by activating the MAPK signaling pathway, which significantly up-regulated the expression of Gadd45a. Furthermore, Gadd45a silencing obviously attenuated bavachin-mediated cell apoptosis. Inhibition of the MAPK signaling pathway by JNK/ERK/p38 inhibitors also weakened the up-regulation of Gadd45a by bavachin. The anticancer effect of bavachin was also validated using a mouse xenograft model of human colorectal cancer. In conclusion, these findings suggest that bavachin induces the apoptosis of colorectal cancer cells through activating the MAPK signaling pathway.

Loss of ATM activity induces GADD45α-dependent apoptosis in cerebellar granular neurons / 中山大学学报(医学科学版)

objectiveTo explore the specific molecular mechanism of neuronal apoptosis induced by ATM inactivation. MethodsCGNs matured 7 days in vitro were cultured 8 h with 25 K， 5 K or 25 K medium containing ATM-specific inhibitors （Ku55933， 10 µmol/L； Ku60019， 15 µmol/L） for Hoechst stain and apoptosis analysis， or cultured for different lengths of time （2， 4， 8 h） to detect the protein expression levels of ATM， caspase-3 and cleaved caspase-3 by Western blotting. ATM and GADD45α specific siRNA was transfected into C6 cells and CGNs， and its interference efficiency was verified by q-PCR and Western blotting. CGNs matured for 5 days in vitro were transfected with ATM specific siRNA and pCMV-EGFP by calcium phosphate for 48 h， Hoechst staining and apoptosis analysis were performed. CGNs matured for 7 days in vitro were treated with 25 K medium containing ATM specific inhibitors for 8 h， transcriptome sequencing， differential expression gene identification and pathway enrichment analysis were performed. CGNs matured for 5 days in vitro were co-transfected with GADD45α specific siRNA and pCMV-EGFP by calcium phosphate for 48 h， then treated with 5 K or 25 K medium containing 15 µmol/L Ku6 for 8 h. Hoechst staining and apoptosis analysis were performed. ResultsCompared with the 25 K， CGNs nuclear pyknosis rate， cleaved Caspase-3 and ATM protein expression level were increased in the 5 K and ATM-specific inhibitor groups. The mRNA and protein expression levels of ATM and GADD45α were effectively reduced after transfection of ATM and GADD45α specific siRNA in C6 cells and CGNs. Compared with control， CGNs transfected with ATM specific siRNA showed a higher nuclear pyknosis rate. Totally 835 genes were identified to be up-regulated and 848 genes to be down-regulated in the Ku55933 treatment group； 454 genes were identified to be up-regulated and 314 genes to be down-regulated in the Ku6 treatment group； 274 genes were co-up regulated in the Ku5 and Ku60019 treatment groups， while 179 genes were co-down-regulated in the Ku5 and Ku6 treatment groups and the expression of ATM downstream target GADD45α was upregulated. The enrichment results showed that TNF signaling pathway， NF-κB signaling pathway and Apoptosis signaling pathway were significantly enriched. Compared with control， mRNA and protein expression levels of GADD45α were increased in inhibitor treatment and 5 K， while knocking down GADD45α resulted in a decrease in nuclear pyknosis rate in the Ku60019 and 5 K treatment group. ConclusionLoss of ATM activity induces GADD45α-dependent cerebellar granular neuronal apoptosis.

Carfilzomib inhibits the growth of lung adenocarcinoma via upregulation of Gadd45a expression / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.

GADD45g exerts anti-tumor effect in acute myeloid leukemia via inhibition of E2F1 / 中国肿瘤生物治疗杂志

@# Objective: To investigate the correlation between the expression of E2F1 and growth arrest and DNA damage inducible protein 45g (GADD45g) in patients with acute myeloid leukemia (AML), and to explore whether GADD45g exerts its induction of DNA damage, cell apoptosis, senescence, cell cycle arrest and drug sensitivity in AML through inhibition of E2F1. Methods: A total of 32 cases of bone marrow specimens from patients initially diagnosed asAML in Hospital of Blood DiseasesAffiliated to ChineseAcademy of Medical Sciences from January 2013 to December 2016, were selected for this study; In addition, AML cell lines (U937, HL60, THP-1, Molm-13) were also collected for this study. The mRNAexpression of GADD45g and E2F1 in above mentioned specimens and cell lines by qPCR,andtheircorrelationwasalsoanalyzed.Thelentiviral vector over-expressing E2F1 (pLV-E2F1-RFP) was constructed to prepare recombinant lentivirus, which was then transfected Molm-13 and THP-1 cells that over-expressing GADD45g. Whether GADD45g exerts tumor inhibition effect on AML cells through inhibition of E2F1 was determined by comet assay, Annexin V/7AAD flow cytometry, β-galactosidase staining and PI staining flow cytometry etc. Results: The mRNA expression of GADD45g was negatively correlated with E2F1 in bone marrow of AML patients and AML cell lines (r=–0.663, P<0.01). Over-expression of GADD45g significantly inhibited the expression of E2F1 in AML cell lines (all P<0.01). Molm-13 and THP-1 cells that simultaneously over-expressing GADD45g and E2F1 were successfully constructed. Compared with the control group, the protein expressions of GADD45g and E2F1 in over-expression groups were significantly increased (all P<0.01). Compared with cells over-expressing GADD45g alone, simultaneous over-expression of both GADD45g and E2F1 significantly reduced the apoptosis, senescence and DNA damage (all P< 0.01), and rescued cell cycle arrest in Molm-13 and THP-1 cells (all P<0.01), thus further reduced the chemo-sensitivity of AML cells caused by GADD45g over-expression (all P<0.01). Conclusion: GADD45g exerts anti-tumor effect inAMLvia inhibition of E2F1.

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

Down-regulated expression and antioncogenetic role of GADD45g in acute myeloid leukemia / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To quantify the expression of growth arrest and DNA damage inducible protein 45g (GADD45g) gene in the bone marrow samples of patients withAML (acute myeloid leukemia) and inAML cell lines, as well as to study the correlation between the GADD45g expression and prognostic outcome in patients withAML and investigate the role of GADD45g over-expression in proliferation, apoptosis, senescence, differentiation, cell cycle arrest, and drug sensitivity in AML cell lines. Methods: In the study, a total of 27 cases of bone marrow specimens were selected from patients initially diagnosed as AML in Hospital of Blood Diseases affiliated to Chinese Academy of Medical Sciences from January 2013 to December 2016. mRNA and protein expression levels of GADD45g in BMMNCs (Bone marrow mononuclear cells) from patients with AML and healthy donors and in AML cell lines were quantified by quantitative real-time PCR and Western blotting. The correlation between GADD45g expression and overall survival (OS), coupled with event-free survival (EFS) in patients with AML was analyzed in two gene expression datasets (GSE10358, GSE425-GPL317). Lentiviral vectors over-expressing GADD45g were constructed and transfected into AML cell lines (U937, THP-1 and Molm-13 cell lines). The role of GADD45g over-expression in cell proliferation, colony formation, senescence, apoptosis, cell cycle arrest, differentiation and drug sensitivity of U937, THP-1 and Molm-13 cells were detected by cell counting, colony-forming assay, β-galactosidase staining and flow cytometric analysis of Annexin V/7AAD staining, PI staining and so on, respectively. Results: Expression of GADD45g was dramatically down-regulated in BMMNCs in AML patients and AML cell lines compared to that from healthy donors (all P<0.01). The OS (P<0.05) and EFS (P<0.05) of AML patients with low GADD45g expression were significantly shorter that those of AML patients with higher GADD45g level. Enforced expression of GADD45g could inhibit cell growth and colony formation, promote senescence and apoptosis, induce cell cycle arrest and differentiation and enhance drug sensitivity in AML cell lines (P<0.05 or P<0.01). Conclusion: GADD45g expression was remarkably silenced in marrow tissues of patients withAML andAML cell lines; it showed remarkable and all-around inhibiting effect onAMLcell lines, indicating that GADD45g expression has prognostic value inAML.

Correlation between GADD45 mRNA expression and exposure dose in peripheral blood of radiation workers / 中国职业医学

OBJECTIVE: To analyze the growth arrest and DNA damage gene 45( GADD45) mRNA expression in peripheral blood of radiation workers,and its relationship with exposure dose. METHODS: Using a typical sampling method,97 radiation workers from a hospital of Guangdong were selected as radiation group,48 hospital administrative staff without exposure to radiation and other occupational hazard factors were selected as control group. The real-time quantitative polymerase chain reaction was used to detect GADD45 mRNA expression of two groups. With the use of thermoluminescence dosimetry,we detected and collected personal dose data of radiation group over the past year. RESULTS: The GADD45 mRNA relative expression of the radiation group was higher than that of the control group( P < 0. 01).There was no correlation between GADD45 mRNA relative expression and factors such as personal dose,sex,age,length of service,smoking,and alcohol drinking( P > 0. 05). CONCLUSION: The ionizing radiation can lead to up-regulation of GADD45 mRNA expression in peripheral blood of radiation workers,but there was no linear relationship between GADD45 mRNA expression and its personal dose.

GADD45αin genotoxicity test:molecular principles and research progress / 中国药理学与毒理学杂志

As a member of growth arrest and DNA damage inducible gene family,GADD45αparticipats in the regulation of cell cycle,cell senescence,cell survival and apoptosis. GADD45αplays a critical role in the responses to cell injury induced by a variety of factors including cell stress and genotoxic chemicals. Different transcription factors and proteins are involved in transcriptional regulation of GADD45αgene. GADD45αprotein has been implicated in the regulation of genomic stability related cellular responses through interaction with other proteins. Genotoxicity test systems based on the char?acteristics of GADD45α in regulation of cell function,can be applied to the detection of potentially genotoxic compounds,which provides new ideas and methods about genotoxicity assessment. The molecular mechanism and research progress of GADD45α in genotoxicity test are summarized in this article.

Effects of 2-methoxyestradiol on the proliferation and apoptosis of B16 malignant melanoma cells / 中华皮肤科杂志

Objective To investigate the effects of 2-methoxyestradiol (2-ME) on the proliferation and apoptosis of a mouse malignant melanoma cell line B16,and to explore their mechanism.Methods B16 cells were cultured in vitro,and divided into a negative control group receiving no treatment and several intervention groups treated with 2-ME at final concentrations of 5,10,20,40 mmol/L,respectively.After different durations of treatment,inverted phase-contrast microscopy was conducted to observe the morphologic change of B16 cells,sulforhodamine B (SRB) assay to evaluate proliferative activity and to draw growth curve of B16 cells according to the absorbance value at 490 nm,flow cytometry to detect cell cycle and apoptosis,and reverse transcription PCR and real-time PCR were performed to measure the expressions of the apoptosis-inducing gene gadd45b and proto-oncogene c-myc.Results As repeated measures analysis of variance showed,there were significant differences in the inhibitory effect on B16 cell proliferation among different concentrations (5,10,20,40 mmol/L) and different treatment durations (24,48,72 hours) of 2-ME (F =1170.94,1843.04,respectively,both P ＜ 0.01),and there was a significant interaction effect between these concentrations and treatment durations (F =272.79,P ＜ 0.01).After 48-hour treatment with 2-ME at 10,20 and 40 mmol/L,the apoptosis rate of B16 cells was increased to (4.13 ± 1.12)％,(11.25 ± 2.380)％ and (19.46 ± 2.9)％ respectively,compared to (0.23 ± 0.5)％ in the negative control group (all P＜ 0.01); the proportion of B16 cells in G0/G1 phase was increased to (59.5 ± 5.6)％,(63.4 ± 8.2)％ and (70.8 ± 4.4)％ respectively,compared to (44.1 ± 3.4)％ in the negative control group.There was a significant difference in the proportion of B16 cells in G0/G1 phase among the negative control group and intervention groups (F =13.56,P ＜ 0.05).Moreover,the mRNA expression of gadd45b was significantly enhanced after 24-hour treatment with 2-ME at concentrations of 20 and 40 mmol/L (both P＜ 0.01),while that of c-myc was significantly weakened after treatment with 2-ME at 10,20 and 40 mmol/L (all ＜ 0.05) compared with the negative control group.Conclusion 2-ME can inhibit the proliferation of B16 cells in vitro,upregulate the expression of gadd45b gene and downregulate the expression of C-myc gene.

Effects of 18F-FDG on P53, XIAP, and GADD45 expressions in transplanted Lewis lung carcinoma in mice / 中华放射医学与防护杂志

Objective To study the effects of internal exposure of 18F-FDG (18F-2-deoxy-2-fiuoroD-glucose) on the protein expressions of P53,XIAP (X-linked inhibitors of apoptosis protein) and GADD (growth arrest and DNA damage)45 in Lewis lung carcinoma,and to explore the possibility of applying 18F-FDG as a radiotherapy drug in vivo.Methods Lewis lung cancer transplanted tumor models were established via subcutaneous injection of 0.2 ml of 2 × 107/ml Lewis lung carcinoma cells at left hind limbs of 48 C57BL/6 mice that were randomly divided into high dose group,low dose group and control group with 16 mice each.After 7 d of cancer cell inoculation,18.5 × 107 Bq and 9.25 × 107 Bq of 18F-FDG in 0.2 ml saline or equal volume of physiological saline was intraperitoneally injected into three group mice,respectively.22 d post inoculation,the protein expressions of P53,XIAP and GADD45 were immuohistochemically detected by using SP approach.Results There was significant difference among the protein expressions in each group (x2 =8.30,16.02,7.68,P ＜0.05).The mean integral optic density of protein expression increased from 0.089 ± 0.033 in control group to 0.315 ± 0.028 in high dose group for P53,and from 0.126 ± 0.023 to 0.383 ±-0.035 for GADD45,but it decreased from 0.422 ± 0.034 to 0.149 ± 0.055 for XIAP.There was significantly difference of these protein expressions in each group (P53:F=5.26,P＜0.05;XIAP:F=4.29,P ＜0.05;GADD45:F=5.98,P ＜0.05).Conclusions 18F-FDG can up-regulate the expressions of P53 and GADD45 proteins and down-regulate the expression of XIAP protein in tumor cells,and it inhibits tumor growth by promoting cell apoptosis in the Lewis lung carcinoma tissue with a P53-dependent manner.

Stress sensor Gadd45G inhibits cell proliferation of human colon cancer cells in vitro / 肿瘤

Objective: To investigate the effect of Gadd45G (growth arrest and DNA-damage-inducible 45 gamma) overexpression on cell proliferation of colon cancer cells and the possible related mechanism. Methods: To explore whether Gadd45G functions in regulating cell proliferation of colon cancer cells, a lentiviral Tet (tetracycline)-inducible expression system was used for ectopic Gadd45G expression in colon cancer HCT116 and SW480 cells. The effects of Gadd45G over-expression on cell proliferation, cell cycle distribution, apoptosis and cellular senescence were analyzed by MTT method, FCM (flow cytometry) and senescence-associated beta-galactosidase assay, respectively. The expression of senescenceassociated secrete phenotype IL-8 (interleukin 8) and cell cycle-regulators were measured by RFQ-PCR (real-time fluorogenic quantitative-PCR) and Western blotting, respectively. Results: After doxycycline induction, Gadd45G expression was validated in HCT116 and SW480 cells. Gadd45G overexpression significantly inhibited the proliferation of HCT116 and SW480 cells (P < 0.001), and resulted in cell cycle arrest at G2/M phage. Intriguingly, Gadd45G over-expression efficiently elicited cellular senescence (P < 0.001) but not apoptosis. Moreover, the protein levels of cell cycle regulators p53, p21/CDKN1a, p16INK4a and Rb were not significantly altered in the colon cancer cells upon Gadd45G induction, while the expressions of IL-8 mRNA and senescence marker γ-H2A protein were significantly increased. Conclusion: The over-expression of stress sensor Gadd45G inhibits the proliferation of colon cancer cells through inducing cellular senescence. Copyright © 2013 by TUMOR.

Effects of trichostatin A on the expression of growth arrest and DNA damage-inducible protein 45 alpha in, proliferation of and apoptosis in a human epithelial carcinoma cell line A431 / 中华皮肤科杂志

Objective To investigate the effect of trichostatin A on the expression of growth arrest and DNA damage-inducible protein 45 alpha (Gadd45α) in,proliferation of and apoptosis in a human epithelial carcinoma cell line A431.Methods Cultured A431 cells were treated with different concentrations (0.05,0.1,0.25,0.5,1 μmogL) of trichostatin A for various durations (6,12,24,48 hours).Subsequently,cell proliferation,cycle and apoptosis were detected by cell counting kit-8 (CCK8) and flow cytometry respectively.The expression of Gadd45eα mRNA and protein in cultured A431 cells was detected by reverse transcription-PCR and Western blot respectively.Results Trichostatin A inhibited the proliferation of A431 cells in a dose (0.05-1.0 μmol/L)-dependent manner at all the 4 time points (F =3554.71,P ＜ 0.05),as well as in a time (6-48 hours)-dependent manner at these tested concentrations (F =1685.18,P ＜ 0.05).A statistical increase was induced in the early apoptosis rate,late apoptosis rate and Gadd45α mRNA expression in A431 cells by the 24 hour-treatment with trichostatin A of 0.1 to 0.5μmol/L.Elevated percentage of cells at G1 phase (26.910％ ± 0.799％,30.406％ ±0.625％,32.896％ ± 0.599％ vs.21.633％ ± 1.144％,F =105.93,P ＜ 0.05) and expression of Gadd45α protein (0.6536 ± 0.2193,0.6990 ± 0.0110,0.9040 ± 0.1971 vs.0.3766 ± 0.0241,F =332.88,P ＜ 0.01) were observed in A431 cells treated with trichostatin A of 0.1,0.25 and 0.5 μmol/L for 24 hours compared with untreated A431 cells.Conclusions Trichostatin A can enhance the mRNA and protein expression of Gadd45α in A431 cells,which may be involved in the suppression of cell proliferation as well as acceleration of apoptosis of A431 cells by trichostatin A.

Construction of Gadd45a expression plasmid and its expression in human T cells / 中南大学学报(医学版)

Objective To construct Gadd45a expression plasmid and induce its expression in human T cells.Methods Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells,and cloned into the pcDNA3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4+T cells using electroporation,and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot.Results Human Gadd45a expression plasmid was constructed successfully.Gadd45a was overexpressed both in Jurkat cells and normal human CD4+T cells after these cells were transfected with pcDNA3.1-Gadd45a.Conclusion The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45a in the epigenetic mechanism.

Effects of narrow-band ultraviolet B(NB-UVB)on the expression of Gadd45α and proliferation of human HaCaT keratinocytes / 中华皮肤科杂志

Objective To investigate the effects of NB-UVB on the expression of Gadd45α as well as cell apoptosis and cycle of human HaCaT keratinocytes.Methods Cultured HaCaT cells were exposed to various doses (100,200,400 mJ/cm2)of NB-UVB followed by an additional culture of 6,12 and 24 hours,respectively.Reverse transcription PCR and Western blot were performed to detect the mRNA and protein expression of Gadd45α respectively in HaCaT cells,cell counting kit 8 (CCK8)to measure the proliferation of cells,and flow cytometry to determine the cell cycle distribution of HaCaT cells before and after the exposure to NB-UVB.Results Gadd45α was expressed in HaCaT cells.After exposure to NB-UVB of the three doses,the mRNA and protein levels of Gadd45α increased at 6 hours and 12 hours,but declined at 24 hours,and significant changes were observed in HaCaT cells at the three time points after exposure to NB-UVB of the three doses (all P<0.05).The Gadd45α/β-actin mRNA ratio was 1.4360±0.6551.1.8633±0.0979,1.9266±0.1724 in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2,respectively,significantly higher than that in unirradiated cells(0.6000±0.1276,all P<0.05).Also,increased Gadd45α/β-actin protein ratio was noted in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2 compared with unirradiated cells (0.0773±0.0005,0.1936±0.0015,0.2373±0.0015 vs.0.0290±0.0010,all P<0.05).NB-UVB inhibited the proliferation of HaCaT cells in a time-and dose-dependent manner.Flow cytometry showed that irradiated HaCaT cells were blocked in G2 phase of the cell cycle.and the percentage of HaCaT cells in G2 phase was 13.53%±1.03%,17.77%±2.25%,30.03%±4.29%afler exposure to NB-UVB of 100,200 and 400 mJ/cm2,respectively,compared to 9.24%±0.97%in unirradiated cells (all P<0.05).Conclusions The expression of Gadd45α is increased in HaCaT cells after exposure to NB-UVB,and Gadd45α may be involved in the NB-UVB-induced suprression of cell proliferation of and cell cycle arrest in HaCaT cells.

Radiation-Induced changes in expression of genes related to DNA damage response in lymphocytes of human peripheral blood / 中华放射医学与防护杂志

Objective To detect the expression of DNA damage response genes induced by radiation in human peripheral blood lymphocyte,and to explore the new biomarkers of radiation.Methods The human peripheral blood cells were irradiated to X-rays at different doses of 0,1,2,3,4,and 5 Gy.The quantitative real.time qPCR wag used to detect the expressions of cyclin-dependent kinase inhibitor l a gene(Cdknl a)and growth arrest and DNA damage inducible gene(Gadd45a)in lymphoeytes at 4 and 24 h post-irradiation,respectively.The method of CB mieronucleus was used to determine the change of micronucleus ratio.Results The expression of Cdknl a in peripheral blood lymphocytes wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy.reached the peak at 4 Gy and began to decrease at 5 Gy,which showed a dose-dependent manner(r=0.946,0.975,P<0.05).Similarly,the expression of Gadd45α in human peripheral blood lymphocytes was also increased significantly at 4 and 24 h post-irradiation to 0-5 Gy in a dose-dependent manner,while the expression of Gadd45a at 4 h wag higher than that at 24 h(r=0.936,0.797,P<0.05).The ratio of micronuclei wag increased significantly at 4 and 24 h post-irradiation to 0-5 Gy(r=0.990,0.984,P<0.05).Conciusions Cdknl a and Gadd45α expression could be increaged significandy at 4 and 24 h post-irradiation to 0-5 Gy,showing a good linear relationship.which might be candidate for radiation biological dosimeter.

ATM modulates transcription in response to histone deacetylase inhibition as part of its DNA damage response

Chromatin structure has a crucial role in a diversity of physiological processes, including development, differentiation and stress responses, via regulation of transcription, DNA replication and DNA damage repair. Histone deacetylase (HDAC) inhibitors regulate chromatin structure and activate the DNA damage checkpoint pathway involving Ataxia-telangiectasia mutated (ATM). Herein, we investigated the impact of histone acetylation/deacetylation modification on the ATM-mediated transcriptional modulation to provide a better understanding of the transcriptional function of ATM. The prototype HDAC inhibitor trichostain A (TSA) reprograms expression of the myeloid cell leukemia-1 (MCL1) and Gadd45alpha genes via the ATM-mediated signal pathway. Transcription of MCL1 and Gadd45alpha is enhanced following TSA treatment in ATM+ cells, but not in isogenic ATM- or kinase-dead ATM expressing cells, in the ATM-activated E2F1 or BRCA1-dependent manner, respectively. These findings suggest that ATM and its kinase activity are essential for the TSA-induced regulation of gene expression. In summary, ATM controls the transcriptional upregulation of MCL1 and Gadd45alpha through the activation of the ATM-mediated signal pathway in response to HDAC inhibition. These findings are important in helping to design combinatory treatment schedules for anticancer radio- or chemo-therapy with HDAC inhibitors.

GADD45α MODULATES DNA METHYLATION INDUCED BY DNA DAMAGE DURING HOMOLOGOUS RECOMBINATION / GADD45α MODULA LA METILACIÓN DEL ADN INDUCIDO POR EL DAÑO DEL ADN DURANTE LA RECOMBINACIÓN HOMÓLOGA

Homologous recombination is one of the major pathways for repairing DNA double strand breaks, the most deleterious of DNA lesions. Recent studies suggest that DNA methylation events target homologous recombination segments; however, the underlying mechanism of DNA methylation during homologous recombination is not understood. In this work, we show that GADD45α, a protein involved in cell cycle control, growth arrest, and apoptosis, plays some role in the epigenetic of homologous recombination. Specifically, it is suggested that dimerization of GADD45α monomers is required. Several point mutants of GADD45α were constructed and analyzed to show defects in self-association. Among them, the GADD45α mutant, CE83AA, lacked the ability to dimerize or oligomerize, which suppressed DNA methylation at homologous recombination sites in vivo. Based on this, we propose a model in which the dimerization (or oligomerization) of GADD45α is involved in strand specific DNA methylation that attends homologous recombination.

La recombinación homóloga es una de las principales vías para la reparación de la ruptura de doble cadena del ADN, la más grave de las lesiones del ADN. Estudios recientes sugieren que la metilación del ADN apunta hacia segmentos de recombinación homóloga; sin embargo, el mecanismo de metilación del ADN durante la recombinación homóloga no es claro. En este estudio, mostramos que GADD45α, una proteína que se encuentra relacionada con el control del ciclo celular, el ceso del crecimiento y la apoptosis, juega un papel en la epigenética de la recombinación homóloga. Específicamente, se ha sugerido que es requerido un dímero de monómeros de GADD45α. Varios puntos mutantes de GADD45α fueron construidos y analizados para mostrar defectos en la libre asociación. Entre ellos, el mutante GADD45α, CE83AA, carecía de la habilidad de dimerización u oligomerización, lo cual suprimió in vivo la metilación del ADN en los sitios de recombinación homóloga. Con base en esto, proponemos un modelo en el cual la dimerización (u oligomerización) de GADD45α está involucrada en la cadena específica de metilación del ADN que lleva a la recombinación homóloga.

The Role of GADD45beta in Acute Myocardial Injury / 대한흉부외과학회지

BACKGROUND: A critical shortage of donor organs has necessitated an investigation of new strategies to increase the availability of additional organs available for human transplantation. We investigated the amount of apoptosis and expression of GADD45beta in two groups, a GADD45beta-transfected group and untransfected group. MATERIAL AND METHOD: The experimental groups consist of a control group (normal H9C2 cell line) and GADD45beta-transfected group. After injury of the each group, we evaluated the expression of GADD45beta and the level of apoptosis in each group. RESULT: There was a significant increase in the expression of GADD45beta in the GADD45beta-transfected group at 1 hour, 2 hours, and 3 hours after stimuli as compared with the control group. The amount of cardiac myoblast cell line apoptosis was significantly lower in the GADD45beta-transfected group as compared with the control group. The concentration of annexin in the GADD45beta-transfected group was significantly lower than that of the control group after cell injury. CONCLUSION: Transfection of a rat myoblast cell line with the GADD45beta gene results in decreased susceptibility to cell injury of human serum.

Expression and significance of Gadd45beta protein in human hepatocellular carcinoma / 临床检验杂志

Objective To investigate the expression of Gadd45beta protein in human hepatocellular carcinoma(HCC) and its clinicopathologic significance.Methods Immunohistochemical method was used to detect the expressions of Gadd45beta in both hepatocellular carcinoma tissues and adjacent non-neoplastic liver tissues form 50 cases.Results The positive rates of Gadd45beta expression in the hepatocellular carcinoma tissues and adjacent non-neoplastic liver tissues were 26.0%(13/50) and 82.0%(41/50) respectively.There were significant difference for the positive rate between the two groups(P0.05).Conclusion Low expression of Gadd45beta protein in hepatocellular carcinoma tissues was exhibited,which may be related to hepatocarcinogenesis as well as biological behaviors and prognosis of HCC.