Does the p53 up-regulated Gadd45 protein have a role in excision repair?
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Cell Line↗
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Does the p53 up-regulated Gadd45 protein have a role in excision repair?
Explore the source record for details and available documents.
Cell Line↗
Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress.
Gadd45 proteins are induced by hyperosmolality in renal inner medullary (IM) cells, but their role for cell adaptation to osmotic stress is not known. We show that a cell line derived from murine renal IM cells responds to moderate hyperosmotic stress (540 mosmol/kg) by activation of G(2)/M arrest without significant apoptosis. If the severity of hyperosmotic stress exceeds the tolerance limit of this cell line (620 mosmol/kg) apoptosis is strongly induced. Using transient overexpression of ectopic Gadd45 proteins and simultaneous analysis of transfected versus non-transfected cells by laser-scanning cytometry, we were able to measure the effects of Gadd45 super-induction during hyperosmolality on G(2)/M arrest and apoptosis. Our results demonstrate that induction of all three Gadd45 isoforms inhibits mitosis and promotes G(2)/M arrest during moderate hyperosmotic stress but not in isosmotic controls. Furthermore, all three Gadd45 proteins are also involved in control of apoptosis during severe hyperosmotic stress. Under these conditions Gadd45gamma induction strongly potentiates apoptosis. In contrast, Gadd45alpha/beta induction transiently increases caspase 3/7 and annexin V binding before 12 h but inhibits later stages of apoptosis during severe hyperosmolality. These results show that Gadd45 isoforms function in common but also in distinct pathways during hyperosmolality and that their increased abundance contributes to the low mitotic index and protection of genomic integrity in cells of the mammalian renal inner medulla.
Animals↗
Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45.
Recently Gadd45, a p53-regulated stress protein, has been implicated in the activation of a G2/M checkpoint after damage by UV radiation and alkylating agents. While inhibitory phosphorylation of Cdc2 and suppression of cyclin B1 levels are known to be involved in G2 delays after genotoxic stress, Gadd45 has now been found to directly inhibit the activity of Cdc2/Cyclin B1 complex, while it had no appreciable effect on Cdk2/ Cyclin E activity even at very high levels of Gadd45. In contrast, p21CiP1/Waf1 is an universal cdk/cyclin inhibitor and inhibited both of the cyclin complexes tested here. Gadd45 was also able to physically interact with Cdc2, but not Cyclin B1. Addition of Gadd45 to immunoprecipitated Cdc2/Cyclin B1 in vitro led to a dissociation of this complex, and thus may represent a new checkpoint mechanism whereby Cdc2/Cyclin B1 can be inhibited. With the use of an antisense approach, reduced Gadd45 expression attenuated the suppression of Cdc2/Cyclin B1 activity in UV-irradiated human cells. Taken together, these results implicate Gadd45 in the control of G2/M cell cycle progression after certain stresses.
Antibodies, Monoclonal↗
DNA repair pathway stimulated by the forkhead transcription factor FOXO3a through the Gadd45 protein.
The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. We show that mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appeared to be a direct target of FOXO3a that mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span.
Animals↗
DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat.
Cerebral ischemia in adult rodents leads to the production of several types of lesions in the genomic DNA, followed by the activation of the damage-response indicator Gadd45. Our purpose was to investigate the structural changes that occur in chromatin DNA and repair processes after ischemic injury in neonatal brain. Neonatal ischemia was induced by the permanent left MCA occlusion in association with 1 h occlusion of the left common carotid artery in 7-day-old Wistar pups. Oligonucleosome fragments that are recognized as the characteristic DNA ladder was observed in a delayed fashion. Double-strand breaks result in high molecular weight fragments of 50- and 300-kbp as demonstrated by pulsed-field gel electrophoresis, and visualized by the TUNEL assay at 24 h of recovery. In contrast, DNA single-strand breaks, shown by the use of DNA polymerase I-mediated biotin-dATP nick translation were not so abundant. Gadd45 immunoreactivity was sequentially increased in vulnerable neurons in the infarct (4 to 24 h) and in sublethally injured neurons in the penumbra (24-48 h). Taken together, these findings suggest that Gadd45 responds to DNA damage following neonatal ischemia. Furthermore, repairing processes seem to be more active in the penumbra and therefore Gadd45 could have also a protective role in cerebral ischemia.
Animals↗
Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen.
GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.
Cell Division↗
Characterization of human Gadd45, a p53-regulated protein.
GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.
Animals↗
A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK.
The stress-responsive p38 and JNK MAPK pathways regulate cell cycle and apoptosis. A human MAPKKK, MTK1 (= MEKK4), mediates activation of both p38 and JNK in response to environmental stresses. Using a yeast two-hybrid method, three related proteins, GADD45alpha (= GADD45), GADD45, (= MyD118), and GADD45gamma, were identified that bound to an N-terminal domain of MTK1. These proteins activated MTK1 kinase activity, both in vivo and in vitro. The GADD45-like genes are induced by environmental stresses, including MMS, UV, and gamma irradiation. Expression of the GADD45-like genes induces p38/JNK activation and apoptosis, which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein. We propose that the GADD45-like proteins mediate activation of the p38/JNK pathway, via MTK1/ MEKK4, in response to environmental stresses.
Amino Acid Sequence↗
[Suppression of HCT116 growth of cells by Gadd45].
OBJECTIVE: To study the mechanism of suppression of growth of HCT116 human colon carcinoma cells by Gadd45 gene. METHODS: HCT116 human colon carcinoma cells were transfected with pTRE-Gadd45 vector so as to establish Gadd45-inducible cell line that was cultured in medium with tetracycline. Then tetracycline was withdrawn. The number of cell clones was counted. Flow cytometry was used to detect the percentages of cells at the G1, S, and G2-M phases. TUNEL technique was used to detect the apoptosis of cells. Western blotting was used to analyze the expression of Gadd45 protein, and apoptosis-related proteins: poly-ADP-ribose polymerase (PARP), and caspase 3 protein. RESULTS: Gadd45 protein was not expressed in the HCT116 cells cultured in the medium with tetracycline, however, it was expressed with a gradually increased level in the cells cultured in the medium from which tetracycline was withdrawn, The clone formation rate of HCT116 cells was 100% in the medium with tetracycline, however, was only 14.2% in the medium with tetracycline withdrawal, with a suppression rate of more than 85%. The percentage of cells in G(2)-M phase was significantly increased in the cells cultured in the medium with tetracycline withdrawal. 24-36 hours after the withdrawal of tetracycline, PARP and caspase 3 protein were activated remarkably. CONCLUSION: High expression of Gadd45 inhibits the growth of HCT116 cells, through inducing G2-M arrest and activating apoptotic pathway.
Blotting, Western↗
Gadd45 family proteins are coactivators of nuclear hormone receptors.
Gadd45 family genes encode nuclear acidic proteins composed of Gadd45, MyD118, and CR6. Sequence analysis showed that Gadd45 family proteins (Gadd45, MyD118, and CR6) contain LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. Interaction between Gadd45 or CR6 and RXR alpha was confirmed by a two-hybrid test in yeast. Results from a series of GST pulldown assays showed that these Gadd45 family proteins interact with several nuclear hormone receptors including RXR alpha, RAR alpha, ER alpha, PPAR alpha, PPAR beta, and PPAR gamma2 in vitro. Interaction between Gadd45 family proteins and nuclear hormone receptors resulted in modest activation of transactivating function of nuclear hormone receptors in reporter systems. When fused to DNA binding domain of GAL4, Gadd45 and CR6 activated the UAS-mediated transcription in mammalian cells. These results suggest that Gadd45 family proteins bind to nuclear hormone receptors and act as nuclear coactivators.
3T3 Cells↗
CR6-interacting factor 1 interacts with Gadd45 family proteins and modulates the cell cycle.
The Gadd45 family of proteins includes Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. These proteins play important roles in maintaining genomic stability and in regulating the cell cycle. This study reports the cloning of a novel protein called CR6-interacting factor 1 (CRIF1) which interacts with Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. CRIF1 binds specifically to the Gadd45 family proteins, as determined by an in vitro glutathione S-transferase pull-down assay and an in vivo mammalian cell two-hybrid assay along with coimmunoprecipitation assays. CRIF1 mRNA is highly expressed in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues. CRIF1 localizes exclusively to the nucleus and colocalizes with Gadd45gamma. Recombinant CRIF1 inhibits the histone H1 kinase activity of immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E, and the inhibitory effects were additive with Gadd45 proteins. Overexpression of CRIF1 increases the percentage of cells in G1, decreases the percentage of cells in S phase, and suppresses growth in NIH3T3 cells. The down-regulation of endogenous CRIF1 by the transfection of the small interfering RNA duplexes resulted in the inactivation of Rb by phosphorylation and decreased the G1 phase cell populations. Expression of CRIF1 is barely detectable in adrenal adenoma and papillary thyroid cancer and much lower than in adjacent normal tissue. The results presented here suggest that CRIF1 is a novel nuclear protein that interacts with Gadd45 and may play a role in negative regulation of cell cycle progression and cell growth.
3T3 Cells↗
Stress-induced JNK activation is independent of Gadd45 induction.
DNA damage and environmental stress activate signaling and induce genes involved in cell cycle and cell death. Expression of the Gadd45 protein is induced following DNA damage and other stress. Gadd45 is believed to play a role in growth arrest and possibly in cell death. The JNK signaling pathway is also activated by some DNA-damaging agents. This activation leads to phosphorylation and activation of transcription factors, such as c-Jun/AP-1 and ATF2, which mediate immediate early gene induction. Recently Gadd45 was suggested to be involved in JNK activation. However, as this suggestion relied on in vitro experiments and ectopic overexpression of Gadd45 protein, we examined whether physiological levels of Gadd45 that are induced following exposure to DNA damaging agents and stress can lead to JNK induction. We found that JNK activation by UV irradiation and anisomycin treatment precedes the induction of gadd45 mRNA by these agents. Gadd45 protein induction by methyl methanesulfonate also lagged behind JNK activation. The use of protein synthesis inhibitors suggested that newly synthesized proteins, including the stress-induced Gadd45, make only a marginal contribution to JNK activation. We also found that stresses such as gamma irradiation induce Gadd45 and do not activate JNK in mouse fibroblasts. Therefore, stress-induced JNK does not depend on Gadd45 induction.
3T3 Cells↗
Kainate induces the expression of the DNA damage-inducible gene, GADD45, in the rat brain.
The expression of the novel growth arrest and DNA damage-inducible gene GADD45 was examined in kainate-induced epileptic brain damage in the rat using in situ hybridization, northern blot analysis, western blot analysis and immunocytochemistry. Systemic administration of kainate resulted in DNA damage and neuronal degeneration in vulnerable neurons of limbic regions, including the amygdala and hippocampal pyramidal layers, as shown by in situ DNA nick end-labelling and histological staining. GADD45 messenger RNA was transiently increased in non-vulnerable neurons (2-8 h after kainate injection) but was persistently elevated in vulnerable neurons (up to 24 h after injection) after kainate injection. GADD45 protein was elevated in both vulnerable and non-vulnerable neurons at 4 h, but levels decreased in vulnerable neurons thereafter, suggesting that translational blockage of GADD45 protein occurred in these cells. GADD45 protein was overexpressed in non-vulnerable neurons up to 72 h after kainate injection. Because GADD45 may participate in the DNA excision repair process and because it has been shown to be overexpressed in neurons that survive focal cerebral ischaemia, these results support the hypothesis that GADD45 may have a protective role in the injured brain.
Animals↗
Characterisation of the interaction between PCNA and Gadd45.
We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45. We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA. We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites. Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding. This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45. Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170. The crystal structure of PCNA provides insight into our genetic and immunochemical data. Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.
Amino Acid Sequence↗
GADD45 is induced in Alzheimer's disease, and protects against apoptosis in vitro.
Expression of the growth arrest DNA damage-inducible protein, GADD45, has recently been reported to be induced by a wide range of stimuli, especially those that produce a high level of base pair damage. We have investigated the expression of GADD45 in brain tissue obtained from patients suffering from Alzheimer's disease (AD). Our results demonstrate that many neurons express the GADD45 protein, and that expression of this protein in neurons is associated with expression of the anti-apoptotic protein Bcl-2, and the presence of DNA damage, but not closely associated with tangle-bearing neurons. Additionally, cell lines overexpressing this protein confer resistance to apoptosis induced by DNA damage agent, suggesting that this protein may participate in cell survival mechanisms.
Alzheimer Disease↗
Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2.
Murine kidney cells of the inner medullary collecting duct (mIMCD) were exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium (isosmotic medium + 150 mM NaCl) after seeding. We determined cell numbers, total nucleic acid, DNA, and RNA contents in both groups every day for a total period of 7 days. Based on all 4 parameters it was evident that growth of mIMCD3 cells is arrested for approximately 18 h following onset of hyperosmolality. However, none of the parameters measured indicated cell death because of hyperosmolality. Growth curves of hyperosmotic samples were shifted compared with isosmotic samples showing a gap of 18 h but had the same shape otherwise. We demonstrated that at 24 and 48 h after onset of hyperosmolality, but not in isosmotic controls, growth arrest and DNA damage-inducible (GADD) proteins GADD45 and GADD153 are strongly induced. This result is consistent with growth arrest observed in hyperosmotic medium. We tested if mitogen- and stress-activated protein kinase (SAPK) cascades are involved in osmosignaling that leads to GADD45 and GADD153 induction. Using phosphospecific antibodies we showed that extracellular signal-regulated kinases 1 and 2 (ERK), SAPK1 (JNK), and SAPK2 (p38) are hyperosmotically activated in mIMCD cells. Hyperosmotic GADD45 induction was significantly decreased by 37.5% following inhibition of the SAPK2 pathway, whereas it was significantly increased (65.2%) after inhibition of the ERK pathway. We observed similar, although less pronounced effects of SAPK2 and ERK inhibition on hyperosmotic GADD153 induction. In conclusion, we demonstrate that mIMCD cells arrest growth following hyperosmotic shock, that this causes strong induction of GADD45 and GADD153, that GADD induction is partially dependent on osmosignaling via SAPK2 and ERK, and that SAPK2 and ERK pathways have opposite effects on GADD expression.
Animals↗
[Gadd45 mediated G2/M cell cycle arrest induced by BRCA1].
BACKGROUND & OBJECTIVE: It is considered that tumor suppressor gene BRCA1 is an important factor in the regulation of cell cycle checkpoint, but the molecular mechanism by which BRCA1 regulates cell cycle G(2)/M arrest is less known. The objective of this study was to investigate the effects of Gadd45 on the BRCA1-induced cell growth suppression. METHODS: BRCA1 induction of Gadd45 protein was analyzed using Western-blot assay following cells transfection with BRCA1 expression vector and cell sorting. Activation of the Gadd45 promoter by BRCA1 was determined by CAT assay. Effect of antisense Gadd45 on the BRCA1-induced cell cycle G(2)-M arrest was examined by flow cytometry analysis. And the effects of antisense Gadd45 on BRCA1-mediated growth suppression in HeLa and HCT116 cell lines was determined by colony formation assay. RESULTS: Gadd45 protein was highly induced after expression of BRCA1. BRCA1 strongly activated the Gadd45 promoter. Antisense Gadd45 substantially abrogated BRCA1-activated cell cycle G(2)-M arrest and BRCA1-induced cell growth suppression on HeLa and HCT116 cell lines. CONCLUSION: Gadd45 is a BRCA1-regulated downstream gene and mediates the role of BRCA1 in the control of cell cycle G(2)/M arrest and growth suppression.
BRCA1 Protein↗