Gadd45 Protein Research Articles

Transient global ischemia triggers expression of the DNA damage-inducible gene GADD45 in the rat brain.

Using in situ hybridization, Northern blot analysis, Western blot analysis, and immunocytochemistry, mRNA and protein expression of the novel DNA damage-inducible gene GADD45 was examined in the rat brain at 0.5, 2, 4, 8, 16, 24, 48, and 72 hours after 15 minutes of transient global ischemia. Transient ischemia produced by the four-vessel occlusion method resulted in DNA double-strand breaks and delayed neuronal cell death in vulnerable neurons of the hippocampal CA1 sector, the hilus, dorsal caudate-putamen, and thalamus, as shown by in situ DNA nick end-labeling and histologic staining. GADD45 mRNA was transiently increased in less-vulnerable regions such as the parietal cortex (up to 8 hours after ischemia) and dentate granule cells (up to 24 hours after ischemia) but was persistently increased in vulnerable neurons such as CA1 pyramidal neurons (up to 48 hours). GADD45 immunoreactivity was increased in both vulnerable and less-vulnerable regions at earlier reperfusion periods (4 to 16 hours), but thereafter immunoreactivity was decreased below control levels in most vulnerable regions before delayed cell death and DNA double-strand breaks. At 72 hours after transient ischemia, a moderate increase in GADD45 immunoreactivity was still detectable in some CA3 neurons and in a few surviving neurons in the CA1 region. Double staining performed at 16 to 72 hours after ischemia revealed that GADD45 immunoreactivity was persistently increased in neurons that did not develop DNA damage. Because GADD45 protein may participate in the DNA excision repair process and because it has been shown that this protein is also overexpressed in neurons that survive focal ischemia and kainate-induced epileptic seizures, the results reported here support the hypothesis that GADD45 could have a protective role in neuronal injury.

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.

Focal ischemia induces expression of the DNA damage-inducible gene GADD45 in the rat brain.

GADD45 is a DNA damage-inducible gene that accelerates DNA excision repair and can be induced by a variety of DNA-damaging stimuli in mammalian cells. We investigated the expression of GADD45 mRNA and protein using in situ hybridization and immunocytochemistry in rat brains after 2 h of temporal focal ischemia. The expression of GADD45 mRNA was induced in neurons throughout ischemic cortex 4 h after the onset of ischemia but was restricted to ischemic penumbra regions at 24 h after ischemia. The expression of GADD45 protein was increased only in sublethally injured neurons in the penumbra regions at both 4 h and 24 h following ischemia. These results suggest that GADD45 could have a protective role in ischemic neurons.

Characterization of the GADD45 response to ionizing radiation in WI-L2-NS cells, a p53 mutant cell line

We have previously reported that WI-L2-NS, a human lymphoblastoid cell line, has very high basal levels of GADD45 mRNA and protein in spite of a p53 mutation at amino acid 237. Regardless of the amount of Gadd45 in this cell line, no growth suppression activity was detected. We report here that in WI-L2-NS, the mutated p53 protein adopts predominantly a wild type (wt) conformation and binds to the p53 binding site in the GADD45 third intron. In this cell line, the already high levels of mutated p53 protein can be induced further by ionizing radiation OR but the response of the p53 downstream effector genes is altered. Induction of GADD45 and CIP1/ WAF1 is reduced compared to p53 wt cell lines but is still substantially higher than the average fold induction obtained from 39 p53 mutant cell lines. Induction of the MDM2 gene was not detected in WI-L2-NS following IR. The induction pattern of the three p53 effector genes by the alkylating agent methylmethane sulfonate (MMS) was also attenuated in WI-L2-NS cells. In TK6 cells, a WI-L2-NS sister cell line having a p53 wt genotype, the induction of the p53 downstream effectors is normal, i.e. induced, both at the protein and the mRNA levels. These results indicate that the DNA binding activity of the mutated p53 protein in WI-L2-NS might be responsible, at least in part, for the high basal levels of GADD45 but can not mediate the full induction of the p53 downstream effector genes. The reason(s) for the inability of Gadd45 to suppress growth in this cell line remains however unknown.

Response: Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

Response: Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

Does the p53 Up-Regulated Gadd45 Protein Have a Role in Excision Repair?

The production and characterization of murine monoclonal antibodies to human Gadd45 raised against a recombinant protein.

The production of two different murine monoclonal antibodies to human Gadd45, a protein that is induced in response to DNA damage, is reported. Antibodies were generated in a SJL mouse using a recombinant form of the human Gadd45 protein. Monoclonal antibody 4TCYA1, which recognizes the denatured form of human Gadd45 in Western blots, was selected based upon the recognition of Gadd45 induced by functional p53 in the human myeloid leukemia cell line, ML-1. A second monoclonal antibody, designated 30T.14, immunoprecipitates native human Gadd45 in lysates produced from RKO cells, a colorectal carcinoma cell line that expresses relatively high basal levels of Gadd45, as well as from cell lysates made from ML-1 cells after exposure to ionizing irradiation (IR). Since 4TCYA1 fails to immunoprecipitate Gadd45, and 30T.14 fails to bind to IR-induced Gadd45 in immunoblotting, these two monoclonal antibodies probably recognize different epitopes.

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.