Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

Abstract

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gaddl53, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gaddl53 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays ofgadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in ataxia telangiectasia.There is increasing evidence that ionizing radiation can induce specific genes in mammalian and other eucaryotic cells. Such genes may have functions like those in procaryotes, in which genes encoding protective functions, such as DNA repair processes, can be induced (39). Prior treatment with a low dose of X rays induces a transient (usually small) protection, manifested as an increased survival or as a reduction in chromosomal damage, against a second, higher dose in human lymphocytes (25), Chinese hamster cells (12), and insect cells (15). These protective effects are blocked by inhibitors of protein (12) and RNA synthesis (15), indicating that such processes may be mediated by the induction of particular genes. The finding that certain proteins increase in abundance after X irradiation of human cells also provides evidence for X-ray-inducible genes (2). Recently, tumor necrosis factor a (TNF) mRNA has been reported to increase in human sarcoma cells after ionizing radiation (9). TNF has a role in a variety of cellular processes, including the acute-phase response and inflammation (1), and thus its induction probably represents a general response to cell injury rather than a specific response to X rays or other DNA-damaging agents. Transcription from the long terminal repeat o...

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation

“…Our observation suggests that TR4 is another essential mediator for inducing GADD45A upon genotoxic stress besides FOXO3a. Previous reports describe faster and transient induction of GADD45A by IR (starting from 30 min and reaching a plateau within 4 h) while IR induction of Gadd45a mRNA level in MEF cells started at 10 h and lasted until 24 h [23, 29]. The difference in time-point of induction observed could be due to lower sensitivity of Q-PCR assay than RNase protection assay applied in previous reports.…”

“…This might be because the amplification efficiency of Q-PCR primers for Gadd45a is not optimal. Another possibility is that there could be variations between species and tissues as IR did not induce GADD45A in CHO as significantly as in human cells [23]. The induction of GADD45A in human fibroblasts is observed in early (1–12 h), intermediate (24–48 h), and late (72 h) phases [30].…”

“…By using DNA Damage Signaling Pathway focused PCR Array (SABiosciences ™ ), we found that expression of several genes controlling cell cycle arrest, including Gadd45a (growth arrest and DNA-damage-inducible, alpha), was reduced in TR4 knockdown cells (data not shown). Gadd45a is reported to be induced by multiple types of genotoxic stress including ultraviolet radiation and ionizing radiation (IR) [22, 23], and is downstream of the FOXO3a stimulated DNA repair pathway when cells are exposed to genotoxic stress [24]. We therefore suggest that TR4 is the mediator through which FOXO3a regulates GADD45A to promote DNA repair and protect cells from IR-induced cell death.…”

Cloning of rat GADD45 gene and induction analysis following ionizing radiation in vivo

“…In mammals, only a few number of genes have been identified that are involved in G1 or G2 checkpoints. One such candidate gene, GADD45 (growth arrest and DNA damage-inducible gene), was originally isolated from CHO cells after UV irradiation [5] and the expression of GADD45 was shown to be regulated by growth arrest [6] and is induced by ionizing radiation in cultured cells [7]. GADD45 protein consists of 165 amino acids, with sequence similarity to the murine MyDll8 cDNA [8].…”

Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation

“…However, the molecular mechanism of wild-type p53 action is not fully understood. The p53 protein has been shown to activate several genes by interacting with specific DNA sequences, such as the promoter of muscle specific creatine kinase (Weintraub et al, 1991) and the third intron of the GADD-45 gene, which is induced after gamma irradiation (Papathanasiou et al, 1991). A consensus p53 DNA-binding site has been derived from these studies (Kern et al, 1991;El-Deiry et al, 1992;Funke et al, 1992).…”