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 gadd153, 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 gadd153 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 of gadd45 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 taxia telangiectasia.

In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm

“…It also revealed potentially new markers such as RREB1, CUX1, IRF9, DDIT3, TFAP2C, PBX3, JUN, and JUND ( Figure 2 E). Notably, RREB1 ( Lee et al., 2012 ) and CUX1 ( Ripka et al., 2010 ) are important for the development of the primitive gut tube and pancreas, while DDIT3 is a known apoptosis inducer ( Papathanasiou et al., 1991 ), and TFAP2C might be involved in mesoderm specification ( Madrigal et al., 2020 ). The identification of DDIT3, IRF, JUN, and TFAP2 was consistent with the previous bulk RNA-seq results ( Figure 1 B).…”

The gadd and MyD Genes Define a Novel Set of Mammalian Genes Encoding Acidic Proteins That Synergistically Suppress Cell Growth

“…As shown by EMSA, p53 activity is DNA damage inducible in the 3T3 line used in our studies. Considering that IR weakly induced GADD153 and that induction of GADD45 and GADD153 was reduced in AT cells after IR or MMS treatment (36), p53 may very well have role in the regulation of multiple gadd genes after stresses that strongly induce p53. This finding and other data to be published elsewhere indicate that p53, while not required for the gadd gene response to agents like MMS and medium depletion, may contribute to this response.…”

“…The partial-length clone pDDIA34 was originally isolated from CHO cDNA library constructed after hybridization subtraction to enrich for UV radiation-inducible transcripts (15). Nearly full-length hamster clones pXR45m (gadd45) and pXR34d (gadd34) were isolated from a custom library constructed by Stratagene (La Jolla, Calif.), which has been described previously (36). For gaddl53 studies, a nearly full-length hamster clone, pASA4 ( 16), or mouse clone, CHOP-10 ( 41), was used.…”

“…RNA isolation and blot analysis. Cells were lysed in situ with 4 M guanidine thiocyanate; poly(A) RNA was prepared and then analyzed by Northern (RNA) blot analysis or by quantitative dot blot hybridization as described previously (20,36) for 3T3, HEC, and CHO cells. The relative poly(A)+ content of each RNA sample in the dot blot analyses was estimated by using a labeled polythymidylate probe (20).…”

“…Interestingly, MMS or medium depletion induced p53 activity much more strongly than IR. Second, induction of GADD45 and GADD153 by MMS and the small response of GADD153 to IR were reduced in AT cells compared with normal cells (36). These results suggest a possible role for p53 and the gadd genes in the responses to a variety of stresses eliciting growth arrest.…”