Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

A polymorphism but no mutations in the GADD45 gene in breast cancers

The p53 gene product is part of a pathway regulating growth arrest at the G₁ checkpoint of the cell cycle. Mutation of other components of this pathway, including the products of the ataxia telangiectasia (AT), GADD45, mdm2, and p21^WAF1/CIP1 genes may have effects comparable to mutations in the p53 gene. The GADD45 gene is induced by ionizing radiation and several DNA-damaging xenobiotics. Induction requires the binding of wild-type p53 to an evoulutionarily highly conserved putative intronic p53 binding site in intron 3 of GADD45. We recently analyzed the entire coding region of the p53 gene in primary breast cancers of Midwestern white women and found 21 mutations among 53 tumors (39,6%). We now have shown by direct sequencing that there are no mutations in the intronic p53 binding site of the GADD45 gene in any of the 53 primary breast cancers and no mutations in the entire coding region of the GADD45 gene in a subset of 26 consecutive tumors (12 with p53 mutation and 14 without p53 mutation). The only sequence variation detected was a common polymorphism in intron 3. The absence of mutations in the GADD45 gene, including the putative p53-binding intronic site, suggests that this gene is not a frequent target of mutations in breast cancer. Although mutations of the p53 gene have been studied in a wide spectrum of human cancers, GADD45 has not been examined in any tumor or cell line to the best of our knowledge. Our results raise the possibility that mutation of the GADD45 gene alone is not functionally equivalent to loss of wild-type p53 activity. Received: 14 September 1995     

Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction.

We have cloned the immediate-early serum-reponsive JE gene from the rat in order to study the regulation of this gene. We show that sequences of the JE promoter region confer serum-inducibility to a reporter gene. Analysis of the promoter in transient assays reveals that: i) the -141/-88 region is required for the response to the phorbol ester TPA, ii) the -70/-38 region is essential for basal activity. This latter region harbors the sequence TGACTCC, which resembles the consensus site for AP-1 binding, TGACTCA. DNA-protein binding assays indicate that the JE AP-1 site and the consensus AP-1 site have an overlapping but not identical binding spectrum for AP-1 proteins. Our data suggest that the inability of some AP-1 sites to respond to TPA is caused by subtle differences in affinity for AP-1 proteins.