A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, whic, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggest a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.

The tumor suppressor p53 can function as a sequence-specific transcription factor and is required for activation by ionizing radiation (IR) of one or more downstream effector genes, such as the human GADD45 gene. One important consequence of IR that is probably mediated by these downstream effector genes is activation of the p53-mediated G1 cell cycle checkpoint. While the induction of reporter constructs containing p53-binding sites has already been demonstrated with p53 expression vectors, we have now demonstrated the direct activation of such a construct after treatment of the human RKO line, which has a normal p53 phenotype, with various types of DNA-damaging agents and also after growth arrest produced by medium depletion (starvation). IR, UV radiation, and methylmethane sulfonate were found to induce p53 activity when a stably integrated reporter construct containing functional p53-binding sites was used and also in mobility shift assays with a p53-binding site from the GADD45 gene, and IR-inducible gene previously associated with growth arrest. The same cell treatments that induced this p53 activity also caused an increase in cellular p53 protein levels. The response in cells lacking normal p53 or in RKO cells expressing a dominant negative mutant p53 was markedly reduced. Interestingly, the spectrum of effective inducing agents for the above-described experiments was similar to that which induces GADD45 either in cells with a normal p53 status or, with the exception of IR, in cells lacking normal p53. These results indicate a role for p53 in the IR pathway, which is completely p53 dependent, and in other genotoxic stress responses, in which p53 has a cooperative effect but is not required.

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.