Arthur G. Balliet

GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress.

Gadd45a (Gadd45), Gadd45b (MyD118), and Gadd45g (CR6) constitute a family of evolutionarily conserved, small, acidic, nuclear proteins, which have been implicated in terminal differentiation, growth suppression, and apoptosis. How Gadd45 proteins function in negative growth control is not fully understood. Recent evidence has implicated Gadd45a in inhibition of cdc2/cyclinB1 kinase and in G2/M cell cycle arrest. Yet, whether Gadd45b and/or Gadd45g function as inhibitors of cdc2/cyclinB1 kinase and/or play a role in G2/M cell cycle arrest has not been fully established. In this work, we show that Gadd45b and Gadd45g specifically interact with the Cdk1/CyclinB1 complex, but not with other Cdk/Cyclin complexes, in vitro and in vivo. Data also has been obtained that Gadd45b and Gadd45g, as well as GADD45a, interact with both Cdk1 and cyclinB1, resulting in inhibition of the kinase activity of the Cdk1/cyclinB1 complex. Inhibition of Cdk1/cyclinB1 kinase activity by Gadd45b and Gadd45a was found to involve disruption of the complex, whereas Gadd45g did not disrupt the complex. Moreover, using RKO lung carcinoma cell lines, which express antisense Gadd45 RNA, data has been obtained, which indicates that all three Gadd45 proteins are likely to cooperate in activation of S and G2/M checkpoints following exposure of cells to UV irradiation.

Hematopoietic cells from Gadd45a- and Gadd45b-deficient mice are sensitized to genotoxic-stress-induced apoptosis

Gadd45a, gadd45b and gadd45g (Gadd45/MyD118/CR6) are genes that are rapidly induced by genotoxic stress. However, the exact function of Gadd45 proteins in the response of mammalian cells to genotoxic stress is unclear. Here, advantage was taken of gadd45a- and gadd45b-deficient mice to determine the role gadd45a and gadd45b play in the response of bone marrow (BM) cells to genotoxic stress. BM cells from gadd45a- and gadd45b-deficient mice were observed to be more sensitive to ultraviolet radiation chemotherapy (UVC), VP-16 and daunorubicin (DNR)-induced apoptosis compared to wild-type (wt) cells. The increased apoptosis in gadd45a- and gadd45b-deficient cells was evident also by enhanced activation of caspase-3 and poly-ADP-ribose polymerase cleavage and decreased expression of c-inhibitor of apoptotic protein-1, Bcl-2, Bcl-xL compared to wt cells. Reintroduction of gadd45 into gadd45-deficient BM cells restored the wt apoptotic phenotype. Both gadd45a- and gadd45b-deficient BM cells also displayed defective G2/M arrest following exposure to UVC and VP-16, but not to DNR, indicating the existence of different G2/M checkpoints that are either dependent or independent of gadd45. Taken together, these findings identify gadd45a and gadd45b as antiapoptotic genes that increase the survival of hematopoietic cells following exposure to UV radiation and certain anticancer drugs.

Comparative analysis of the genetic structure and chromosomal location of the murine MyD118 (GADD45β) gene

The MyD118 (Gadd45β) protein is a member of a family of structurally related proteins, including Gadd45 (Gadd45α) and CR6 (Gadd45γ), that have critical roles in regulating growth arrest and apoptosis. The MyD118 and other members of its family display distinct patterns of expression in response to stimuli that induce differentiation, growth arrest, or apoptosis. Species-blot analysis showed that MyD118 is an evolutionarily conserved gene, and comparative sequence analysis showed that MyD118 has a gene structure similar to that of other members of its gene family. Comparison of putative transcription factor-binding sites found in sequences of this gene family provides evidence that p53 is involved in regulating the expression of MyD118 and that NF-κB may play a role in differential expression of MyD118 and Gadd45(Gadd45α). Fluorescence in situ hybridization localized the MyD118 gene to mouse chromosome band 10B5.3, correcting a previous assignment to mouse chromosome 9.

Comparative Analysis of the Genetic Structure and Chromosomal Mapping of the Murine Gadd45g/CR6 Gene

Gadd45g/CR6, Gadd45b/MyD118, and Gadd45a/Gadd45 are members of a gene family that displays distinct patterns of gene expression in response to stimuli that induce differentiation, growth arrest, and/or apoptosis. All three of these highly conserved proteins interact with a number of critical cell cycle and cell survival regulatory proteins such as PCNA, p21(WAF1/CIP1), CDK1 (cdc2-p34), and MTK1/MEKK4, and have been reported to influence the activity of the p38 and JNK kinases. Species-blot analysis showed that Gadd45g is an evolutionarily conserved gene and sequence analysis showed that Gadd45g has a gene structure conserved with that of other members of its gene family. A comparison of the putative transcription factor binding sites found in the sequences of the gene family members suggests, that like Gadd45b, NF-kappaB and STATs may be responsible for the differences in regulation of expression observed between Gadd45g and Gadd45a. Analysis of the Gadd45b/MyD118 promoter shows that there are three different enhanceosome-like regions that may allow cell-type specific responses to TGF-beta1 by the Gadd45b/MyD118 promoter. Fluorescent in situ hybridization (FISH) confirmed the localization of the Gadd45g gene to mouse chromosome band 13A5-B, which has been reported to contain a quantitative trait locus that regulates body weight in mice. This suggests that alleles of the Gadd45g gene may function in the regulation of body weight, in addition to its currently recognized roles in differentiation and stress responses.