Barbara Hoffman

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.

Gadd45 in stress signaling

Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome.

Conserved DNA methylation in Gadd45a-/- mice

Gadd45a (growth arrest and DNA-damage-inducible protein 45 alpha) plays a pivotal role in cellular stress responses and is implicated in DNA repair, cell cycle arrest and apoptosis. Recently, it was proposed that GADD45A is a key regulator of active DNA demethylation by way of its role in DNA repair. Barreto et al. reported that Gadd45a overexpression activated transcription from methylation-silenced reporter plasmids and promoted global DNA demethylation. siRNA-mediated knockdown of Gadd45a levels resulted in increased levels of DNA methylation at specific endogenous loci. Based on these exciting results, Gadd45a-/- mice might be predicted to have a hypermethylation phenotype. We report here that neither global nor locus-specific methylation is increased in Gadd45a-/- mice.

Myeloid differentiation (MyD) primary response genes in hematopoiesis

Myeloid Differentiation (MyD) primary response and Growth Arrest DNA-Damage (Gadd) genes comprise a set of overlapping genes, including known (IRF-1, EGR-1, Jun) and novel (MyD88, Gadd45alpha MyD118/Gadd45beta, GADD45gamma, MyD116/Gadd34) genes, that have been cloned by virtue of there being co-ordinately induced upon the onset of terminal myeloid differentiation. This review delineates the role MyD genes play in blood cell development, where they function as positive regulators of terminal differentiation, lineage specific blood cell development and control of blood cell homeostasis, including growth inhibition and apoptosis.

Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and terminal differentiation

Members of the cdc25 family are protein phosphatases that play pivotal roles in cell cycle progression. Cdc25A has been shown to be a critical regulator of the G1/S transition of mammalian cells and to be a myc-target gene with oncongenic properties. We investigated the regulation of cdc25A during terminal differentiation using myeloblastic leukemia M1 cells, that can be induced to undergo differentiation into macrophages by interleukin-6 (IL-6) treatment. In this report it is shown that cdc25A protein is degraded by the ubiquitin-proteasome machinery in both terminally differentiating and cycling cells. Cdc25A was found to have two major peaks of accumulation during cell cycle progression, one in G1 and the other in S/G2. Evidence was obtained that degradation of cdc25A by the ubiquitin-proteasome machinery in terminally differentiating myeloid cells is accelerated compared to cycling cells. Moreover, deregulated expression of c-myc in M1 cells, which had been previously shown to block terminal differentiation, was also found to block IL-6 induced degradation of cdc25A.

Gadd45 modulation of intrinsic and extrinsic stress responses in myeloid cells

Gadd45 proteins modulate signaling in response to physiological and environmental stressors. Expression of gadd45 genes is rapidly induced by different stressors, including differentiation‐inducing cytokines and genotoxic stress. Induction of gadd45 genes at the onset of myeloid differentiation suggested that Gadd45 protein(s) play a role in hematopoiesis, yet no apparent abnormalities were observed in either the bone marrow (BM) or peripheral blood compartments of mice deficient for either gadd45a or gadd45b. However, under conditions of hematological stress, including acute stimulation with cytokines, myelo‐ablation and inflammation, both gadd45a‐deficient and gadd45b‐deficient mice exhibited deficiencies. This is discussed within the context of what is known about Gadd45 proteins in stress signaling, hematopoietic development and the innate immune response. Furthermore, myeloid enriched BM cells from gadd45a and gadd45b deficient mice were observed to be more sensitive to ultraviolet radiation (UVC), VP‐16 and daunorubicin (DNR) induced apoptosis compared to wild‐type (WT) cells, displaying defective G2/M arrest following exposure to UVC and VP‐16, but not to DNR. Novel mechanisms that mediate the pro‐survival functions of Gadd45 in hematopoietic cells following UV irradiation were demonstrated, involving activation of the Gadd45a‐p38‐NF‐κB survival pathway and Gadd45b mediated inhibition of the stress response MKK4‐JNK apoptotic pathway. The ramifications regarding the pathogenesis of different leukemias and the response of normal and malignant hematopoietic cells to chemo‐ and radiation‐therapy, as well as other challenges to the hematopoietic compartment, are discussed. J. Cell. Physiol. 218: 26–31, 2009.

Egr-1 abrogates the E2F-1 block in terminal myeloid differentiation and suppresses leukemia

Deregulated growth and blocks in differentiation collaborate in the multistage process of leukemogenesis. Previously, we have shown that ectopic expression of the zinc finger transcription factor Egr-1 in M1 myeloblastic leukemia cells promotes terminal differentiation with interleukin-6 (IL-6). In addition, we have shown that deregulated expression of the oncogene E2F-1 blocks the myeloid terminal differentiation program, resulting in proliferation of immature cells in the presence of IL-6. Here it is shown that the positive regulator of differentiation Egr-1 abrogates the E2F-1-driven block in myeloid terminal differentiation. The M1E2F-1/Egr-1 cells underwent G0/G1 arrest and functional macrophage maturation following treatment with IL-6. Furthermore, Egr-1 diminished the aggressiveness of M1E2F-1 leukemias and abrogated the leukemic potential of IL-6-treated M1E2F-1 cells. Previously, we reported that Egr-1 abrogated the block in terminal myeloid differentiation imparted by deregulated c-myc, which blocks differentiation at a later stage than E2F-1, resulting in cells that have the characteristics of functionally mature macrophages that did not undergo G0/G1 arrest. Taken together, this work extends and highlights the tumor suppressor role of Egr-1, with Egr-1 behaving as a tumor suppressor against two oncogenes, each blocking myeloid differentiation by a different mechanism. These findings suggest that Egr-1 and/or Egr-1 target genes may be useful tools to treat or suppress oncogene-driven hematological malignancies.

Myeloid differentiation (MyD) primary response genes in hematopoiesis.

Myeloid Differentiation (MyD) primary response and Growth Arrest DNA-Damage (Gadd) genes comprise a set of overlapping genes, including known (IRF-1, EGR-1, Jun) and novel (MyD88, Gadd45α MyD118/Gadd45β, GADD45γ, MyD116/Gadd34) genes, that have been cloned by virtue of there being co-ordinately induced upon the onset of terminal myeloid differentiation. This review delineates the role MyD genes play in blood cell development, where they function as positive regulators of terminal differentiation, lineage specific blood cell development and control of blood cell homeostasis, including growth inhibition and apoptosis.

Hematopoietic cells from gadd45a-deficient and gadd45b-deficient mice exhibit impaired stress responses to acute stimulation with cytokines, myeloablation and inflammation

The gadd45 family of gene(s) is rapidly induced by genotoxic stress or by differentiation-inducing cytokines. Using bone marrow (BM) from gadd45a−/−, gadd45b−/− and wild-type (wt) mice, we investigated their role in stress responses of myeloid cells to acute stimulation with differentiating cytokines, myelotoxic agents and inflammatory substances. Bone marrow cells from gadd45a−/− and gadd45b−/− mice displayed compromised myeloid differentiation and higher apoptosis in vitro, following acute stimulation with a variety of differentiating cytokines. Intriguingly, gadd45a−/− and gadd45b−/− colony forming units granulocyte/macrophage progenitors displayed prolonged proliferation capacity compared to wt controls upon re-plating in methylcellulose supplemented with interleukin-3. The recovery of the BM myeloid compartment following 5-Fluorouracil-induced myelo-ablation was much slower in gadd45a−/− and gadd45b−/− mice compared to wt controls. Furthermore, the response of myeloid cells to inflammatory stress, inflicted via intraperitoneal administration of sodium caseinate was impaired in gadd45a−/− and gadd45b−/− mice compared to age-matched wt mice, as indicated by lower percentage of Gr-1-positive cells in the BM and lower number of myeloid cells in peritoneal exudates. Overall, these data indicate that both gadd45a and gadd45b play a role in modulating physiological stress responses of myeloid cells to acute stimulation with differentiating cytokines, myelo-ablation and inflammation. These findings should aid in understanding the response of normal and malignant hematopoietic cells to physiological and chemical stressors including anticancer agents.

p53-independent apoptosis associated with c-Myc-mediated block in myeloid cell differentiation.

Previously we have shown that deregulated expression of c-myc in M1 myeloid leukemic cells blocked IL-6-induced differentiation and its associated growth arrest; however, the cells proliferated at a significantly reduced rate compared to untreated cells. The basis for the increased doubling time of IL-6-treated M1myc cells was found to be due to the induction of a p53-independent apoptotic pathway. The apoptotic response was not completely penetrant; in the same population of cells both proliferation and apoptosis were continuously ongoing. Down-regulation of Bcl-2 was insufficient to account for the apoptotic response, since deregulated expression of Bcl-2 delayed, but did not block, the onset of apoptosis. Furthermore, our results indicated that the IL-6-induced partial hypophosphorylation of the retinoblastoma gene product (Rb), observed in M1myc cells, was not responsible for the apoptotic response. Finally, the findings in M1 cells were extended to myeloid cells derived from the bone marrow of wild type and p53-deficient mice, where the deregulated expression of c-myc was also shown to block terminal differentiation and induce apoptosis independent of p53. These findings provide new insights into how myc participates in the neoplastic process, and how additional mutations can promote more aggressive tumors.